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Sample records for stability maintenance proteins

  1. Cops2 promotes pluripotency maintenance by Stabilizing Nanog Protein and Repressing Transcription

    PubMed Central

    Zhang, Weiyu; Ni, Peiling; Mou, Chunlin; Zhang, Yanqin; Guo, Hongchao; Zhao, Tong; Loh, Yuin-Han; Chen, Lingyi

    2016-01-01

    The COP9 signalosome has been implicated in pluripotency maintenance of human embryonic stem cells. Yet, the mechanism for the COP9 signalosome to regulate pluripotency remains elusive. Through knocking down individual COP9 subunits, we demonstrate that Cops2, but not the whole COP9 signalosome, is essential for pluripotency maintenance in mouse embryonic stem cells. Down-regulation of Cops2 leads to reduced expression of pluripotency genes, slower proliferation rate, G2/M cell cycle arrest, and compromised embryoid differentiation of embryonic stem cells. Cops2 also facilitates somatic cell reprogramming. We further show that Cops2 binds to Nanog protein and prevent the degradation of Nanog by proteasome. Moreover, Cops2 functions as transcriptional corepressor to facilitate pluripotency maintenance. Altogether, our data reveal the essential role and novel mechanisms of Cops2 in pluripotency maintenance. PMID:27226076

  2. Cops2 promotes pluripotency maintenance by Stabilizing Nanog Protein and Repressing Transcription.

    PubMed

    Zhang, Weiyu; Ni, Peiling; Mou, Chunlin; Zhang, Yanqin; Guo, Hongchao; Zhao, Tong; Loh, Yuin-Han; Chen, Lingyi

    2016-01-01

    The COP9 signalosome has been implicated in pluripotency maintenance of human embryonic stem cells. Yet, the mechanism for the COP9 signalosome to regulate pluripotency remains elusive. Through knocking down individual COP9 subunits, we demonstrate that Cops2, but not the whole COP9 signalosome, is essential for pluripotency maintenance in mouse embryonic stem cells. Down-regulation of Cops2 leads to reduced expression of pluripotency genes, slower proliferation rate, G2/M cell cycle arrest, and compromised embryoid differentiation of embryonic stem cells. Cops2 also facilitates somatic cell reprogramming. We further show that Cops2 binds to Nanog protein and prevent the degradation of Nanog by proteasome. Moreover, Cops2 functions as transcriptional corepressor to facilitate pluripotency maintenance. Altogether, our data reveal the essential role and novel mechanisms of Cops2 in pluripotency maintenance. PMID:27226076

  3. Protein Degradation Pathways Regulate the Functions of Helicases in the DNA Damage Response and Maintenance of Genomic Stability

    PubMed Central

    Sommers, Joshua A.; Suhasini, Avvaru N.; Brosh, Robert M.

    2015-01-01

    Degradation of helicases or helicase-like proteins, often mediated by ubiquitin-proteasomal pathways, plays important regulatory roles in cellular mechanisms that respond to DNA damage or replication stress. The Bloom’s syndrome helicase (BLM) provides an example of how helicase degradation pathways, regulated by post-translational modifications and protein interactions with components of the Fanconi Anemia (FA) interstrand cross-link (ICL) repair pathway, influence cell cycle checkpoints, DNA repair, and replication restart. The FANCM DNA translocase can be targeted by checkpoint kinases that exert dramatic effects on FANCM stability and chromosomal integrity. Other work provides evidence that degradation of the F-box DNA helicase (FBH1) helps to balance translesion synthesis (TLS) and homologous recombination (HR) repair at blocked replication forks. Degradation of the helicase-like transcription factor (HLTF), a DNA translocase and ubiquitylating enzyme, influences the choice of post replication repair (PRR) pathway. Stability of the Werner syndrome helicase-nuclease (WRN) involved in the replication stress response is regulated by its acetylation. Turning to transcription, stability of the Cockayne Syndrome Group B DNA translocase (CSB) implicated in transcription-coupled repair (TCR) is regulated by a CSA ubiquitin ligase complex enabling recovery of RNA synthesis. Collectively, these studies demonstrate that helicases can be targeted for degradation to maintain genome homeostasis. PMID:25906194

  4. Protein degradation pathways regulate the functions of helicases in the DNA damage response and maintenance of genomic stability.

    PubMed

    Sommers, Joshua A; Suhasini, Avvaru N; Brosh, Robert M

    2015-04-21

    Degradation of helicases or helicase-like proteins, often mediated by ubiquitin-proteasomal pathways, plays important regulatory roles in cellular mechanisms that respond to DNA damage or replication stress. The Bloom's syndrome helicase (BLM) provides an example of how helicase degradation pathways, regulated by post-translational modifications and protein interactions with components of the Fanconi Anemia (FA) interstrand cross-link (ICL) repair pathway, influence cell cycle checkpoints, DNA repair, and replication restart. The FANCM DNA translocase can be targeted by checkpoint kinases that exert dramatic effects on FANCM stability and chromosomal integrity. Other work provides evidence that degradation of the F-box DNA helicase (FBH1) helps to balance translesion synthesis (TLS) and homologous recombination (HR) repair at blocked replication forks. Degradation of the helicase-like transcription factor (HLTF), a DNA translocase and ubiquitylating enzyme, influences the choice of post replication repair (PRR) pathway. Stability of the Werner syndrome helicase-nuclease (WRN) involved in the replication stress response is regulated by its acetylation. Turning to transcription, stability of the Cockayne Syndrome Group B DNA translocase (CSB) implicated in transcription-coupled repair (TCR) is regulated by a CSA ubiquitin ligase complex enabling recovery of RNA synthesis. Collectively, these studies demonstrate that helicases can be targeted for degradation to maintain genome homeostasis.

  5. Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments

    PubMed Central

    Yu, Lan; Shang, Zeng-Fu; Abdisalaam, Salim; Lee, Kyung-Jong; Gupta, Arun; Hsieh, Jer-Tsong; Asaithamby, Aroumougame; Chen, Benjamin P.C.; Saha, Debabrata

    2016-01-01

    Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability. PMID:27568005

  6. Stabilization Pond Operation and Maintenance Manual.

    ERIC Educational Resources Information Center

    Sexauer, Willard N.; Karn, Roger V.

    This manual provides the waste stabilization pond operator with the basics necessary for the treatment of wastewater in stabilization ponds. The material is organized as a comprehensive guide that follows the normal operation and maintenance procedures from the time the wastewater enters the left station until it leaves the pond. A comprehensive…

  7. Forces stabilizing proteins.

    PubMed

    Nick Pace, C; Scholtz, J Martin; Grimsley, Gerald R

    2014-06-27

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. (1) Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a -CH2- group on folding contributes 1.1±0.5 kcal/mol to protein stability. (2) The burial of non-polar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. (3) Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1±0.8 kcal/mol to protein stability. (4) The contribution of hydrogen bonds to protein stability is strongly context dependent. (5) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (6) Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. (7) Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability.

  8. Forces Stabilizing Proteins

    PubMed Central

    Pace, C. Nick; Scholtz, J. Martin; Grimsley, Gerald R.

    2014-01-01

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. 1. Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH2– group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. 2. The burial of nonpolar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. 3. Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. 4. The contribution of hydrogen bonds to protein stability is strongly context dependent. 5. Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 6. Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. 7. Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability. PMID:24846139

  9. Protein stability in ice.

    PubMed

    Strambini, Giovanni B; Gonnelli, Margherita

    2007-03-15

    This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (DeltaG degrees ) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from -8 to -16 degrees C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (V(L)) in equilibrium with the solid phase. The data establish a threshold, around V(L)=1.5%, below which in ice DeltaG degrees decreases progressively relative to liquid state, up to 3 kcal/mole for V(L)=0.285%. The sharp dependence of DeltaG degrees on V(L) is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in DeltaG degrees is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins.

  10. Stabilized polyacrylic saccharide protein conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1996-01-01

    This invention is directed to water soluble protein polymer conjugates which are stabile in hostile environments. The conjugate comprises a protein which is linked to an acrylic polymer at multiple points through saccharide linker groups.

  11. Chromatin maintenance by a molecular motor protein

    PubMed Central

    Sung, Myong-Hee; Misteli, Tom

    2011-01-01

    The kinesin motor protein KIF4 performs essential functions in mitosis. Like other mitotic kinesins, loss of KIF4 causes spindle defects, aneuploidy, genomic instability and ultimately tumor formation. However, KIF4 is unique among molecular motors in that it resides in the cell nucleus throughout interphase, suggesting a non-mitotic function as well. Here we identify a novel cellular function for a molecular motor protein by demonstrating that KIF4 acts as a modulator of large-scale chromatin architecture during interphase. KIF4 binds globally to chromatin and its absence leads to chromatin decondensation and loss of heterochromatin domains. KIF4-dependent chromatin decondensation has functional consequences by causing replication defects and global mis-regulation of gene expression programs. KIF4 exerts its function in chromatin architecture via regulation of ADP-ribosylation of core and linker histones and by physical interaction and recruitment of chromatin assembly proteins during S-phase. These observations document a novel function for a molecular motor protein in establishment and maintenance of higher order chromatin structure. PMID:22130187

  12. Role of polycomb group protein cbx2/m33 in meiosis onset and maintenance of chromosome stability in the Mammalian germline.

    PubMed

    Baumann, Claudia; De La Fuente, Rabindranath

    2011-01-01

    Polycomb group proteins (PcG) are major epigenetic regulators, essential for establishing heritable expression patterns of developmental control genes. The mouse PcG family member M33/Cbx2 (Chromobox homolog protein 2) is a component of the Polycomb-Repressive Complex 1 (PRC1). Targeted deletion of Cbx2/M33 in mice results in homeotic transformations of the axial skeleton, growth retardation and male-to-female sex reversal. In this study, we tested whether Cbx2 is involved in the control of chromatin remodeling processes during meiosis. Our analysis revealed sex reversal in 28.6% of XY(-/-) embryos, in which a hypoplastic testis and a contralateral ovary were observed in close proximity to the kidney, while the remaining male mutant fetuses exhibited bilateral testicular hypoplasia. Notably, germ cells recovered from Cbx2((XY-/-)) testes on day 18.5 of fetal development exhibited premature meiosis onset with synaptonemal complex formation suggesting a role for Cbx2 in the control of meiotic entry in male germ cells. Mutant females exhibited small ovaries with significant germ cell loss and a high proportion of oocytes with abnormal synapsis and non-homologous interactions at the pachytene stage as well as formation of univalents at diplotene. These defects were associated with failure to resolve DNA double strand breaks marked by persistent γH2AX and Rad51 foci at the late pachytene stage. Importantly, two factors required for meiotic silencing of asynapsed chromatin, ubiquitinated histone H2A (ubH2A) and the chromatin remodeling protein BRCA1, co-localized with fully synapsed chromosome axes in the majority of Cbx2((-/-)) oocytes. These results provide novel evidence that Cbx2 plays a critical and previously unrecognized role in germ cell viability, meiosis onset and homologous chromosome synapsis in the mammalian germline. PMID:22200029

  13. The human Rothmund-Thomson syndrome gene product, RECQL4, localizes to distinct nuclear foci that coincide with proteins involved in the maintenance of genome stability.

    PubMed

    Petkovic, Maja; Dietschy, Tobias; Freire, Raimundo; Jiao, Renjie; Stagljar, Igor

    2005-09-15

    Rothmund-Thomson syndrome (RTS) is a human genetic disorder characterized by genome instability, cancer susceptibility and premature aging. The gene defective in a subset of RTS cases, RECQL4, encodes a member of the RecQ family of DNA helicases. To better define the function of the RECQL4 protein, we have determined its subcellular localization. We have raised antibodies against the N- and C-terminal parts of RECQL4 and could show that in various human cells endogenous RECQL4 forms discrete nuclear foci that colocalize with promyelotic leukaemia protein (PML). The number of foci and their colocalization with PML does not significantly change after induction of different types of DNA damages. Silencing of RECQL4 expression by siRNA causes a significant reduction in RECQL4 nuclear foci formation. Furthermore, we demonstrate that RECQL4 foci coincide with foci formed by human Rad51 and regions of single-stranded DNA after induction of DNA double-strand breaks. In agreement with this, we also show that RECQL4 and Rad51 form a complex in human cells. Our findings suggest a role for RECQL4 in the repair of DNA double-strand breaks by homologous recombination and shed new light onto RECQL4's function in human cells. PMID:16141230

  14. The role of stabilization centers in protein thermal stability.

    PubMed

    Magyar, Csaba; Gromiha, M Michael; Sávoly, Zoltán; Simon, István

    2016-02-26

    The definition of stabilization centers was introduced almost two decades ago. They are centers of noncovalent long range interaction clusters, believed to have a role in maintaining the three-dimensional structure of proteins by preventing their decay due to their cooperative long range interactions. Here, this hypothesis is investigated from the viewpoint of thermal stability for the first time, using a large protein thermodynamics database. The positions of amino acids belonging to stabilization centers are correlated with available experimental thermodynamic data on protein thermal stability. Our analysis suggests that stabilization centers, especially solvent exposed ones, do contribute to the thermal stabilization of proteins.

  15. The role of stabilization centers in protein thermal stability.

    PubMed

    Magyar, Csaba; Gromiha, M Michael; Sávoly, Zoltán; Simon, István

    2016-02-26

    The definition of stabilization centers was introduced almost two decades ago. They are centers of noncovalent long range interaction clusters, believed to have a role in maintaining the three-dimensional structure of proteins by preventing their decay due to their cooperative long range interactions. Here, this hypothesis is investigated from the viewpoint of thermal stability for the first time, using a large protein thermodynamics database. The positions of amino acids belonging to stabilization centers are correlated with available experimental thermodynamic data on protein thermal stability. Our analysis suggests that stabilization centers, especially solvent exposed ones, do contribute to the thermal stabilization of proteins. PMID:26845354

  16. Protein stability: a crystallographer’s perspective

    PubMed Central

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed. PMID:26841758

  17. Ras enhances Myc protein stability.

    PubMed

    Sears, R; Leone, G; DeGregori, J; Nevins, J R

    1999-02-01

    Various experiments have demonstrated a collaborative action of Myc and Ras, both in normal cell growth control as well as during oncogenesis. We now show that Ras enhances the accumulation of Myc activity by stabilizing the Myc protein. Whereas Myc has a very short half-life when produced in the absence of mitogenic signals, due to degradation by the 26S proteasome, the half-life of Myc increases markedly in growth-stimulated cells. This stabilization is dependent on the Ras/Raf/MAPK pathway and is not augmented by proteasome inhibition, suggesting that Ras inhibits the proteasome-dependent degradation of Myc. We propose that one aspect of Myc-Ras collaboration is an ability of Ras to enhance the accumulation of transcriptionally active Myc protein.

  18. Cosolvent Effects on Protein Stability

    NASA Astrophysics Data System (ADS)

    Canchi, Deepak R.; García, Angel E.

    2013-04-01

    Proteins are marginally stable, and the folding/unfolding equilibrium of proteins in aqueous solution can easily be altered by the addition of small organic molecules known as cosolvents. Cosolvents that shift the equilibrium toward the unfolded ensemble are termed denaturants, whereas those that favor the folded ensemble are known as protecting osmolytes. Urea is a widely used denaturant in protein folding studies, and the molecular mechanism of its action has been vigorously debated in the literature. Here we review recent experimental as well as computational studies that show an emerging consensus in this problem. Urea has been shown to denature proteins through a direct mechanism, by interacting favorably with the peptide backbone as well as the amino acid side chains. In contrast, the molecular mechanism by which the naturally occurring protecting osmolyte trimethylamine N-oxide (TMAO) stabilizes proteins is not clear. Recent studies have established the strong interaction of TMAO with water. Detailed molecular simulations, when used with force fields that incorporate these interactions, can provide insight into this problem. We present the development of a model for TMAO that is consistent with experimental observations and that provides physical insight into the role of cosolvent-cosolvent interaction in determining its preferential interaction with proteins.

  19. Recombination and the maintenance of plant organelle genome stability.

    PubMed

    Maréchal, Alexandre; Brisson, Normand

    2010-04-01

    Like their nuclear counterpart, the plastid and mitochondrial genomes of plants have to be faithfully replicated and repaired to ensure the normal functioning of the plant. Inability to maintain organelle genome stability results in plastid and/or mitochondrial defects, which can lead to potentially detrimental phenotypes. Fortunately, plant organelles have developed multiple strategies to maintain the integrity of their genetic material. Of particular importance among these processes is the extensive use of DNA recombination. In fact, recombination has been implicated in both the replication and the repair of organelle genomes. Revealingly, deregulation of recombination in organelles results in genomic instability, often accompanied by adverse consequences for plant fitness. The recent identification of four families of proteins that prevent aberrant recombination of organelle DNA sheds much needed mechanistic light on this important process. What comes out of these investigations is a partial portrait of the recombination surveillance machinery in which plants have co-opted some proteins of prokaryotic origin but have also evolved whole new factors to keep their organelle genomes intact. These new features presumably optimized the protection of plastid and mitochondrial genomes against the particular genotoxic stresses they face.

  20. ATM is required for telomere maintenance and chromosome stability during Drosophila development.

    PubMed

    Silva, Elizabeth; Tiong, Stanley; Pedersen, Michael; Homola, Ellen; Royou, Anne; Fasulo, Barbara; Siriaco, Giorgia; Campbell, Shelagh D

    2004-08-10

    ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia.

  1. Protein interactions in genome maintenance as novel antibacterial targets.

    PubMed

    Marceau, Aimee H; Bernstein, Douglas A; Walsh, Brian W; Shapiro, Walker; Simmons, Lyle A; Keck, James L

    2013-01-01

    Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction "hubs" that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.

  2. Selection maintaining protein stability at equilibrium.

    PubMed

    Miyazawa, Sanzo

    2016-02-21

    The common understanding of protein evolution has been that neutral mutations are fixed by random drift, and a proportion of neutral mutations depending on the strength of structural and functional constraints primarily determines evolutionary rate. Recently it was indicated that fitness costs due to misfolded proteins are a determinant of evolutionary rate and selection originating in protein stability is a driving force of protein evolution. Here we examine protein evolution under the selection maintaining protein stability. Protein fitness is a generic form of fitness costs due to misfolded proteins; s=κexp(ΔG/kT)(1-exp(ΔΔG/kT)), where s and ΔΔG are selective advantage and stability change of a mutant protein, ΔG is the folding free energy of the wildtype protein, and κ is a parameter representing protein abundance and indispensability. The distribution of ΔΔG is approximated to be a bi-Gaussian distribution, which represents structurally slightly- or highly-constrained sites. Also, the mean of the distribution is negatively proportional to ΔG. The evolution of this gene has an equilibrium point (ΔGe) of protein stability, the range of which is consistent with observed values in the ProTherm database. The probability distribution of Ka/Ks, the ratio of nonsynonymous to synonymous substitution rate per site, over fixed mutants in the vicinity of the equilibrium shows that nearly neutral selection is predominant only in low-abundant, non-essential proteins of ΔGe>-2.5 kcal/mol. In the other proteins, positive selection on stabilizing mutations is significant to maintain protein stability at equilibrium as well as random drift on slightly negative mutations, although the average 〈Ka/Ks〉 is less than 1. Slow evolutionary rates can be caused by both high protein abundance/indispensability and large effective population size, which produces positive shifts of ΔΔG through decreasing ΔGe, and strong structural constraints, which directly make

  3. Stability and solubility of proteins from extremophiles.

    PubMed

    Greaves, Richard B; Warwicker, Jim

    2009-03-13

    Charges are important for hyperthermophile protein structure and function. However, the number of charges and their predicted contributions to folded state stability are not correlated, implying that more charge does not imply greater stability. The charge properties that distinguish hyperthermophile proteins also differentiate psychrophile proteins from mesophile proteins, but in the opposite direction and to a smaller extent. We conclude that charge number relates to solubility, whereas protein stability is determined by charge location. Most other structural properties are poorly separated over the ambient temperature range, apart from the burial of certain amino acids. Of particular interest are large non-polar sidechains that tend to increased exposure in proteins evolved to function at higher temperatures. Looking at tryptophan in more detail, this increase is often located close to the termini of secondary structure elements, and is discussed in terms of a novel potential role in protein thermostabilisation.

  4. Stabilized polyacrylic saccharide protein conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1996-02-20

    This invention is directed to water soluble protein polymer conjugates which are stable in hostile environments. The conjugate comprises a protein which is linked to an acrylic polymer at multiple points through saccharide linker groups. 16 figs.

  5. Amphiphiles for protein solubilization and stabilization

    DOEpatents

    Gellman, Samuel Helmer; Chae, Pil Seok; Laible, Philip D.; Wander, Marc J.

    2012-09-11

    The invention provides amphiphiles for manipulating membrane proteins. The amphiphiles can feature carbohydrate-derived hydrophilic groups and branchpoints in the hydrophilic moiety and/or in a lipophilic moiety. Such amphiphiles are useful as detergents for solubilization and stabilization of membrane proteins, including photosynthetic protein superassemblies obtained from bacterial membranes.

  6. Amphiphiles for protein solubilization and stabilization

    DOEpatents

    Gellman, Samuel Helmer; Chae, Pil Seok; Laible, Phillip D; Wander, Marc J

    2014-11-04

    The invention provides amphiphiles for manipulating membrane proteins. The amphiphiles can feature carbohydrate-derived hydrophilic groups and branchpoints in the hydrophilic moiety and/or in a lipophilic moiety. Such amphiphiles are useful as detergents for solubilization and stabilization of membrane proteins, including photosynthetic protein superassemblies obtained from bacterial membranes.

  7. Identification of protein stability determinants in chloroplasts

    PubMed Central

    Apel, Wiebke; Schulze, Waltraud X; Bock, Ralph

    2010-01-01

    Although chloroplast protein stability has long been recognised as a major level of post-translational regulation in photosynthesis and gene expression, the factors determining protein stability in plastids are largely unknown. Here, we have identified stability determinants in vivo by producing plants with transgenic chloroplasts that express a reporter protein whose N- and C-termini were systematically modified. We found that major stability determinants are located in the N-terminus. Moreover, testing of all 20 amino acids in the position after the initiator methionine revealed strong differences in protein stability and indicated an important role of the penultimate N-terminal amino acid residue in determining the protein half life. We propose that the stability of plastid proteins is largely determined by three factors: (i) the action of methionine aminopeptidase (the enzyme that removes the initiator methionine and exposes the penultimate N-terminal amino acid residue), (ii) an N-end rule-like protein degradation pathway, and (iii) additional sequence determinants in the N-terminal region. PMID:20545891

  8. Stability of proteins inside a hydrophobic cavity

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Sharma, Sumit; Kumar, Sanat K.

    2011-03-01

    Previous studies have shown that enclosing a protein in an athermal cavity stabilizes the protein against reversible unfolding by virtue of eliminating many open chain conformations. Examples of such confined spaces include pores in chromatographic columns, Anfinsen's cage in Chaperonins, interiors of Ribosomes or regions of steric occlusion inside cells. However, the situation is more complex inside a hydrophobic cavity. The protein has a tendency to adsorb on the surface of the hydrophobic cavity, but at the same time it loses conformational entropy because of confinement. We study this system using a simple Hydrophobic Polar (HP) lattice protein model. Canonical Monte Carlo (MC) simulations at different temperatures and surface hydrophobicity show that proteins are stabilized at low and moderate hydrophobicity upon adsorption. The range of surface hydrophobicity over which a protein is stable increases with a decrease in radius of the cavity.

  9. Probing protein stability with unnatural amino acids

    SciTech Connect

    Mendel, D.; Ellman, J.A.; Zhiyuh Chang; Veenstra, D.L.; Kollman, P.A.; Schultz, P.G. )

    1992-06-26

    Unnatural amino acid mutagenesis, in combination with molecular modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (1) leucine (wt), norvaline, ethylglycine, and alanine to measure the cost of stepwise removal of methyl groups from the hydrophobic core, (2) norvaline and O-methyl serine to evaluate the effects of side chain solvation, and (3) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing density and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.

  10. Cytosolic Selection Systems To Study Protein Stability

    PubMed Central

    Malik, Ajamaluddin; Mueller-Schickert, Antje

    2014-01-01

    Here we describe biosensors that provide readouts for protein stability in the cytosolic compartment of prokaryotes. These biosensors consist of tripartite sandwich fusions that link the in vitro stability or aggregation susceptibility of guest proteins to the in vivo resistance of host cells to the antibiotics kanamycin, spectinomycin, and nourseothricin. These selectable markers confer antibiotic resistance in a wide range of hosts and are easily quantifiable. We show that mutations within guest proteins that affect their stability alter the antibiotic resistances of the cells expressing the biosensors in a manner that is related to the in vitro stabilities of the mutant guest proteins. In addition, we find that polyglutamine tracts of increasing length are associated with an increased tendency to form amyloids in vivo and, in our sandwich fusion system, with decreased resistance to aminoglycoside antibiotics. We demonstrate that our approach allows the in vivo analysis of protein stability in the cytosolic compartment without the need for prior structural and functional knowledge. PMID:25266385

  11. Selenium and its' role in the maintenance of genomic stability.

    PubMed

    Ferguson, Lynnette R; Karunasinghe, Nishi; Zhu, Shuotun; Wang, Alice H

    2012-05-01

    Selenium (Se) is an essential micronutrient for humans, acting as a component of the unusual amino acids, selenocysteine (Se-Cys) and selenomethionine (Se-Met). Where Se levels are low, the cell cannot synthesise selenoproteins, although some selenoproteins and some tissues are prioritised over others. Characterised functions of known selenoproteins, include selenium transport (selenoprotein P), antioxidant/redox properties (glutathione peroxidases (GPxs), thioredoxin reductases and selenoprotein P) and anti-inflammatory properties (selenoprotein S and GPx4). Various forms of Se are consumed as part of a normal diet, or as a dietary supplement. Supplementation of tissue culture media, animal or human diets with moderate levels of certain Se compounds may protect against the formation of DNA adducts, DNA or chromosome breakage, and chromosome gain or loss. Protective effects have also been shown on mitochondrial DNA, and on telomere length and function. Some of the effects of Se compounds on gene expression may relate to modulation of DNA methylation or inhibition of histone deacetylation. Despite a large number of positive effects of selenium and selenoproteins in various model systems, there have now been some human clinical trials that have shown adverse effects of Se supplementation, according to various endpoints. Too much Se is as harmful as too little, with animal models showing a "U"-shaped efficacy curve. Current recommended daily allowances differ among countries, but are generally based on the amount of Se necessary to saturate GPx enzymes. However, increasing evidence suggests that other enzymes may be more important than GPx for Se action, that optimal levels may depend upon the form of Se being ingested, and vary according to genotype. New paradigms, possibly involving nutrigenomic tools, will be necessary to optimise the forms and levels of Se desirable for maximum protection of genomic stability in all humans.

  12. Impact of reconstituted cytosol on protein stability

    PubMed Central

    Sarkar, Mohona; Smith, Austin E.; Pielak, Gary J.

    2013-01-01

    Protein stability is usually studied in simple buffered solutions, but most proteins function inside cells, where the heterogeneous and crowded environment presents a complex, nonideal system. Proteins are expected to behave differently under cellular crowding owing to two types of contacts: hard-core repulsions and weak, chemical interactions. The effect of hard-core repulsions is purely entropic, resulting in volume exclusion owing to the mere presence of the crowders. The weak interactions can be repulsive or attractive, thus enhancing or diminishing the excluded volume, respectively. We used a reductionist approach to assess the effects of intracellular crowding. Escherichia coli cytoplasm was dialyzed, lyophilized, and resuspended at two concentrations. NMR-detected amide proton exchange was then used to quantify the stability of the globular protein chymotrypsin inhibitor 2 (CI2) in these crowded solutions. The cytosol destabilizes CI2, and the destabilization increases with increasing cytosol concentration. This observation shows that the cytoplasm interacts favorably, but nonspecifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. The effects of the cytosol are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions. PMID:24218610

  13. Effects of confinement on protein folding and protein stability

    NASA Astrophysics Data System (ADS)

    Ping, G.; Yuan, J. M.; Vallieres, M.; Dong, H.; Sun, Z.; Wei, Y.; Li, F. Y.; Lin, S. H.

    2003-05-01

    In a cell, proteins exist in crowded environments; these environments influence their stability and dynamics. Similarly, for an enzyme molecule encapsulated in an inorganic cavity as in biosensors or biocatalysts, confinement and even surface effects play important roles in its stability and dynamics. Using a minimalist model (two-dimensional HP lattice model), we have carried out Monte Carlo simulations to study confinement effects on protein stability. We have calculated heat capacity as a function of temperature using the histogram method and results obtained show that confinement tends to stabilize the folded conformations, consistent with experimental results (some reported here) and previous theoretical analyses. Furthermore, for a protein molecule tethered to a solid surface the stabilization effect can be even greater. We have also investigated the effects of confinement on the kinetics of the refolding and unfolding processes as functions of temperature and box size. As expected, unfolding time increases as box size decreases, however, confinement affects folding times in a more complicated way. Our theoretical results agree with our experimentally observed trends that thermal stability of horseradish peroxidase and acid phosphatase, encapsulated in mesoporous silica, increases as the pore size of the silica matrix decreases.

  14. Enhancing protein stability with extended disulfide bonds.

    PubMed

    Liu, Tao; Wang, Yan; Luo, Xiaozhou; Li, Jack; Reed, Sean A; Xiao, Han; Young, Travis S; Schultz, Peter G

    2016-05-24

    Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.

  15. Trehalose glycopolymers as excipients for protein stabilization.

    PubMed

    Lee, Juneyoung; Lin, En-Wei; Lau, Uland Y; Hedrick, James L; Bat, Erhan; Maynard, Heather D

    2013-08-12

    Herein, the synthesis of four different trehalose glycopolymers and investigation of their ability to stabilize proteins to heat and lyophilization stress are described. The disaccharide, α,α-trehalose, was modified with a styrenyl acetal, methacrylate acetal, styrenyl ether, or methacrylate moiety resulting in four different monomers. These monomers were then separately polymerized using free radical polymerization with azobisisobutyronitrile (AIBN) as an initiator to synthesize the glycopolymers. Horseradish peroxidase and glucose oxidase were incubated at 70 and 50 °C, respectively, and β-galactosidase was lyophilized multiple times in the presence of various ratios of the polymers or trehalose. The protein activities were subsequently tested and found to be significantly higher when the polymers were present during the stress compared to no additive and to equivalent amounts of trehalose. Different molecular weights (10 kDa, 20 kDa, and 40 kDa) were tested, and all were equivalent in their stabilization ability. However, some subtle differences were observed regarding stabilization ability between the different polymer samples, depending on the stress. Small molecules such as benzyl ether trehalose were not better stabilizers than trehalose, and the trehalose monomer decreased protein activity, suggesting that hydrophobized trehalose was not sufficient and that the polymeric structure was required. In addition, cytotoxicity studies with NIH 3T3 mouse embryonic fibroblast cells, RAW 264.7 murine macrophages, human dermal fibroblasts (HDFs), and human umbilical vein endothelial cells (HUVECs) were conducted with polymer concentrations up to 8 mg/mL. The data showed that all four polymers were noncytotoxic for all tested concentrations. The results together suggest that trehalose glycopolymers are promising as additives to protect proteins from a variety of stressors. PMID:23777473

  16. Axonal maintenance, glia, exosomes, and heat shock proteins.

    PubMed

    Tytell, Michael; Lasek, Raymond J; Gainer, Harold

    2016-01-01

    Of all cellular specializations, the axon is especially distinctive because it is a narrow cylinder of specialized cytoplasm called axoplasm with a length that may be orders of magnitude greater than the diameter of the cell body from which it originates. Thus, the volume of axoplasm can be much greater than the cytoplasm in the cell body. This fact raises a logistical problem with regard to axonal maintenance. Many of the components of axoplasm, such as soluble proteins and cytoskeleton, are slowly transported, taking weeks to months to travel the length of axons longer than a few millimeters after being synthesized in the cell body. Furthermore, this slow rate of supply suggests that the axon itself might not have the capacity to respond fast enough to compensate for damage to transported macromolecules. Such damage is likely in view of the mechanical fragility of an axon, especially those innervating the limbs, as rapid limb motion with high impact, like running, subjects the axons in the limbs to considerable mechanical force. Some researchers have suggested that local, intra-axonal protein synthesis is the answer to this problem. However, the translational state of axonal RNAs remains controversial. We suggest that glial cells, which envelop all axons, whether myelinated or not, are the local sources of replacement and repair macromolecules for long axons. The plausibility of this hypothesis is reinforced by reviewing several decades of work on glia-axon macromolecular transfer, together with recent investigations of exosomes and other extracellular vesicles, as vehicles for the transmission of membrane and cytoplasmic components from one cell to another. PMID:26962444

  17. Axonal maintenance, glia, exosomes, and heat shock proteins

    PubMed Central

    Tytell, Michael; Lasek, Raymond J.; Gainer, Harold

    2016-01-01

    Of all cellular specializations, the axon is especially distinctive because it is a narrow cylinder of specialized cytoplasm called axoplasm with a length that may be orders of magnitude greater than the diameter of the cell body from which it originates. Thus, the volume of axoplasm can be much greater than the cytoplasm in the cell body. This fact raises a logistical problem with regard to axonal maintenance. Many of the components of axoplasm, such as soluble proteins and cytoskeleton, are slowly transported, taking weeks to months to travel the length of axons longer than a few millimeters after being synthesized in the cell body. Furthermore, this slow rate of supply suggests that the axon itself might not have the capacity to respond fast enough to compensate for damage to transported macromolecules. Such damage is likely in view of the mechanical fragility of an axon, especially those innervating the limbs, as rapid limb motion with high impact, like running, subjects the axons in the limbs to considerable mechanical force. Some researchers have suggested that local, intra-axonal protein synthesis is the answer to this problem. However, the translational state of axonal RNAs remains controversial. We suggest that glial cells, which envelop all axons, whether myelinated or not, are the local sources of replacement and repair macromolecules for long axons. The plausibility of this hypothesis is reinforced by reviewing several decades of work on glia-axon macromolecular transfer, together with recent investigations of exosomes and other extracellular vesicles, as vehicles for the transmission of membrane and cytoplasmic components from one cell to another. PMID:26962444

  18. Maintenance of native-like protein dynamics may not be required for engineering functional proteins.

    PubMed

    Gobeil, Sophie M C; Clouthier, Christopher M; Park, Jaeok; Gagné, Donald; Berghuis, Albert M; Doucet, Nicolas; Pelletier, Joelle N

    2014-10-23

    Proteins are dynamic systems, and understanding dynamics is critical for fully understanding protein function. Therefore, the question of whether laboratory engineering has an impact on protein dynamics is of general interest. Here, we demonstrate that two homologous, naturally evolved enzymes with high degrees of structural and functional conservation also exhibit conserved dynamics. Their similar set of slow timescale dynamics is highly restricted, consistent with evolutionary conservation of a functionally important feature. However, we also show that dynamics of a laboratory-engineered chimeric enzyme obtained by recombination of the two homologs exhibits striking difference on the millisecond timescale, despite function and high-resolution crystal structure (1.05 Å) being conserved. The laboratory-engineered chimera is thus functionally tolerant to modified dynamics on the timescale of catalytic turnover. Tolerance to dynamic variation implies that maintenance of native-like protein dynamics may not be required when engineering functional proteins.

  19. How Hofmeister ion interactions affect protein stability.

    PubMed Central

    Baldwin, R L

    1996-01-01

    Model compound studies in the literature show how Hofmeister ion interactions affect protein stability. Although model compound results are typically obtained as salting-out constants, they can be used to find out how the interactions affect protein stability. The null point in the Hofmeister series, which divides protein denaturants from stabilizers, arises from opposite interactions with different classes of groups: Hofmeister ions salt out nonpolar groups and salt in the peptide group. Theories of how Hofmeister ion interactions work need to begin by explaining the mechanisms of these two classes of interactions. Salting-out nonpolar groups has been explained by the cavity model, but its use is controversial. When applied to model compound data, the cavity model 1) uses surface tension increments to predict the observed values of the salting-out constants, within a factor of 3, and 2) predicts that the salting-out constant should increase with the number of carbon atoms in the aliphatic side chain of an amino acid, as observed. The mechanism of interaction between Hofmeister ions and the peptide group is not well understood, and it is controversial whether this interaction is ion-specific, or whether it is nonspecific and the apparent specificity resides in interactions with nearby nonpolar groups. A nonspecific salting-in interaction is known to occur between simple ions and dipolar molecules; it depends on ionic strength, not on position in the Hofmeister series. A theory by Kirkwood predicts the strength of this interaction and indicates that it depends on the first power of the ionic strength. Ions interact with proteins in various ways besides the Hofmeister ion interactions discussed here, especially by charge interactions. Much of what is known about these interactions comes from studies by Serge Timasheff and his co-workers. A general model, suitable for analyzing diverse ion-protein interactions, is provided by the two-domain model of Record and co

  20. Multifunctional role of ATM/Tel1 kinase in genome stability: from the DNA damage response to telomere maintenance.

    PubMed

    Di Domenico, Enea Gino; Romano, Elena; Del Porto, Paola; Ascenzioni, Fiorentina

    2014-01-01

    The mammalian protein kinase ataxia telangiectasia mutated (ATM) is a key regulator of the DNA double-strand-break response and belongs to the evolutionary conserved phosphatidylinositol-3-kinase-related protein kinases. ATM deficiency causes ataxia telangiectasia (AT), a genetic disorder that is characterized by premature aging, cerebellar neuropathy, immunodeficiency, and predisposition to cancer. AT cells show defects in the DNA damage-response pathway, cell-cycle control, and telomere maintenance and length regulation. Likewise, in Saccharomyces cerevisiae, haploid strains defective in the TEL1 gene, the ATM ortholog, show chromosomal aberrations and short telomeres. In this review, we outline the complex role of ATM/Tel1 in maintaining genomic stability through its control of numerous aspects of cellular survival. In particular, we describe how ATM/Tel1 participates in the signal transduction pathways elicited by DNA damage and in telomere homeostasis and its importance as a barrier to cancer development.

  1. Flavor and stability of milk proteins.

    PubMed

    Smith, T J; Campbell, R E; Jo, Y; Drake, M A

    2016-06-01

    A greater understanding of the nature and source of dried milk protein ingredient flavor(s) is required to characterize flavor stability and identify the sources of flavors. The objective of this study was to characterize the flavor and flavor chemistry of milk protein concentrates (MPC 70, 80, 85), isolates (MPI), acid and rennet caseins, and micellar casein concentrate (MCC) and to determine the effect of storage on flavor and functionality of milk protein concentrates using instrumental and sensory techniques. Spray-dried milk protein ingredients (MPC, MPI, caseins, MCC) were collected in duplicate from 5 commercial suppliers or manufactured at North Carolina State University. Powders were rehydrated and evaluated in duplicate by descriptive sensory analysis. Volatile compounds were extracted by solid phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry. Compounds were identified by comparison of retention indices, odor properties, and mass spectra against reference standards. A subset of samples was selected for further analysis using direct solvent extraction with solvent-assisted flavor extraction, and aroma extract dilution analysis. External standard curves were created to quantify select volatile compounds. Pilot plant manufactured MPC were stored at 3, 25, and 40°C (44% relative humidity). Solubility, furosine, sensory properties, and volatile compound analyses were performed at 0, 1, 3, 6, and 12 mo. Milk proteins and caseins were diverse in flavor and exhibited sweet aromatic and cooked/milky flavors as well as cardboard, brothy, tortilla, soapy, and fatty flavors. Key aroma active compounds in milk proteins and caseins were 2-aminoacetophenone, nonanal, 1-octen-3-one, dimethyl trisulfide, 2-acetyl-1-pyrroline, heptanal, methional, 1-hexen-3-one, hexanal, dimethyl disulfide, butanoic acid, and acetic acid. Stored milk proteins developed animal and burnt sugar flavors over time. Solubility of

  2. Stability analysis of an autocatalytic protein model

    NASA Astrophysics Data System (ADS)

    Lee, Julian

    2016-05-01

    A self-regulatory genetic circuit, where a protein acts as a positive regulator of its own production, is known to be the simplest biological network with a positive feedback loop. Although at least three components—DNA, RNA, and the protein—are required to form such a circuit, stability analysis of the fixed points of this self-regulatory circuit has been performed only after reducing the system to a two-component system, either by assuming a fast equilibration of the DNA component or by removing the RNA component. Here, stability of the fixed points of the three-component positive feedback loop is analyzed by obtaining eigenvalues of the full three-dimensional Hessian matrix. In addition to rigorously identifying the stable fixed points and saddle points, detailed information about the system can be obtained, such as the existence of complex eigenvalues near a fixed point.

  3. Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer

    PubMed Central

    Miller, Kyle M.

    2016-01-01

    Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer. PMID:27631103

  4. Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer.

    PubMed

    Gong, Fade; Chiu, Li-Ya; Miller, Kyle M

    2016-09-01

    Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer.

  5. Monitoring prion protein stability by NMR.

    PubMed

    Julien, Olivier; Graether, Steffen P; Sykes, Brian D

    2009-01-01

    Prion diseases, or transmissible spongiform encephalopathies (TSE), are a group of fatal neurological diseases that affect both humans and animals. At the end of the 20th century, bovine spongiform encephalopathy (BSE), better known as mad cow disease, was shown to be transmissible to humans. This resulted in considerable concern for public health and a number of questions for scientists. The first question answered was the possible source of the disease, which appears to be the prion protein (PrP). There are two major forms of this protein: the native, noninfectious form (PrP(C)), and the misfolded infectious form (PrP(Sc)). PrP(C) is mainly alpha-helical in structure, whereas PrP(Sc) aggregates into an assembly of beta-sheets, forming amyloid fibrils. Since the first solution structure of the noninfectious form of the mouse prion protein, about 30 structures of the globular portion of PrP(C) have been characterized from different organisms. However, only a few minor differences are observed when comparing one PrP(C) structure to another. The key to understanding prion formation may then be not in the structure of PrP(C), but in the mechanism underlying PrP(C) unfolding and then conversion into a misfolded fibril state. To identify the possible region(s) of PrP(C) responsible for initiating the conversion into the amyloid fibril formation, nuclear magnetic resonance (NMR) was applied to characterize the stability and structure of PrP(C) and intermediate states during the conversion from PrP(C) to PrP(Sc). Subsequently urea was used to induce unfolding, and data analysis revealed region-specific structural stabilities that may bring insights into the mechanisms underlying conversion of protein into an infectious prion. PMID:19697241

  6. Invaded grassland communities have altered stability-maintenance mechanisms but equal stability compared to native communities.

    PubMed

    Wilsey, Brian J; Daneshgar, Pedram P; Hofmockel, Kirsten; Polley, H Wayne

    2014-01-01

    Theory predicts that stability should increase with diversity via several mechanisms. We tested predictions in a 5-year experiment that compared low-diversity exotic to high-diversity native plant mixtures under two irrigation treatments. The study included both wet and dry years. Variation in biomass across years (CV) was 50% lower in mixtures than monocultures of both native and exotic species. Growth among species was more asynchronous and overyielding values were greater during and after a drought in native than exotic mixtures. Mean-variance slopes indicated strong portfolio effects in both community types, but the intercept was higher for exotics than for natives, suggesting that exotics were inherently more variable than native species. However, this failed to result in higher CV's in exotic communities because species that heavily dominated plots tended to have lower than expected variance. Results indicate that diversity-stability mechanisms are altered in invaded systems compared to native ones they replaced. PMID:24325664

  7. Invaded grassland communities have altered stability-maintenance mechanisms but equal stability compared to native communities.

    PubMed

    Wilsey, Brian J; Daneshgar, Pedram P; Hofmockel, Kirsten; Polley, H Wayne

    2014-01-01

    Theory predicts that stability should increase with diversity via several mechanisms. We tested predictions in a 5-year experiment that compared low-diversity exotic to high-diversity native plant mixtures under two irrigation treatments. The study included both wet and dry years. Variation in biomass across years (CV) was 50% lower in mixtures than monocultures of both native and exotic species. Growth among species was more asynchronous and overyielding values were greater during and after a drought in native than exotic mixtures. Mean-variance slopes indicated strong portfolio effects in both community types, but the intercept was higher for exotics than for natives, suggesting that exotics were inherently more variable than native species. However, this failed to result in higher CV's in exotic communities because species that heavily dominated plots tended to have lower than expected variance. Results indicate that diversity-stability mechanisms are altered in invaded systems compared to native ones they replaced.

  8. Protein hydrolysis by immobilized and stabilized trypsin.

    PubMed

    Marques, Daniela; Pessela, Benavides C; Betancor, Lorena; Monti, Rubens; Carrascosa, Alfonso V; Rocha-Martin, Javier; Guisán, Jose M; Fernandez-Lorente, Gloria

    2011-01-01

    The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pK(a) amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pK(a) amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins.

  9. Cumulative versus Stabilizing Effects of Methadone Maintenance: A Quasi-Experimental Study Using Longitudinal Self-Report Data.

    ERIC Educational Resources Information Center

    Powers, Keiko Ichikawa; Anglin, M. Douglas

    1993-01-01

    Whether methadone maintenance treatment demonstrates cumulative (rehabilitative) or stabilizing effects on behavior of narcotics addicts over multiple treatment episodes was studied involving 993 addicts in a quasi-experimental design. Observed behavioral changes and longitudinal self-reports indicate stabilizing, but not cumulative, effects. (SLD)

  10. Characterisation of protein stability in rod-insert vaginal rings.

    PubMed

    Pattani, Aditya; Lowry, Deborah; Curran, Rhonda M; McGrath, Stephanie; Kett, Vicky L; Andrews, Gavin P; Malcolm, R Karl

    2012-07-01

    A major goal in vaccine development is elimination of the 'cold chain', the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4 °C, but not when stored at 40 °C/75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40 °C/75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation compared to the original formulation when stored at 40 °C/75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general. PMID

  11. Road Maintenance Experience Using Polyurethane (PU) Foam Injection System and Geocrete Soil Stabilization as Ground Rehabilitation

    NASA Astrophysics Data System (ADS)

    Fakhar, A. M. M.; Asmaniza, A.

    2016-07-01

    There are many types of ground rehabilation and improvement that can be consider and implement in engineering construction works for soil improvement in order to prevent road profile deformation in later stage. However, when comes to road maintenance especially on operated expressways, not all method can be apply directly as it must comply to opreation's working window and lane closure basis. Key factors that considering ideal proposal for ground rehabilitation are time, cost, quality and most importantly practicality. It should provide long lifespan structure in order to reduce continuous cycle of maintenance. Thus, this paper will present two approaches for ground rehabilitation, namely Polyurethane (PU) Foam Injection System and Geocrete Soil Stabilization. The first approach is an injection system which consists two-parts chemical grout of Isocynate and Polyol when mixed together within soil structure through injection will polymerized with volume expansion. The strong expansion of grouting causes significant compression and compacting of the surrounding soil and subsequently improve ground properties and uplift sunken structure. The later is a cold in-place recyclying whereby mixture process that combines in-situ soil materials, cement, white powder (alkaline) additive and water to produce hard yet flexible and durable ground layer that act as solid foundation with improved bearing capacity. The improvement of the mechanical behaviour of soil through these two systems is investigated by an extensive testing programme which includes in-situ and laboratory test in determining properties such as strength, stiffness, compressibility, bearing capacity, differential settlement and etc.

  12. Designed Modular Proteins as Scaffolds To Stabilize Fluorescent Nanoclusters.

    PubMed

    Couleaud, Pierre; Adan-Bermudez, Sergio; Aires, Antonio; Mejías, Sara H; Sot, Begoña; Somoza, Alvaro; Cortajarena, Aitziber L

    2015-12-14

    Proteins have been used as templates to stabilize fluorescent metal nanoclusters thus obtaining stable fluorescent structures, and their fluorescent properties being modulated by the type of protein employed. Designed consensus tetratricopeptide repeat (CTPR) proteins are suited candidates as templates for the stabilization of metal nanoclusters due to their modular structural and functional properties. Here, we have studied the ability of CTPR proteins to stabilize fluorescent gold nanoclusters giving rise to designed functional hybrid nanostructures. First, we have investigated the influence of the number of CTPR units, as well as the presence of cysteine residues in the CTPR protein, on the fluorescent properties of the protein-stabilized gold nanoclusters. Synthetic protocols to retain the protein structure and function have been developed, since the structural and functional integrity of the protein template is critical for further applications. Finally, as a proof-of-concept, a CTPR module with specific binding capabilities has been used to stabilize gold nanoclusters with positive results. Remarkably, the protein-stabilized gold nanocluster obtained combines both the fluorescence properties of the nanoclusters and the functional properties of the protein. The fluorescence changes in nanoclusters fluorescence have been successfully used as a sensor to detect when the specific ligand was recognized by the CTPR module.

  13. SPLINTS: small-molecule protein ligand interface stabilizers.

    PubMed

    Fischer, Eric S; Park, Eunyoung; Eck, Michael J; Thomä, Nicolas H

    2016-04-01

    Regulatory protein-protein interactions are ubiquitous in biology, and small molecule protein-protein interaction inhibitors are an important focus in drug discovery. Remarkably little attention has been given to the opposite strategy-stabilization of protein-protein interactions, despite the fact that several well-known therapeutics act through this mechanism. From a structural perspective, we consider representative examples of small molecules that induce or stabilize the association of protein domains to inhibit, or alter, signaling for nuclear hormone, GTPase, kinase, phosphatase, and ubiquitin ligase pathways. These SPLINTS (small-molecule protein ligand interface stabilizers) drive interactions that are in some cases physiologically relevant, and in others entirely adventitious. The diverse structural mechanisms employed suggest approaches for a broader and systematic search for such compounds in drug discovery. PMID:26829757

  14. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  15. Cold denaturation as a tool to measure protein stability.

    PubMed

    Sanfelice, Domenico; Temussi, Piero Andrea

    2016-01-01

    Protein stability is an important issue for the interpretation of a wide variety of biological problems but its assessment is at times difficult. The most common parameter employed to describe protein stability is the temperature of melting, at which the populations of folded and unfolded species are identical. This parameter may yield ambiguous results. It would always be preferable to measure the whole stability curve. The calculation of this curve is greatly facilitated whenever it is possible to observe cold denaturation. Using Yfh1, one of the few proteins whose cold denaturation occurs at neutral pH and low ionic strength, we could measure the variation of its full stability curve under several environmental conditions. Here we show the advantages of gauging stability as a function of external variables using stability curves.

  16. Designing Whey Protein-Polysaccharide Particles for Colloidal Stability.

    PubMed

    Wagoner, Ty; Vardhanabhuti, Bongkosh; Foegeding, E Allen

    2016-01-01

    Interactions between whey proteins and polysaccharides, in particular the formation of food-grade soluble complexes, are of interest because of potential functional and health benefits. A specific application that has not received much attention is the use of complexes for enhanced colloidal stability of protein sols, such as protein-containing beverages. In beverages, the primary goal is the formation of complexes that remain dispersed after thermal processing and extended storage. This review highlights recent progress in the area of forming whey protein-polysaccharide soluble complexes that would be appropriate for beverage applications. Research in this area indicates that soluble complexes can be formed and stabilized that are reasonably small in size and possess a large surface charge that would predict colloidal stability. Selection of specific proteins and polysaccharides can be tailored to desired conditions. The principal challenges involve overcoming restrictions on protein concentration and ensuring that protein remains bioavailable.

  17. Protein kinase M ζ and the maintenance of long-term memory.

    PubMed

    Zhang, Yang; Zong, Wei; Zhang, Lei; Ma, Yuanye; Wang, Jianhong

    2016-10-01

    Although various molecules have been found to mediate the processes of memory acquisition and consolidation, the molecular mechanism to maintain memory still remains elusive. In recent years, a molecular pathway focusing on protein kinase Mζ (PKMζ) has become of interest to researchers because of its potential role in long-term memory maintenance. PKMζ is an isoform of protein kinase C (PKC) and has a related structure that influences its function in maintaining memory. Considerable evidence has been gathered on PKMζ activity, including loss of function studies using PKMζ inhibitors, such as PKMζ inhibitory peptide (ZIP), suggesting PKMζ plays an important role in long-term memory maintenance. This review provides an overview of the role of PKMζ in long-term memory and outlines the molecular structure of PKMζ, the molecular mechanism of PKMζ in long-term memory maintenance and future directions of PKMζ research.

  18. INCREASING PROTEIN STABILITY BY IMPROVING BETA-TURNS

    PubMed Central

    Fu, Hailong; Grimsley, Gerald R.; Razvi, Abbas; Scholtz, J. Martin; Pace, C. Nick

    2009-01-01

    Our goal was to gain a better understanding of how protein stability can be increased by improving β-turns. We studied 22 β-turns in nine proteins with 66 to 370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some β-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein (HPr), Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase α-subunit (TSα), and Maltose binding protein (MBP). Of the fifteen single proline mutations, 11increased stability (Average = 0.8 ± 0.3; Range = 0.3 – 1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. Based on this and our previous work, we conclude that proteins can generally be stabilized by replacing non-proline residues with proline residues at the i + 1 position of Type I and II β-turns and at the i position in Type II β-turns. Other turn positions can sometimes be used if the φ angle is near −60° for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in β-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in β-turns that could be replaced by Gly to increase protein stability. Improving β-turns by substituting Pro residues is a generally useful way of increasing protein stability. PMID:19626709

  19. Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

    PubMed

    Oulhen, Nathalie; Wessel, Gary M

    2016-10-01

    Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability.

  20. Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

    PubMed

    Oulhen, Nathalie; Wessel, Gary M

    2016-10-01

    Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. PMID:27424271

  1. Regulation of TET Protein Stability by Calpains

    PubMed Central

    Wang, Yu; Zhang, Yi

    2014-01-01

    SUMMARY DNA methylation at the fifth position of cytosine (5mC) is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (Tet) family of proteins in regulating DNA methylation dynamics. Three Tet genes have been identified in mammals, and they all encode for proteins capable of oxidizing 5mC as part of the DNA demethylation process. While regulation of Tet expression at the transcriptional level is well documented, how TET proteins are regulated at post-translational level is poorly understood. In this study, we report that all three TET proteins are direct substrates of calpains, a family of calcium-dependent proteases. Specifically, calpain1 mediates TET1 and TET2 turnover in mouse ES cells, and calpain2 regulates TET3 level during differentiation. This study provides the first evidence that TET proteins are subject to calpain-mediated degradation. PMID:24412366

  2. Two Putative Polysaccharide Deacetylases Are Required for Osmotic Stability and Cell Shape Maintenance in Bacillus anthracis.

    PubMed

    Arnaouteli, Sofia; Giastas, Petros; Andreou, Athina; Tzanodaskalaki, Mary; Aldridge, Christine; Tzartos, Socrates J; Vollmer, Waldemar; Eliopoulos, Elias; Bouriotis, Vassilis

    2015-05-22

    Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) β-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis. PMID:25825488

  3. Two Putative Polysaccharide Deacetylases Are Required for Osmotic Stability and Cell Shape Maintenance in Bacillus anthracis*

    PubMed Central

    Arnaouteli, Sofia; Giastas, Petros; Andreou, Athina; Tzanodaskalaki, Mary; Aldridge, Christine; Tzartos, Socrates J.; Vollmer, Waldemar; Eliopoulos, Elias; Bouriotis, Vassilis

    2015-01-01

    Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) β-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis. PMID:25825488

  4. RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens.

    PubMed

    Odahara, Masaki; Inouye, Takayuki; Nishimura, Yoshiki; Sekine, Yasuhiko

    2015-11-01

    Chloroplast DNA (cpDNA) encodes essential genes for chloroplast functions, including photosynthesis. Homologous recombination occurs frequently in cpDNA; however, its significance and underlying mechanism remain poorly understood. In this study, we analyzed the role of a nuclear-encoded chloroplast-localized homolog of RecA recombinase, which is a key factor in homologous recombination in bacteria, in the moss Physcomitrella patens. Complete knockout (KO) of the P. patens chloroplast RecA homolog RECA2 caused a modest growth defect and conferred sensitivity to methyl methanesulfonate and UV. The KO mutant exhibited low recovery of cpDNA from methyl methanesulfonate damage, suggesting that RECA2 knockout impairs repair of damaged cpDNA. The RECA2 KO mutant also exhibited reduced cpDNA copy number and an elevated level of cpDNA molecule resulting from aberrant recombination between short dispersed repeats (13-63 bp), indicating that the RECA2 KO chloroplast genome was destabilized. Taken together, these data suggest a dual role for RECA2 in the maintenance of chloroplast genome stability: RECA2 suppresses aberrant recombination between short dispersed repeats and promotes repair of damaged DNA.

  5. Stabilization of supercooled fluids by thermal hysteresis proteins.

    PubMed Central

    Wilson, P W; Leader, J P

    1995-01-01

    It has been reported that thermal hysteresis proteins found in many cold-hardy, freeze-avoiding arthropods stabilize their supercooled body fluids. We give evidence that fish antifreeze proteins, which also produce thermal hysteresis, bind to and reduce the efficiency of heterogenous nucleation sites, rather than binding to embryonic ice nuclei. We discuss both possible mechanisms for stabilization of supercooled body fluids and also describe a new method for measuring and defining the supercooling point of small volumes of liquid. PMID:7612853

  6. Effects of Glycosylation on the Stability of Protein Pharmaceuticals

    PubMed Central

    SOLÁ, RICARDO J.; GRIEBENOW, KAI

    2008-01-01

    In recent decades, protein-based therapeutics have substantially expanded the field of molecular pharmacology due to their outstanding potential for the treatment of disease. Unfortunately, protein pharmaceuticals display a series of intrinsic physical and chemical instability problems during their production, purification, storage, and delivery that can adversely impact their final therapeutic efficacies. This has prompted an intense search for generalized strategies to engineer the long-term stability of proteins during their pharmaceutical employment. Due to the well known effect that glycans have in increasing the overall stability of glycoproteins, rational manipulation of the glycosylation parameters through glycoengineering could become a promising approach to improve both the in vitro and in vivo stability of protein pharmaceuticals. The intent of this review is therefore to further the field of protein glycoengineering by increasing the general understanding of the mechanisms by which glycosylation improves the molecular stability of protein pharmaceuticals. This is achieved by presenting a survey of the different instabilities displayed by protein pharmaceuticals, by addressing which of these instabilities can be improved by glycosylation, and by discussing the possible mechanisms by which glycans induce these stabilization effects. PMID:18661536

  7. Energy and protein requirements for maintenance and growth of Boer crossbred kids.

    PubMed

    Fernandes, M H M R; Resende, K T; Tedeschi, L O; Fernandes, J S; Silva, H M; Carstens, G E; Berchielli, T T; Teixeira, I A M A; Akinaga, L

    2007-04-01

    Meat production by goats has become an important livestock enterprise in several parts of the world. Nonetheless, energy and protein requirements of meat goats have not been defined thoroughly. The objective of this study was to determine the energy and protein requirements for maintenance and growth of 34 (3/4) Boer x (1/4) Saanen crossbred, intact male kids (20.5 +/- 0.24 kg of initial BW). The baseline group was 7 randomly selected kids, averaging 21.2 +/- 0.36 kg of BW. An intermediate group consisted of 6 randomly selected kids, fed for ad libitum intake, that were slaughtered when they reached an average BW of 28.2 +/- 0.39 kg. The remaining kids (n = 21) were allocated randomly on d 0 to 3 levels of DMI (treatments were ad libitum or restricted to 70 or 40% of the ad libitum intake) within 7 slaughter groups. A slaughter group contained 1 kid from each treatment, and kids were slaughtered when the ad libitum treatment kid reached 35 kg of BW. Individual body components (head plus feet, hide, internal organs plus blood, and carcass) were weighed, ground, mixed, and subsampled for chemical analyses. Initial body composition was determined using equations developed from the composition of the baseline kids. The calculated daily maintenance requirement for NE was 77.3 +/- 1.05 kcal/kg(0.75) of empty BW (EBW) or 67.4 +/- 1.04 kcal/kg(0.75) of shrunk BW. The daily ME requirement for maintenance (118.1 kcal/kg(0.75) of EBW or 103.0 kcal/kg(0.75) of shrunk BW) was calculated by iteration, assuming that the heat produced was equal to the ME intake at maintenance. The partial efficiency of use of ME for NE below maintenance was 0.65. A value of 2.44 +/- 0.4 g of net protein/kg(0.75) of EBW for daily maintenance was determined. Net energy requirements for growth ranged from 2.55 to 3.0 Mcal/kg of EBW gain at 20 and 35 kg of BW, and net protein requirements for growth ranged from 178.8 to 185.2 g/kg of EBW gain. These results suggest that NE and net protein requirements

  8. Stabilized helical peptides: a strategy to target protein-protein interactions.

    PubMed

    Klein, Mark A

    2014-08-14

    Protein-protein interactions are critical for cell proliferation, differentiation, and function. Peptides hold great promise for clinical applications focused on targeting protein-protein interactions. Advantages of peptides include a large chemical space and potential diversity of sequences and structures. However, peptides do present well-known challenges for drug development. Progress has been made in the development of stabilizing alpha helices for potential therapeutic applications. Advantages and disadvantages of different methods of helical peptide stabilization are discussed.

  9. Protein encapsulation in mesoporous silicate: the effects of confinement on protein stability, hydration, and volumetric properties.

    PubMed

    Ravindra, Revanur; Zhao, Shuang; Gies, Hermann; Winter, Roland

    2004-10-01

    On the basis of the predictions of statistical-thermodynamic models, it is postulated that excluded volume effects may play a significant role in the stability, interaction, and function of proteins. We studied the effects of confinement on protein un/refolding and stability. Our approach was to encapsulate a model protein, RNase A, in a mesoporous silica, MCM-48, with glasslike wall structure and with well-defined pores to create a crowded microenvironment. To the best of our knowledge, this is the first report where pressure perturbation and differential scanning calorimetric techniques are employed to evaluate the stability, hydration, and volumetric properties of the confined protein. A drastic increase in protein stability ( approximately 30 degrees C increase in unfolding temperature) is observed. The increase in stability is probably not only due to a restriction in conformational space (excluded volume effect due to nonspecific interactions) but also due to an increased strength of hydration of the protein within the narrow silica pores.

  10. Inferring Stabilizing Mutations from Protein Phylogenies: Application to Influenza Hemagglutinin

    PubMed Central

    Bloom, Jesse D.; Glassman, Matthew J.

    2009-01-01

    One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution. PMID:19381264

  11. The HU Protein Is Important for Apicoplast Genome Maintenance and Inheritance in Toxoplasma gondii

    PubMed Central

    Reiff, Sarah B.; Vaishnava, Shipra

    2012-01-01

    The apicoplast, a chloroplast-like organelle, is an essential cellular component of most apicomplexan parasites, including Plasmodium and Toxoplasma. The apicoplast maintains its own genome, a 35-kb DNA molecule that largely encodes proteins required for organellar transcription and translation. Interference with apicoplast genome maintenance and function is a validated target for drug therapy for malaria and toxoplasmosis. However, the many proteins required for genome maintenance and inheritance remain largely unstudied. Here we genetically characterize a nucleus-encoded homolog to the bacterial HU protein in Toxoplasma gondii. In bacteria, HU is a DNA-binding structural protein with fundamental roles in transcription, replication initiation, and DNA repair. Immunofluorescence assays reveal that in T. gondii this protein localizes to the apicoplast. We have found that the HU protein from Toxoplasma can successfully complement bacterial ΔhupA mutants, supporting a similar function. We were able to construct a genetic knockout of HU in Toxoplasma. This Δhu mutant is barely viable and exhibits significant growth retardation. Upon further analysis of the mutant phenotype, we find that this mutant has a dramatically reduced apicoplast genome copy number and, furthermore, suffers defects in the segregation of the apicoplast organelle. Our findings not only show that the HU protein is important for Toxoplasma cell biology but also demonstrate the importance of the apicoplast genome in the biogenesis of the organelle. PMID:22611021

  12. Electrostatic Stabilization Of Growing Protein Crystals

    NASA Technical Reports Server (NTRS)

    Shlichta, Paul J.

    1991-01-01

    Proposed technique produces large crystals in compact, economical apparatus. Report presents concept for supporting protein crystals during growth in microgravity. Yields crystals larger and more-nearly perfect than those grown on Earth. Combines best features of sandwich-drop and electrostatic-levitation methods of support. Drop of protein solution inserted between pair of glass or plastic plates, as in sandwich-drop-support method. Electrostatically charged ring confines drop laterally and shapes it, as in electrostatic technique. Apparatus also made to accommodate several drops simultaneously between same pair of supporting plates. Drops can be inserted and crystals removed through ducts in plates.

  13. Structural basis for the enhanced stability of highly fluorinated proteins

    PubMed Central

    Buer, Benjamin C.; Meagher, Jennifer L.; Stuckey, Jeanne A.; Marsh, E. Neil G.

    2012-01-01

    Noncanonical amino acids have proved extremely useful for modifying the properties of proteins. Among them, extensively fluorinated (fluorous) amino acids seem particularly effective in increasing protein stability; however, in the absence of structural data, the basis of this stabilizing effect remains poorly understood. To address this problem, we solved X-ray structures for three small proteins with hydrophobic cores that are packed with either fluorocarbon or hydrocarbon side chains and compared their stabilities. Although larger, the fluorinated residues are accommodated within the protein with minimal structural perturbation, because they closely match the shape of the hydrocarbon side chains that they replace. Thus, stability increases seem to be better explained by increases in buried hydrophobic surface area that accompany fluorination than by specific fluorous interactions between fluorinated side chains. This finding is illustrated by the design of a highly fluorinated protein that, by compensating for the larger volume and surface area of the fluorinated side chains, exhibits similar stability to its nonfluorinated counterpart. These structure-based observations should inform efforts to rationally modulate protein function using noncanonical amino acids. PMID:22411812

  14. Heat stability of reconstituted, protein-standardized skim milk powders.

    PubMed

    Sikand, V; Tong, P S; Walker, J

    2010-12-01

    We determined the effects of standardization material, protein content, and pH on the heat stability of reconstituted milk made from low-heat (LH) and medium-heat (MH) nonfat dry milk (NDM). Low-heat and MH NDM were standardized downward from 35.5% to 34, 32, and 30% protein by adding either edible lactose powder (ELP) or permeate powder (PP) from skim milk ultrafiltration. These powders were called standardized skim milk powders (SSMP). The LH and MH NDM and SSMP were reconstituted to 9% total solids. Furthermore, subsamples of reconstituted NDM and SSMP samples were set aside to measure heat stability at native (unadjusted) pH, and the rest were adjusted to pH 6.3 to 7.0. Heat stability is defined as heat coagulation time at 140°C of the reconstituted LH or MH NDM and SSMP samples. The entire experiment was replicated 3 times at unadjusted pH values and 2 times at adjusted pH values. At an unadjusted pH, powder type, standardization material, and protein content influenced the heat stability of the samples. Heat stability for reconstituted LH NDM and SSMP was higher than reconstituted MH NDM and SSMP. Generally, decreased heat stability was observed in reconstituted LH or MH SSMP as protein content was decreased by standardization. However, adding ELP to MH SSMP did not significantly change its heat stability. When pH was adjusted to values between 6.3 and 7.0, powder type, standardization material, and pH had a significant effect on heat stability, whereas protein content did not. Maximum heat stability was noted at pH 6.7 for both reconstituted LH NDM and SSMP samples, and at pH 6.6 for both reconstituted MH NDM and SSMP samples. Furthermore, for samples with adjusted pH, higher heat stability was observed for reconstituted LH SSMP containing PP compared with reconstituted milk from LH SSMP containing ELP. However, no statistical difference was observed in the heat stability of reconstituted milk from MH NDM and MH SSMP samples. We conclude that powder type

  15. USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock

    PubMed Central

    Yoshitane, Hikari; Oyama, Masaaki; Kozuka-Hata, Hiroko; Lanjakornsiripan, Darin; Fukada, Yoshitaka

    2016-01-01

    Mammalian Cryptochromes, CRY1 and CRY2, function as principal regulators of a transcription-translation-based negative feedback loop underlying the mammalian circadian clockwork. An F-box protein, FBXL3, promotes ubiquitination and degradation of CRYs, while FBXL21, the closest paralog of FBXL3, ubiquitinates CRYs but leads to stabilization of CRYs. Fbxl3 knockout extremely lengthened the circadian period, and deletion of Fbxl21 gene in Fbxl3-deficient mice partially rescued the period-lengthening phenotype, suggesting a key role of CRY protein stability for maintenance of the circadian periodicity. Here, we employed a proteomics strategy to explore regulators for the protein stability of CRYs. We found that ubiquitin-specific protease 7 (USP7 also known as HAUSP) associates with CRY1 and CRY2 and stabilizes CRYs through deubiquitination. Treatment with USP7-specific inhibitor or Usp7 knockdown shortened the circadian period of the cellular rhythm. We identified another CRYs-interacting protein, TAR DNA binding protein 43 (TDP-43), an RNA-binding protein. TDP-43 stabilized CRY1 and CRY2, and its knockdown also shortened the circadian period in cultured cells. The present study identified USP7 and TDP-43 as the regulators of CRY1 and CRY2, underscoring the significance of the stability control process of CRY proteins for period determination in the mammalian circadian clockwork. PMID:27123980

  16. Robust enzyme design: bioinformatic tools for improved protein stability.

    PubMed

    Suplatov, Dmitry; Voevodin, Vladimir; Švedas, Vytas

    2015-03-01

    The ability of proteins and enzymes to maintain a functionally active conformation under adverse environmental conditions is an important feature of biocatalysts, vaccines, and biopharmaceutical proteins. From an evolutionary perspective, robust stability of proteins improves their biological fitness and allows for further optimization. Viewed from an industrial perspective, enzyme stability is crucial for the practical application of enzymes under the required reaction conditions. In this review, we analyze bioinformatic-driven strategies that are used to predict structural changes that can be applied to wild type proteins in order to produce more stable variants. The most commonly employed techniques can be classified into stochastic approaches, empirical or systematic rational design strategies, and design of chimeric proteins. We conclude that bioinformatic analysis can be efficiently used to study large protein superfamilies systematically as well as to predict particular structural changes which increase enzyme stability. Evolution has created a diversity of protein properties that are encoded in genomic sequences and structural data. Bioinformatics has the power to uncover this evolutionary code and provide a reproducible selection of hotspots - key residues to be mutated in order to produce more stable and functionally diverse proteins and enzymes. Further development of systematic bioinformatic procedures is needed to organize and analyze sequences and structures of proteins within large superfamilies and to link them to function, as well as to provide knowledge-based predictions for experimental evaluation.

  17. Stabilization of the Virulence Plasmid pSLT of Salmonella Typhimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test

    PubMed Central

    Lobato-Márquez, Damián; Molina-García, Laura; Moreno-Córdoba, Inma; García-del Portillo, Francisco; Díaz-Orejas, Ramón

    2016-01-01

    Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50–90 kb). All of these plasmids share the spv operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system (parAB) and two TA systems (ccdABST and vapBC2ST). The TA module ccdABST has previously been shown to contribute to pSLT plasmid stability and vapBC2ST to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of parAB in pSLT maintenance. We also showed that vapBC2ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdABST encodes an inactive CcdBST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdABST variant containing a single mutation (R99W) that restores the toxicity of CcdBST. The “activation” of CcdBST (R99W) did not increase pSLT stability by ccdABST. In contrast, ccdABST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdABST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdBST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdAST antitoxin more than on its toxic activity. PMID

  18. Impact of alcohols on the formation and stability of protein-stabilized nanoemulsions.

    PubMed

    Zeeb, Benjamin; Herz, Eva; McClements, David Julian; Weiss, Jochen

    2014-11-01

    Nanoemulsions are increasingly being used for encapsulation, protection, and delivery of bioactive lipids, however, their formation from natural emulsifiers is still challenging. We investigated the impact of alcohol on the formation and stability of protein-stabilized oil-in-water nanoemulsions prepared by high-pressure homogenization. The influence of different alcohols (ethanol, 1-propanol, and 1-butanol) at various concentrations (0-25% w/w) on the formation and stability of emulsions stabilized by sodium caseinate, whey protein isolate, and fish gelatin was investigated. The mean particle diameter decreased with increasing alcohol concentrations from 0 to 10%w/w, but extensive droplet aggregation occurred at higher levels. This phenomenon was attributed to enhanced protein-protein interactions between the adsorbed emulsifier molecules in the presence of alcohol leading to droplet flocculation. The smallest droplets (d<100nm) were obtained when 10%w/w 1-butanol was added to sodium caseinate-stabilized nanoemulsions, but relatively small droplets (d<150nm) could also be obtained in the presence of a food-grade alcohol (ethanol). This study demonstrated that alcohol addition might be a useful tool for producing protein-stabilized nanoemulsions suitable for use as delivery systems of lipophilic bioactive agents. PMID:25129338

  19. Development of a simple assay system for protein-stabilizing efficiency based on hemoglobin protection against denaturation and measurement of the cooperative effect of mixing protein stabilizers.

    PubMed

    Chen, Siyu; Manabe, Yoshiyuki; Minamoto, Naoya; Saiki, Naoka; Fukase, Koichi

    2016-10-01

    We have elucidated the cooperative stabilization of proteins by sugars, amino acids, and other protein-stabilizing agents using a new and simple assay system. Our system determines the protein-stabilizing ability of various compounds by measuring their ability to protect hemoglobin from denaturation. Hemoglobin denaturation was readily measured by quantitative changes in its ultraviolet-visible absorption spectrum. The efficiency of our assay was confirmed using various sugars such as trehalose and sucrose that are known to be good protein stabilizers. We have also found that mixtures of two different types of protein stabilizers resulted in a cooperative stabilizing effect on protein. PMID:27253914

  20. Nitrogen metabolism, digestive parameters, and protein requirements for the maintenance of buffalo growth.

    PubMed

    Machado, Erica; Yoshimura, Emerson Henri; Santos, Nadine Woruby; Agustinho, Bruna Calvo; Pereira, Lucelia de Moura; Samensari, Rafael Barreiros; de Aguiar, Silvia Cristina; Zeoula, Lucia Maria

    2016-02-01

    The objectives of this study were to evaluate the effect of crude protein (CP) levels in the diet of growing female buffaloes on nitrogen metabolism and estimate protein requirements for maintenance. Four female buffaloes were used, cannulated in the rumen, with an average initial body weight (BW) of 355 ± 3.5 kg, in a Latin square (4 × 4) with four animals and four levels of CP in the diet (70, 90, 110, and 130 g/kg dry matter (DM)) composed of corn silage and concentrate. The increase in protein intake with increasing levels of dietary CP resulted in a higher concentration of ammonia in the rumen and higher ruminal disappearance of PB. However, omasal flow of protein increased linearly as did the efficiency of microbial protein synthesis. The CP levels affected DM intake and other nutrients positively, but there was no effect on nutrient total digestibility. Nitrogen (N) balance, when expressed relative to N intake, had an average value of 48.5 % observed across. The protein requirement for the maintenance of growing female buffaloes was 4.6 g CP/kg BW(0.75). PMID:26590610

  1. Stabilization of a protein nanocage through the plugging of a protein-protein interfacial water pocket.

    PubMed

    Ardejani, Maziar S; Li, Noel X; Orner, Brendan P

    2011-05-17

    The unique structural properties of the ferritin protein cages have provided impetus to focus on the methodical study of these self-assembling nanosystems. Among these proteins, Escherichia coli bacterioferritin (EcBfr), although architecturally very similar to other members of the family, shows structural instability and an incomplete self-assembly behavior by populating two oligomerization states. Through computational analysis and comparison to its homologues, we have found that this protein has a smaller than average dimeric interface on its 2-fold symmetry axis mainly because of the existence of an interfacial water pocket centered around two water-bridged asparagine residues. To investigate the possibility of engineering EcBfr for modified structural stability, we have used a semiempirical computational method to virtually explore the energy differences of the 480 possible mutants at the dimeric interface relative to that of wild-type EcBfr. This computational study also converged on the water-bridged asparagines. Replacing these two asparagines with hydrophobic amino acids resulted in proteins that folded into α-helical monomers and assembled into cages as evidenced by circular dichroism and transmission electron microscopy. Both thermal and chemical denaturation confirmed that, in all cases, these proteins, in agreement with the calculations, possessed increased stability. One of the three mutations shifts the population in favor of the higher-order oligomerization state in solution as evidenced by both size exclusion chromatography and native gel electrophoresis. These results taken together suggest that our low-level design was successful and that it may be possible to apply the strategy of targeting water pockets at protein--protein interfaces to other protein cage and self-assembling systems. More generally, this study further demonstrates the power of jointly employing in silico and in vitro techniques to understand and enhance biostructural

  2. Defining the role of salt bridges in protein stability.

    PubMed

    Jelesarov, Ilian; Karshikoff, Andrey

    2009-01-01

    Although the energetic balance of forces stabilizing proteins has been established qualitatively over the last decades, quantification of the energetic contribution of particular interactions still poses serious problems. The reasons are the strong cooperativity and the interdependence ofnoncovalent interactions. Salt bridges are a typical example. One expects that ionizable side chains frequently form ion pairs in innumerable crystal structures. Since electrostatic attraction between opposite charges is strong per se, salt bridges can intuitively be regarded as an important factor stabilizing the native structure. Is that really so? In this chapter we critically reassess the available methods to delineate the role ofelectrostatic interactions and salt bridges to protein stability, and discuss the progress and the obstacles in this endeavor. The basic problem is that formation of salt bridges depends on the ionization properties of the participating groups, which is significantly influenced by the protein environment. Furthermore, salt bridges experience thermal fluctuations, continuously break and re-form, and their lifespan in solution is governed by the flexibility of the protein. Finally, electrostatic interactions are long-range and might be significant in the unfolded state, thus seriously influencing the energetic profile. Elimination of salt bridges by protonation/deprotonation at extreme pH or by mutation provides only rough energetic estimates, since there is no way to account for the nonadditive response of the protein moiety. From what we know so far, the strength of electrostatic interactions is strongly context-dependent, yet it is unlikely that salt bridges are dominant factors governing protein stability. Nevertheless, proteins from thermophiles and hyperthermophiles exhibit more, and frequently networked, salt bridges than proteins from the mesophilic counterparts. Increasing the thermal (not the thermodynamic) stability of proteins by optimization

  3. SRide: a server for identifying stabilizing residues in proteins.

    PubMed

    Magyar, Csaba; Gromiha, M Michael; Pujadas, Gerard; Tusnády, Gábor E; Simon, István

    2005-07-01

    Residues expected to play key roles in the stabilization of proteins [stabilizing residues (SRs)] are selected by combining several methods based mainly on the interactions of a given residue with its spatial, rather than its sequential neighborhood and by considering the evolutionary conservation of the residues. A residue is selected as a stabilizing residue if it has high surrounding hydrophobicity, high long-range order, high conservation score and if it belongs to a stabilization center. The definition of all these parameters and the thresholds used to identify the SRs are discussed in detail. The algorithm for identifying SRs was originally developed for TIM-barrel proteins [M. M. Gromiha, G. Pujadas, C. Magyar, S. Selvaraj, and I. Simon (2004), Proteins, 55, 316-329] and is now generalized for all proteins of known 3D structure. SRs could be applied in protein engineering and homology modeling and could also help to explain certain folds with significant stability. The SRide server is located at http://sride.enzim.hu.

  4. Role of arginine in the stabilization of proteins against aggregation.

    PubMed

    Baynes, Brian M; Wang, Daniel I C; Trout, Bernhardt L

    2005-03-29

    The amino acid arginine is frequently used as a solution additive to stabilize proteins against aggregation, especially in the process of protein refolding. Despite arginine's prevalence, the mechanism by which it stabilizes proteins is not presently understood. We propose that arginine deters aggregation by slowing protein-protein association reactions, with only a small concomitant effect on protein folding. The associated rate effect was observed experimentally in association of globular proteins (insulin and a monoclonal anti-insulin) and in refolding of carbonic anhydrase. We suggest that this effect arises because arginine is preferentially excluded from protein-protein encounter complexes but not from dissociated protein molecules. Such an effect is predicted by our gap effect theory [Baynes and Trout (2004) Biophys. J. 87, 1631] for "neutral crowder" additives such as arginine which are significantly larger than water but have only a small effect on the free energies of isolated protein molecules. The effect of arginine on refolding of carbonic anhydrase was also shown to be consistent with this hypothesis.

  5. Effects of sugars on the thermal stability of a protein

    NASA Astrophysics Data System (ADS)

    Oshima, Hiraku; Kinoshita, Masahiro

    2013-06-01

    It is experimentally known that the heat-denaturation temperature of a protein is raised (i.e., its thermal stability is enhanced) by sugar addition. In earlier works, we proposed a physical picture of thermal denaturation of proteins in which the measure of the thermal stability is defined as the solvent-entropy gain upon protein folding at 298 K normalized by the number of residues. A multipolar-model water was adopted as the solvent. The polyatomic structures of the folded and unfolded states of a protein were taken into account in the atomic detail. A larger value of the measure implies higher thermal stability. First, we show that the measure remains effective even when the model water is replaced by the hard-sphere solvent whose number density and molecular diameter are set at those of real water. The physical picture is then adapted to the elucidation of the effects of sugar addition on the thermal stability of a protein. The water-sugar solution is modeled as a binary mixture of hard spheres. The thermal stability is determined by a complex interplay of the diameter of sugar molecules dC and the total packing fraction of the solution η: dC is estimated from the volume per molecule in the sugar crystal and η is calculated using the experimental data of the solution density. We find that the protein is more stabilized as the sucrose or glucose concentration becomes higher and the stabilization effect is stronger for sucrose than for glucose. These results are in accord with the experimental observations. Using a radial-symmetric integral equation theory and the morphometric approach, we decompose the change in the measure upon sugar addition into two components originating from the protein-solvent pair and protein-solvent many-body correlations, respectively. Each component is further decomposed into the excluded-volume and solvent-accessible-surface terms. These decompositions give physical insights into the microscopic origin of the thermal-stability

  6. Rational stabilization of complex proteins: a divide and combine approach

    PubMed Central

    Lamazares, Emilio; Clemente, Isabel; Bueno, Marta; Velázquez-Campoy, Adrián; Sancho, Javier

    2015-01-01

    Increasing the thermostability of proteins is often crucial for their successful use as analytic, synthetic or therapeutic tools. Most rational thermostabilization strategies were developed on small two-state proteins and, unsurprisingly, they tend to fail when applied to the much more abundant, larger, non-fully cooperative proteins. We show that the key to stabilize the latter is to know the regions of lower stability. To prove it, we have engineered apoflavodoxin, a non-fully cooperative protein on which previous thermostabilizing attempts had failed. We use a step-wise combination of structure-based, rationally-designed, stabilizing mutations confined to the less stable structural region, and obtain variants that, according to their van't Hoff to calorimetric enthalpy ratios, exhibit fully-cooperative thermal unfolding with a melting temperature of 75°C, 32 degrees above the lower melting temperature of the non-cooperative wild type protein. The ideas introduced here may also be useful for the thermostabilization of complex proteins through formulation or using specific stabilizing ligands (e.g. pharmacological chaperones). PMID:25774740

  7. EKylation: Addition of an Alternating-Charge Peptide Stabilizes Proteins.

    PubMed

    Liu, Erik J; Sinclair, Andrew; Keefe, Andrew J; Nannenga, Brent L; Coyle, Brandon L; Baneyx, François; Jiang, Shaoyi

    2015-10-12

    For nearly 40 years, therapeutic proteins have been stabilized by chemical conjugation of polyethylene glycol (PEG), but recently zwitterionic materials have proved to be a more effective substitute. In this work, we demonstrate that genetic fusion of alternating-charge extensions consisting of anionic glutamic acid (E) and cationic lysine (K) is an effective strategy for protein stabilization. This bioinspired "EKylation" method not only confers the stabilizing benefits of poly(zwitterions) but also allows for rapid biosynthesis of target constructs. Poly(EK) peptides of different predetermined lengths were appended to the C-terminus of a native β-lactamase and its destabilized TEM-19 mutant. The EK-modified enzymes retained biological activity and exhibited increased stability to environmental stressors such as high temperature and high-salt solutions. This one-step strategy provides a broadly applicable alternative to synthetic polymer conjugation that is biocompatible and degradable. PMID:26407134

  8. Stability of ALS-related Superoxide Dismutase Protein variants

    NASA Astrophysics Data System (ADS)

    Lusebrink, Daniel; Plotkin, Steven

    Superoxide dismutase (SOD1) is a metal binding, homodimeric protein, whose misfolding is implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Monomerization is believed to be a key step in the propagation of the disease. The dimer stability is often difficult to measure experimentally however, because it is entangled with protein unfolding and metal loss. We thus computationally investigate the dimer stability of mutants of SOD1 known to be associated with ALS. We report on systematic trends in dimer stability, as well as intriguing allosteric communication between mutations and the dimer interface. We study the dimer stabilities in molecular dynamics simulations and obtain the binding free energies of the dimers from pulling essays. Mutations are applied in silicoand we compare the differences of binding free energies compared to the wild type.

  9. Low stability of nucleocapsid protein in SARS virus.

    PubMed

    Wang, Yulong; Wu, Xiaoyu; Wang, Yihua; Li, Bing; Zhou, Hao; Yuan, Guiyong; Fu, Yan; Luo, Yongzhang

    2004-08-31

    The nucleocapsid protein (N protein) is one of the major virion structural proteins of a newly identified coronavirus, which has been confirmed as the causative agent of severe acute respiratory syndrome (SARS). The major function of N protein is to assemble the RNA of coronavirus. In the present study, the gene encoding the N protein was cloned and the protein was expressed, purified, and refolded as shown by (1)H NMR measurement. The maximal Trp emission wavelength occurs near 331 nm, suggesting substantial burial of Trp residues. Circular dichroism measurements indicate that N protein contains little alpha-helical structure. Acid titration shows that N protein begins to unfold near pH 5 and is fully denatured near pH 2.7, and the acid unfolding process is reversible. The physical and chemical properties of N protein indicate that its stability is low. N protein is denatured reversibly at pH 7.4 either by urea (with C(m) of 2.77 M and m value of 2.74 kcal mol(-1) M(-1)) or GdmCl (with C(m) of 1.46 M and m value of 4.50 kcal mol(-1) M(-1)). In the heat-induced denaturation in phosphate-buffered saline buffer, N-protein starts to unfold at 35 degrees C and is completely denatured at 55 degrees C, where SARS virus was also reported to be inactivated. We propose that the low stability of N protein may be critical for the stability and function of SARS virus.

  10. Interactions of phospholipase D and cytochrome P450 protein stability

    SciTech Connect

    Zangar, Richard C.; Fan, Yang-Yi; Chapkin, Robert S.

    2004-08-01

    Previous studies have suggested a relationship between cytochrome P450 (P450) 3A (CYP3A) conformation and the phospholipid composition of the associated membrane. In this study, we utilized a novel microsomal incubation system that mimics many of the characteristics of CYP3A degradation pathway that have been observed in vivo and in cultured cells to study the effects of phospholipid composition on protein stability. We found that addition of phosphatidylcholine-specific phospholipase D (PLD) stabilized CYP3A in this system, but that phosphatidylinositol-specific phospholipase C (PLC) was without effect. Addition of phosphatidic acid also stabilized CYP3A protein in the microsomes. The use of 1,10-phenanthroline (phenanthroline), an inhibitor of PLD activity, decreased CYP3A stability in incubated microsomes. Similarly, 6-h treatment of primary cultures of rat hepatocytes with phenanthroline resulted in nearly complete loss of CYP3A protein. Treatment of rats with nicardipine or dimethylsulfoxide (DMSO), which have been shown to affect CYP3A stability, altered the phospholipid composition of hepatic microsomes. It did not appear, though, that the changes in phospholipid composition that resulted from these in vivo treatments accounted for the change in CYP3A stability observed in hepatic microsomes from these animals.

  11. Stabilization of G protein-coupled receptors by point mutations

    PubMed Central

    Heydenreich, Franziska M.; Vuckovic, Ziva; Matkovic, Milos; Veprintsev, Dmitry B.

    2015-01-01

    G protein-coupled receptors (GPCRs) are flexible integral membrane proteins involved in transmembrane signaling. Their involvement in many physiological processes makes them interesting targets for drug development. Determination of the structure of these receptors will help to design more specific drugs, however, their structural characterization has so far been hampered by the low expression and their inherent instability in detergents which made protein engineering indispensable for structural and biophysical characterization. Several approaches to stabilize the receptors in a particular conformation have led to breakthroughs in GPCR structure determination. These include truncations of the flexible regions, stabilization by antibodies and nanobodies, fusion partners, high affinity and covalently bound ligands as well as conformational stabilization by mutagenesis. In this review we focus on stabilization of GPCRs by insertion of point mutations, which lead to increased conformational and thermal stability as well as improved expression levels. We summarize existing mutagenesis strategies with different coverage of GPCR sequence space and depth of information, design and transferability of mutations and the molecular basis for stabilization. We also discuss whether mutations alter the structure and pharmacological properties of GPCRs. PMID:25941489

  12. A topological and conformational stability alphabet for multipass membrane proteins.

    PubMed

    Feng, Xiang; Barth, Patrick

    2016-03-01

    Multipass membrane proteins perform critical signal transduction and transport across membranes. How transmembrane helix (TMH) sequences encode the topology and conformational flexibility regulating these functions remains poorly understood. Here we describe a comprehensive analysis of the sequence-structure relationships at multiple interacting TMHs from all membrane proteins with structures in the Protein Data Bank (PDB). We found that membrane proteins can be deconstructed in interacting TMH trimer units, which mostly fold into six distinct structural classes of topologies and conformations. Each class is enriched in recurrent sequence motifs from functionally unrelated proteins, revealing unforeseen consensus and evolutionary conserved networks of stabilizing interhelical contacts. Interacting TMHs' topology and local protein conformational flexibility were remarkably well predicted in a blinded fashion from the identified binding-hotspot motifs. Our results reveal universal sequence-structure principles governing the complex anatomy and plasticity of multipass membrane proteins that may guide de novo structure prediction, design, and studies of folding and dynamics. PMID:26780406

  13. Requirements of protein for maintenance and growth in ram hair lambs.

    PubMed

    Pereira, Elzania Sales; Fontenele, Rildson Melo; de Medeiros, Ariosvaldo Nunes; Oliveira, Ronaldo Lopes; Campos, Ana Claudia Nascimento; Heinzen, Eduardo Luiz; Bezerra, Leilson Rocha

    2016-10-01

    The experiment was conducted to determine the protein requirements for maintenance (PM) and weight gain (NPg) in Brazilian Somali lambs (n = 48). A database of information on non-castrated rams (n = 48) with an initial body weight (BW) of 13.47 ± 1.76 kg was obtained, and the comparative slaughter technique was used to assess protein requirements. Eight lambs were initially slaughtered and used as a reference for estimation of the empty body weight (EBW) and initial body composition of the remaining animals. The lambs (n = 40) were distributed in a completely randomized block design involving five experimental diets, containing 4.93, 8.65, 9.41, 10.12, and 11.24 MJ/kg dry matter (DM). Daily nitrogen retention (DNR) was calculated as the difference between the final N content in the body minus the initial content estimated from the reference animals, divided by the number of experimental days. PM was estimated from the retained N regression equation as a function of ingested N (g per kg(0.75) EBW/day), extrapolating the equation to the zero level of N intake, followed by multiplication by a factor of 6.25. The protein content of the EBW was reduced from 119.72 to 102.07 g/kg of empty body weight gain (EBG) when BW increased from 13 to 28.70 kg, respectively. The requirement of protein for maintenance was 0.80 g per kg(0.75) EBW/day. Based on information described in the present study, we recommend the value of PM 0.80 g per kg(0.75) EBW/day, for hair sheep Brazilian Somalis raised in tropical regions. We also suggest that hair sheep have different requirements for protein maintenance and gain.

  14. Protein Stability, Folding and Misfolding in Human PGK1 Deficiency.

    PubMed

    Valentini, Giovanna; Maggi, Maristella; Pey, Angel L

    2013-01-01

    Conformational diseases are often caused by mutations, altering protein folding and stability in vivo. We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein folding and misfolding. Expression analyses and in vitro biophysical studies indicate that disease-causing mutations enhance protein aggregation propensity. We found a strong correlation among protein aggregation propensity, thermodynamic stability, cooperativity and dynamics. Comparison of folding and unfolding properties with previous reports in PGKs from other species suggests that hPGK1 is very sensitive to mutations leading to enhance protein aggregation through changes in protein folding cooperativity and the structure of the relevant denaturation transition state for aggregation. Overall, we provide a mechanistic framework for protein misfolding of hPGK1, which is insightful to develop new therapeutic strategies aimed to target native state stability and foldability in hPGK1 deficient patients. PMID:24970202

  15. RNF4-Dependent Oncogene Activation by Protein Stabilization.

    PubMed

    Thomas, Jane J; Abed, Mona; Heuberger, Julian; Novak, Rostislav; Zohar, Yaniv; Beltran Lopez, Angela P; Trausch-Azar, Julie S; Ilagan, Ma Xenia G; Benhamou, David; Dittmar, Gunnar; Kopan, Raphael; Birchmeier, Walter; Schwartz, Alan L; Orian, Amir

    2016-09-20

    Ubiquitylation regulates signaling pathways critical for cancer development and, in many cases, targets proteins for degradation. Here, we report that ubiquitylation by RNF4 stabilizes otherwise short-lived oncogenic transcription factors, including β-catenin, Myc, c-Jun, and the Notch intracellular-domain (N-ICD) protein. RNF4 enhances the transcriptional activity of these factors, as well as Wnt- and Notch-dependent gene expression. While RNF4 is a SUMO-targeted ubiquitin ligase, protein stabilization requires the substrate's phosphorylation, rather than SUMOylation, and binding to RNF4's arginine-rich motif domain. Stabilization also involves generation of unusual polyubiquitin chains and docking of RNF4 to chromatin. Biologically, RNF4 enhances the tumor phenotype and is essential for cancer cell survival. High levels of RNF4 mRNA correlate with poor survival of a subgroup of breast cancer patients, and RNF4 protein levels are elevated in 30% of human colon adenocarcinomas. Thus, RNF4-dependent ubiquitylation translates transient phosphorylation signal(s) into long-term protein stabilization, resulting in enhanced oncoprotein activation. PMID:27653698

  16. Temperature compensation via cooperative stability in protein degradation

    NASA Astrophysics Data System (ADS)

    Peng, Yuanyuan; Hasegawa, Yoshihiko; Noman, Nasimul; Iba, Hitoshi

    2015-08-01

    Temperature compensation is a notable property of circadian oscillators that indicates the insensitivity of the oscillator system's period to temperature changes; the underlying mechanism, however, is still unclear. We investigated the influence of protein dimerization and cooperative stability in protein degradation on the temperature compensation ability of two oscillators. Here, cooperative stability means that high-order oligomers are more stable than their monomeric counterparts. The period of an oscillator is affected by the parameters of the dynamic system, which in turn are influenced by temperature. We adopted the Repressilator and the Atkinson oscillator to analyze the temperature sensitivity of their periods. Phase sensitivity analysis was employed to evaluate the period variations of different models induced by perturbations to the parameters. Furthermore, we used experimental data provided by other studies to determine the reasonable range of parameter temperature sensitivity. We then applied the linear programming method to the oscillatory systems to analyze the effects of protein dimerization and cooperative stability on the temperature sensitivity of their periods, which reflects the ability of temperature compensation in circadian rhythms. Our study explains the temperature compensation mechanism for circadian clocks. Compared with the no-dimer mathematical model and linear model for protein degradation, our theoretical results show that the nonlinear protein degradation caused by cooperative stability is more beneficial for realizing temperature compensation of the circadian clock.

  17. Storage Stability of Food Protein Hydrolysates-A Review.

    PubMed

    Rao, Qinchun; Klaassen Kamdar, Andre; Labuza, Theodore P

    2016-05-18

    In recent years, mainly due to the specific health benefits associated with (1) the discovery of bioactive peptides in protein hydrolysates, (2) the reduction of protein allergenicity by protein hydrolysis, and (3) the improved protein digestibility and absorption of protein hydrolysates, the utilization of protein hydrolysates in functional foods and beverages has significantly increased. Although the specific health benefits from different hydrolysates are somewhat proven, the delivery and/or stability of these benefits is debatable during distribution, storage, and consumption. In this review, we discuss (1) the quality changes in different food protein hydrolysates during storage; (2) the resulting changes in the structure and texture of three food matrices, i.e., low moisture foods (LMF, aw < 0.6), intermediate moisture foods (IMF, 0.6 ≤ aw < 0.85), and high moisture foods (HMF, aw ≥ 0.85); and (3) the potential solutions to improve storage stability of food protein hydrolysates. In addition, we note there is a great need for evaluation of biofunction availability of bioactive peptides in food protein hydrolysates during storage.

  18. Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability

    PubMed Central

    Lee, Jae-Young; Lee, Se-Hwan; Heo, Sun-Hee; Kim, Kwang-Soo; Kim, Changhoon; Kim, Dae-Kwan; Ko, Jeong-Jae; Park, Kyung-Soon

    2015-01-01

    G9a is a lysine methyltransferase (KMTase) for histone H3 lysine 9 that plays critical roles in a number of biological processes. Emerging evidence suggests that aberrant expression of G9a contributes to tumor metastasis and maintenance of a malignant phenotype in cancer by inducing epigenetic silencing of tumor suppressor genes. Here, we show that G9a regulates Sox2 protein stability in breast cancer cells. When G9a lysine methyltransferase activity was chemically inhibited in the ER(+) breast cancer cell line MCF7, Sox2 protein levels were decreased. In addition, ectopic overexpression of G9a induced accumulation of Sox2. Changes in cell migration, invasion, and mammosphere formation by MCF7 cells were correlated with the activity or expression level of G9a. Ectopic expression of G9a also increased Sox2 protein levels in another ER(+) breast cancer cell line, ZR-75-1, whereas it did not affect Sox2 expression in MDA-MB-231 cells, an ER(-) breast cancer cell line, or in glioblastoma cell lines. Furthermore, treatment of mouse embryonic stem cells with a KMT inhibitor, BIX-01294, resulted in a rapid reduction in Sox2 protein expression despite increased Sox2 transcript levels. This finding suggests that G9a has a novel function in the regulation of Sox2 protein stability in a cell type-dependent manner. PMID:26492085

  19. Development of dextran nanoparticles for stabilizing delicate proteins

    NASA Astrophysics Data System (ADS)

    Wu, Fei; Zhou, Zhihua; Su, Jing; Wei, Liangming; Yuan, Weien; Jin, Tuo

    2013-04-01

    One of the most challenging problems in the development of protein pharmaceuticals is to deal with stabilities of proteins due to its complicated structures. This study aims to develop a novel approach to stabilize and encapsulate proteins into dextran nanoparticles without contacting the interface between the aqueous phase and the organic phase. The bovine serum albumin, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), β-galactosidase, and myoglobin were selected as model proteins. The proteins were added into an aqueous solution containing the dextran and polyethylene glycol, and then encapsulated into dextran nanoparticles by aqueous-aqueous freezing-induced phase separation. The encapsulation efficiency and recovery of dextran nanoparticles were determined. The dextran nanoparticles loaded with proteins were characterized by scanning electron microscopy and particle size analysis. The protein aggregation was determined by size-exclusion chromatography-high-performance chromatography, and the bioactivity of proteins recovered during formulation steps was determined. The bioactivity of GM-CSF, G-CSF, and β-galactosidase were examined by the proliferation of TF-1 cell, NSF-60 cell, and ortho-nitrophenyl- β-galactoside assay, respectively. The results of bioactivity recovered show that this novel dextran nanoparticle can preserve the protein's bioactivity during the preparation process. LysoSensor™ Yellow/Blue dextran, a pH-sensitive indicator with fluorescence excited at two channels, was encapsulated into dextran nanoparticles to investigate the ability of dextran nanoparticles to resist the acidic microenvironment (pH < 2.5). The result shows that the dextran nanoparticles attenuate the acidic microenvironment in the poly (lactic-co-glycolic acid) microsphere by means of the dilution effect. These novel dextran nanoparticles provided an appealing approach to stabilize the delicate proteins for

  20. Yeast hnRNP-related proteins contribute to the maintenance of telomeres

    SciTech Connect

    Lee-Soety, Julia Y.; Jones, Jennifer; MacGibeny, Margaret A.; Remaly, Erin C.; Daniels, Lynsey; Ito, Andrea; Jean, Jessica; Radecki, Hannah; Spencer, Shannon

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Yeast hnRNP-related proteins are able to prevent faster senescence in telomerase-null cells. Black-Right-Pointing-Pointer The conserved RRMs in Npl3 are important for telomere maintenance. Black-Right-Pointing-Pointer Human hnRNP A1 is unable to complement the lack of NPL3 in yeast. Black-Right-Pointing-Pointer Npl3 and Cbc2 may work as telomere capping proteins. -- Abstract: Telomeres protect the ends of linear chromosomes, which if eroded to a critical length can become uncapped and lead to replicative senescence. Telomerase maintains telomere length in some cells, but inappropriate expression facilitates the immortality of cancer cells. Recently, proteins involved in RNA processing and ribosome assembly, such as hnRNP (heterogeneous nuclear ribonucleoprotein) A1, have been found to participate in telomere maintenance in mammals. The Saccharomyces cerevisiae protein Npl3 shares significant amino acid sequence similarities with hnRNP A1. We found that deleting NPL3 accelerated the senescence of telomerase null cells. The highly conserved RNA recognition motifs (RRM) in Npl3 appear to be important for preventing faster senescence. Npl3 preferentially binds telomere sequences in vitro, suggesting that Npl3 may affect telomeres directly. Despite similarities between the two proteins, human hnRNP A1 is unable to complement the lack of Npl3 to rescue accelerated senescence in tlc1 npl3 cells. Deletion of CBC2, which encodes another hnRNP-related protein that associates with Npl3, also accelerates senescence. Potential mechanisms by which hnRNP-related proteins maintain telomeres are discussed.

  1. Positively Selected Sites in Cetacean Myoglobins Contribute to Protein Stability

    PubMed Central

    Dasmeh, Pouria; Serohijos, Adrian W. R.; Kepp, Kasper P.; Shakhnovich, Eugene I.

    2013-01-01

    Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10–20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity of Mbs is not significantly different among mammals (with typical oxygenation constants of ∼0.8–1.2 µM−1), folding stabilities of cetacean Mbs are ∼2–4 kcal/mol higher than for terrestrial Mbs. Using ancestral sequence reconstruction, maximum likelihood and Bayesian tests to describe the evolution of cetacean Mbs, and experimentally calibrated computation of stability effects of mutations, we observe accelerated evolution in cetaceans and identify seven positively selected sites in Mb. Overall, these sites contribute to Mb stabilization with a conditional probability of 0.8. We observe a correlation between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected sites that occur later act against other destabilizing mutations to maintain stability across the clade, except for the shallow divers, where late stability relaxation occurs, probably due to the shorter aerobic dive limits of these species. The three main positively selected sites 66, 5, and 35 undergo changes that favor hydrophobic folding, structural integrity, and intra-helical hydrogen bonds. PMID:23505347

  2. Separation of stem cell maintenance and transposon silencing functions of Piwi protein

    PubMed Central

    Klenov, Mikhail S.; Sokolova, Olesya A.; Yakushev, Evgeny Y.; Stolyarenko, Anastasia D.; Mikhaleva, Elena A.; Lavrov, Sergey A.; Gvozdev, Vladimir A.

    2011-01-01

    Piwi-interacting RNAs (piRNAs) and Piwi proteins have the evolutionarily conserved function of silencing of repetitive genetic elements in germ lines. The founder of the Piwi subfamily, Drosophila nuclear Piwi protein, was also shown to be required for the maintenance of germ-line stem cells (GSCs). Hence, null mutant piwi females exhibit two types of abnormalities, overexpression of transposons and severely underdeveloped ovaries. It remained unknown whether the failure of GSC maintenance is related to transposon derepression or if GSC self-renewal and piRNA silencing are two distinct functions of the Piwi protein. We have revealed a mutation, piwiNt, removing the nuclear localization signal of the Piwi protein. piwiNt females retain the ability of GSC self-renewal and a near-normal number of egg chambers in the ovarioles but display a drastic transposable element derepression and nuclear accumulation of their transcripts in the germ line. piwiNt mutants are sterile most likely because of the disturbance of piRNA-mediated transposon silencing. Analysis of chromatin modifications in the piwiNt ovaries indicated that Piwi causes chromatin silencing only of certain types of transposons, whereas others are repressed in the nuclei without their chromatin modification. Thus, Piwi nuclear localization that is required for its silencing function is not essential for the maintenance of GSCs. We suggest that the Piwi function in GSC self-renewal is independent of transposon repression and is normally realized in the cytoplasm of GSC niche cells. PMID:22065765

  3. Separation of stem cell maintenance and transposon silencing functions of Piwi protein.

    PubMed

    Klenov, Mikhail S; Sokolova, Olesya A; Yakushev, Evgeny Y; Stolyarenko, Anastasia D; Mikhaleva, Elena A; Lavrov, Sergey A; Gvozdev, Vladimir A

    2011-11-15

    Piwi-interacting RNAs (piRNAs) and Piwi proteins have the evolutionarily conserved function of silencing of repetitive genetic elements in germ lines. The founder of the Piwi subfamily, Drosophila nuclear Piwi protein, was also shown to be required for the maintenance of germ-line stem cells (GSCs). Hence, null mutant piwi females exhibit two types of abnormalities, overexpression of transposons and severely underdeveloped ovaries. It remained unknown whether the failure of GSC maintenance is related to transposon derepression or if GSC self-renewal and piRNA silencing are two distinct functions of the Piwi protein. We have revealed a mutation, piwi(Nt), removing the nuclear localization signal of the Piwi protein. piwi(Nt) females retain the ability of GSC self-renewal and a near-normal number of egg chambers in the ovarioles but display a drastic transposable element derepression and nuclear accumulation of their transcripts in the germ line. piwi(Nt) mutants are sterile most likely because of the disturbance of piRNA-mediated transposon silencing. Analysis of chromatin modifications in the piwi(Nt) ovaries indicated that Piwi causes chromatin silencing only of certain types of transposons, whereas others are repressed in the nuclei without their chromatin modification. Thus, Piwi nuclear localization that is required for its silencing function is not essential for the maintenance of GSCs. We suggest that the Piwi function in GSC self-renewal is independent of transposon repression and is normally realized in the cytoplasm of GSC niche cells. PMID:22065765

  4. A high-throughput assay of membrane protein stability.

    PubMed

    Postis, Vincent L G; Deacon, Sarah E; Roach, Peter C J; Wright, Gareth S A; Xia, Xiaobing; Ingram, Jean C; Hadden, Jonathan M; Henderson, Peter J F; Phillips, Simon E V; McPherson, Michael J; Baldwin, Stephen A

    2008-12-01

    The preparation of purified, detergent-solubilized membrane proteins in a monodisperse and stable form is usually a prerequisite for investigation not only of their function but also for structural studies by X-ray crystallography and other approaches. Typically, it is necessary to explore a wide range of conditions, including detergent type, buffer pH, and the presence of additives such as glycerol, in order to identify those optimal for stability. Given the difficulty of expressing and purifying membrane proteins in large amounts, such explorations must ideally be performed on as small a scale as practicable. To achieve this objective in the UK Membrane Protein Structure Initiative, we have developed a rapid, economical, light-scattering assay of membrane protein aggregation that allows the testing of 48 buffer conditions in parallel on 6 protein targets, requiring less than 2 mg protein for each target. Testing of the assay on a number of unrelated membrane transporters has shown that it is of generic applicability. Proteins of sufficient purity for this plate-based assay are first rapidly prepared using simple affinity purification procedures performed in batch mode. Samples are then transferred by microdialysis into each of the conditions to be tested. Finally, attenuance at 340 nm is monitored in a 384-well plate using a plate reader. Optimal conditions for protein stability identified in the assay can then be exploited for the tailored purification of individual targets in as stable a form as possible.

  5. Sde2: A novel nuclear protein essential for telomeric silencing and genomic stability in Schizosaccharomyces pombe

    SciTech Connect

    Sugioka-Sugiyama, Rie; Sugiyama, Tomoyasu

    2011-03-18

    Research highlights: {yields} Sde2 is essential for telomere silencing. {yields} Sde2 is involved in the maintenance of genomic stability. {yields} Sde2 promotes the recruitment of SHREC, a histone deacetylase complex, to telomeres. -- Abstract: Telomeres, specialized domains assembled at the ends of linear chromosomes, are essential for genomic stability in eukaryotes. The formation and maintenance of telomeres are governed by numerous factors such as telomeric repeats, telomere-binding proteins, heterochromatin proteins, and telomerase. Here, we report Sde2, a novel nuclear protein essential for telomeric silencing and genomic stability in the fission yeast Schizosaccharomyces pombe. A deficiency in sde2 results in the derepression of the ura4{sup +} gene inserted near telomeric repeats, and the noncoding transcripts from telomeric regions accumulate in sde2{Delta} cells. The loss of Sde2 function compromises transcriptional silencing at telomeres, and this silencing defect is accompanied by increased levels of acetylated histone H3K14 and RNA polymerase II occupancy at telomeres as well as reduced recruitment of the SNF2 ATPase/histone deacetylase-containing complex SHREC to telomeres. Deletion of sde2 also leads to a higher frequency of mitotic minichromosome loss, and sde2{Delta} cells often form asci that contain spores in abnormal numbers, shapes, or both. In addition, sde2{Delta} cells are highly sensitive to several stresses, including high/low temperatures, bleomycin, which induces DNA damage, and thiabendazole, a microtubule-destabilizing agent. Furthermore, Sde2 genetically interacts with the telomere regulators Taz1, Pof3, and Ccq1. These findings demonstrate that Sde2 cooperates with other telomere regulators to maintain functional telomeres, thereby preventing genomic instability.

  6. Sample preservation through heat stabilization of proteins: principles and examples.

    PubMed

    Borén, Mats

    2015-01-01

    Due to post-sampling changes, caused by residual enzyme activity in the sample, levels of analytes can change from their in vivo levels so that they no longer accurately reflect conditions in the living system. The Stabilizor(™) system accomplishes elimination of enzyme activity through heat-induced denaturation of enzymes by permanently altering the 3D protein structure of the enzymes. Heat stabilization can be introduced in the workflow either directly after sampling, with the instrument just next to where the sample is taken, or prior to sample homogenization and extraction, when samples are heat denatured directly from a frozen state. Initially, heat stabilization was developed to enable mass spectrometric analysis of neuropeptides. Heat stabilization has since been further developed and applied to a range of samples and downstream protein analysis techniques such as western blot, 2D gels and phosphorylation analysis with LC-MS.

  7. Some implications of colloid stability theory for protein crystallization

    NASA Technical Reports Server (NTRS)

    Young, C. C.; De Mattei, R. C.; Feigelson, R. S.; Tiller, W. A.

    1988-01-01

    Colloid stability theory has been applied to protein crystallization and predicts a narrow range of conditions under which crystals can be grown without the agglomeration of protein molecules (colloids) in the bulk solution. It also predicts a critical electrolyte concentration above which agglomeration will always occur. Using this theory, the rapid protein agglomeration occurring during Schlieren experiments as well as a terminal crystal size effect in a fixed container were explained. Following this concept, the supposed 'terminal' crystal size has been at least doubled.

  8. The structural stability of wild-type horse prion protein.

    PubMed

    Zhang, Jiapu

    2011-10-01

    Prion diseases (e.g. Creutzfeldt-Jakob disease (CJD), variant CJD (vCJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal Familial Insomnia (FFI) and Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE or 'mad-cow' disease) and chronic wasting disease (CWD) in cattles) are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches or medications to treat all these prion diseases. Rabbits, dogs, and horses are the only mammalian species reported to be resistant to infection from prion diseases isolated from other species. Recently, the β2-α2 loop has been reported to contribute to their protein structural stabilities. The author has found that rabbit prion protein has a strong salt bridge ASP177-ARG163 (like a taut bow string) keeping this loop linked. This paper confirms that this salt bridge also contributes to the structural stability of horse prion protein. Thus, the region of β2-α2 loop might be a potential drug target region. Besides this very important salt bridge, other four important salt bridges GLU196-ARG156-HIS187, ARG156-ASP202 and GLU211-HIS177 are also found to greatly contribute to the structural stability of horse prion protein. Rich databases of salt bridges, hydrogen bonds and hydrophobic contacts for horse prion protein can be found in this paper.

  9. The structural stability of wild-type horse prion protein.

    PubMed

    Zhang, Jiapu

    2011-10-01

    Prion diseases (e.g. Creutzfeldt-Jakob disease (CJD), variant CJD (vCJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal Familial Insomnia (FFI) and Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE or 'mad-cow' disease) and chronic wasting disease (CWD) in cattles) are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches or medications to treat all these prion diseases. Rabbits, dogs, and horses are the only mammalian species reported to be resistant to infection from prion diseases isolated from other species. Recently, the β2-α2 loop has been reported to contribute to their protein structural stabilities. The author has found that rabbit prion protein has a strong salt bridge ASP177-ARG163 (like a taut bow string) keeping this loop linked. This paper confirms that this salt bridge also contributes to the structural stability of horse prion protein. Thus, the region of β2-α2 loop might be a potential drug target region. Besides this very important salt bridge, other four important salt bridges GLU196-ARG156-HIS187, ARG156-ASP202 and GLU211-HIS177 are also found to greatly contribute to the structural stability of horse prion protein. Rich databases of salt bridges, hydrogen bonds and hydrophobic contacts for horse prion protein can be found in this paper. PMID:21875155

  10. Detergent Stabilized Nanopore Formation Kinetics of an Anthrax Protein

    NASA Astrophysics Data System (ADS)

    Peterson, Kelby

    2015-03-01

    This summer research project funded through the Society of Physics Students Internship Program and The National Institute of Standards and Technology focused on optimization of pore formation of Protective Antigen protein secreted by Bacillus Anthraces. This experiment analyzes the use of N-tetradecylphosphocholine (FOS-14 Detergent) to stabilize the water soluble protein, protective antigen protein (PA63) to regulate the kinetics of pore formation in a model bilayer lipid membrane. The FOS-14 Detergent was tested under various conditions to understand its impact on the protein pore formation. The optimization of this channel insertion is critical in preparing samples of oriented for neutron reflectometry that provide new data to increase the understanding of the protein's structure.

  11. Expression, function, and targeting of the nuclear exporter chromosome region maintenance 1 (CRM1) protein.

    PubMed

    Ishizawa, Jo; Kojima, Kensuke; Hail, Numsen; Tabe, Yoko; Andreeff, Michael

    2015-09-01

    Nucleocytoplasmic trafficking of proteins/RNAs is essential to normal cellular function. Indeed, accumulating evidence suggests that cancer cells escape anti-neoplastic mechanisms and benefit from pro-survival signals via the dysregulation of this system. The nuclear exporter chromosome region maintenance 1 (CRM1) protein is the only protein in the karyopherin-β protein family that contributes to the trafficking of numerous proteins and RNAs from the nucleus. It is considered to be an oncogenic, anti-apoptotic protein in transformed cells, since it reportedly functions as a gatekeeper for cell survival, including affecting p53 function, and ribosomal biogenesis. Furthermore, abnormally high expression of CRM1 is correlated with poor patient prognosis in various malignancies. Therapeutic targeting of CRM1 has emerged as a novel cancer treatment strategy, starting with a clinical trial with leptomycin B, the original specific inhibitor of CRM1, followed by development of several next-generation small molecules. KPT-330, a novel member of the CRM1-selective inhibitors of nuclear export (SINE) class of compounds, is currently undergoing clinical evaluation for the therapy of various malignancies. Results from these trials suggest that SINE compounds may be particularly useful against hematological malignancies, which often become refractory to standard chemotherapeutic agents.

  12. Expression, function, and targeting of the nuclear exporter chromosome region maintenance 1 (CRM1) protein

    PubMed Central

    Ishizawa, Jo; Kojima, Kensuke; Hail, Numsen; Tabe, Yoko; Andreeff, Michael

    2015-01-01

    Nucleocytoplasmic trafficking of proteins/RNAs is essential to normal cellular function. Indeed, accumulating evidence suggests that cancer cells escape anti-neoplastic mechanisms and benefit from pro-survival signals via the dysregulation of this system. The nuclear exporter chromosome region maintenance 1 (CRM1) protein is the only protein in the karyopherin-β protein family that contributes to the trafficking of numerous proteins and RNAs from the nucleus. It is considered to be an oncogenic, anti-apoptotic protein in transformed cells, since it reportedly functions as a gatekeeper for cell survival, including affecting p53 function, and ribosomal biogenesis. Furthermore, abnormally high expression of CRM1 is correlated with poor patient prognosis in various malignancies. Therapeutic targeting of CRM1 has emerged as a novel cancer treatment strategy, starting with a clinical trial with leptomycin B, the original specific inhibitor of CRM1, followed by development of several next-generation small molecules. KPT-330, a novel member of the CRM1-selective inhibitors of nuclear export (SINE) class of compounds, is currently undergoing clinical evaluation for the therapy of various malignancies. Results from these trials suggest that SINE compounds may be particularly useful against hematological malignancies, which often become refractory to standard chemotherapeutic agents. PMID:26048327

  13. Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices.

    PubMed

    Xiong, Yijia; Ford, Nicole R; Hecht, Karen A; Roesijadi, Guritno; Squier, Thomas C

    2016-05-18

    Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

  14. Protein stabilization by macromolecular crowding through enthalpy rather than entropy.

    PubMed

    Senske, Michael; Törk, Lisa; Born, Benjamin; Havenith, Martina; Herrmann, Christian; Ebbinghaus, Simon

    2014-06-25

    The interior of the cell is a densely crowded environment in which protein stability is affected differently than in dilute solution. Macromolecular crowding is commonly understood in terms of an entropic volume exclusion effect based on hardcore repulsions among the macromolecules. We studied the thermal unfolding of ubiquitin in the presence of different cosolutes (glucose, dextran, poly(ethylene glycol), KCl, urea). Our results show that for a correct dissection of the cosolute-induced changes of the free energy into its enthalpic and entropic contributions, the temperature dependence of the heat capacity change needs to be explicitly taken into account. In contrast to the prediction by the excluded volume theory, we observed an enthalpic stabilization and an entropic destabilization for glucose, dextran, and poly(ethylene glycol). The enthalpic stabilization mechanism induced by the macromolecular crowder dextran was similar to the enthalpic stabilization mechanism of its monomeric building block glucose. In the case of poly(ethylene glycol), entropy is dominating over enthalpy leading to an overall destabilization. We propose a new model to classify cosolute effects in terms of their enthalpic contributions to protein stability.

  15. Thermodynamic stability and kinetic foldability of a lattice protein model

    NASA Astrophysics Data System (ADS)

    Li, Jie; Wang, Jun; Zhang, Jian; Wang, Wei

    2004-04-01

    By using serial mutations, i.e., a residue replaced by 19 kinds of naturally occurring residues, the stability of native conformation and folding behavior of mutated sequences are studied. The 3×3×3 lattice protein model with two kinds of interaction potentials between the residues, namely the original Miyazawa and Jernigan (MJ) potentials and the modified MJ potentials (MMJ), is used. Effects of various sites in the mutated sequences on the stability and foldability are characterized through the Z-score and the folding time. It is found that the sites can be divided into three types, namely the hydrophobic-type (H-type), the hydrophilic-type (P-type) and the neutral-type (N-type). These three types of sites relate to the hydrophobic core, the hydrophilic surface and the parts between them. The stability of the native conformation for the serial mutated sequences increases (or decreases) as the increasing in the hydrophobicity of the mutated residues for the H-type sites (or the P-type sites), while varies randomly for the N-type sites. However, the foldability of the mutated sequences is not always consistent with the thermodynamic stability, and their relationship depends on the site types. Since the hydrophobic tendency of the MJ potentials is strong, the ratio between the number of the H-type sites and the number of the P-type sites is found to be 1:2. Differently, for the MJJ potentials it is found that such a ratio is about 1:1 which is relevant to that of real proteins. This suggests that the modification of the MJ potentials is rational in the aspect of thermodynamic stability. The folding of model proteins with the MMJ potentials is fast. However, the relationship between the foldability and the thermodynamic stability of the mutated sequences is complex.

  16. Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

    PubMed Central

    Sanchez-Romero, Inmaculada; Ariza, Antonio; Wilson, Keith S.; Skjøt, Michael; Vind, Jesper; De Maria, Leonardo; Skov, Lars K.; Sanchez-Ruiz, Jose M.

    2013-01-01

    The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein

  17. Stability of Protein-Specific Hydration Shell on Crowding.

    PubMed

    Huang, Kuo-Ying; Kingsley, Carolyn N; Sheil, Ryan; Cheng, Chi-Yuan; Bierma, Jan C; Roskamp, Kyle W; Khago, Domarin; Martin, Rachel W; Han, Songi

    2016-04-27

    We demonstrate that the effect of protein crowding is critically dependent on the stability of the protein's hydration shell, which can dramatically vary between different proteins. In the human eye lens, γS-crystallin (γS-WT) forms a densely packed transparent hydrogel with a high refractive index, making it an ideal system for studying the effects of protein crowding. A single point mutation generates the cataract-related variant γS-G18V, dramatically altering the optical properties of the eye lens. This system offers an opportunity to explore fundamental questions regarding the effect of protein crowding, using γS-WT and γS-G18V: (i) how do the diffusion dynamics of hydration water change as a function of protein crowding?; and (ii) upon hydrogel formation of γS-WT, has a dynamic transition occurred generating a single population of hydration water, or do populations of bulk and hydration water coexist? Using localized spin probes, we separately probe the local translational diffusivity of both surface hydration and interstitial water of γS-WT and γS-G18V in solution. Surprisingly, we find that under the influence of hydrogel formation at highly crowded γS-WT concentrations up to 500 mg/mL, the protein hydration shell remains remarkably dynamic, slowing by less than a factor of 2, if at all, compared to that in dilute protein solutions of ∼5 mg/mL. Upon self-crowding, the population of this robust surface hydration water increases, while a significant bulk-like water population coexists even at ∼500 mg/mL protein concentrations. In contrast, surface water of γS-G18V irreversibly dehydrates with moderate concentration increases or subtle alterations to the solution conditions, demonstrating that the effect of protein crowding is highly dependent on the stability of the protein-specific hydration shell. The core function of γS-crystallin in the eye lens may be precisely its capacity to preserve a robust hydration shell, whose stability is abolished

  18. Genome-health nutrigenomics and nutrigenetics: nutritional requirements or 'nutriomes' for chromosomal stability and telomere maintenance at the individual level.

    PubMed

    Bull, Caroline; Fenech, Michael

    2008-05-01

    It is becoming increasingly evident that (a) risk for developmental and degenerative disease increases with more DNA damage, which in turn is dependent on nutritional status, and (b) the optimal concentration of micronutrients for prevention of genome damage is also dependent on genetic polymorphisms that alter the function of genes involved directly or indirectly in the uptake and metabolism of micronutrients required for DNA repair and DNA replication. The development of dietary patterns, functional foods and supplements that are designed to improve genome-health maintenance in individuals with specific genetic backgrounds may provide an important contribution to an optimum health strategy based on the diagnosis and individualised nutritional prevention of genome damage, i.e. genome health clinics. The present review summarises some of the recent knowledge relating to micronutrients that are associated with chromosomal stability and provides some initial insights into the likely nutritional factors that may be expected to have an impact on the maintenance of telomeres. It is evident that developing effective strategies for defining nutrient doses and combinations or 'nutriomes' for genome-health maintenance at the individual level is essential for further progress in this research field.

  19. Genome-health nutrigenomics and nutrigenetics: nutritional requirements or 'nutriomes' for chromosomal stability and telomere maintenance at the individual level.

    PubMed

    Bull, Caroline; Fenech, Michael

    2008-05-01

    It is becoming increasingly evident that (a) risk for developmental and degenerative disease increases with more DNA damage, which in turn is dependent on nutritional status, and (b) the optimal concentration of micronutrients for prevention of genome damage is also dependent on genetic polymorphisms that alter the function of genes involved directly or indirectly in the uptake and metabolism of micronutrients required for DNA repair and DNA replication. The development of dietary patterns, functional foods and supplements that are designed to improve genome-health maintenance in individuals with specific genetic backgrounds may provide an important contribution to an optimum health strategy based on the diagnosis and individualised nutritional prevention of genome damage, i.e. genome health clinics. The present review summarises some of the recent knowledge relating to micronutrients that are associated with chromosomal stability and provides some initial insights into the likely nutritional factors that may be expected to have an impact on the maintenance of telomeres. It is evident that developing effective strategies for defining nutrient doses and combinations or 'nutriomes' for genome-health maintenance at the individual level is essential for further progress in this research field. PMID:18412988

  20. Thermal stability of matrix protein from Newcastle disease virus.

    PubMed

    Morán, Irene Sánchez; Cuadrado-Castano, Sara; Barroso, Isabel Muñoz; Kostetsky, Eduard Ya; Zhadan, Galina; Gómez, Javier; Shnyrov, Valery L; Villar, Enrique

    2013-10-01

    The thermal stability of the matrix protein (M protein) of Newcastle disease virus (NDV) has been investigated using high-sensitivity differential scanning calorimetry (DSC) at pH 7.4. The thermal folding/unfolding of M protein at this pH value is a reversible process involving a highly cooperative transition between folded and unfolded monomers with a transition temperature (Tm) of 63 °C, an unfolding enthalpy, ΔH(Tm), of 340 kcal mol(-1), and the difference in heat capacity between the native and denatured states of the protein, ΔCp, of 5.1 kcal K(-1) mol(-1). The heat capacity of the native state of the protein is in good agreement with the values calculated using a structure-based parameterization, whereas the calculated values for the hypothetical fully-unfolded state of the protein is higher than those determined experimentally. This difference between the heat capacity of denatured M protein and the heat capacity expected for an unstructured polypeptide of the same sequence, together with the data derived from the heat-induced changes in the steady-state fluorescence of the protein, indicates that the polypeptide chain maintains a significant amount of residual structure after thermal denaturation. PMID:23916643

  1. Stability of β-carotene in protein-stabilized oil-in-water delivery systems.

    PubMed

    Cornacchia, Leonardo; Roos, Yrjo H

    2011-07-13

    Inclusion of liposoluble bioactive compounds in fortified foods represents a complex challenge due to the labile nature of such compounds and the instability of oil-in-water emulsion-based delivery systems. In the present study, dispersions prepared with 10% (w/w) sunflower oil (SO) or hydrogenated palm kernel oil (HPKO) containing 0.05% (w/w) β-carotene were stabilized by various concentrations of whey protein isolate (WPI) or sodium caseinate (NaCas) (0.1 to 2.0% w/w) in 30% (w/w) sucrose aqueous solutions. Physicochemical characterization of emulsions was done considering the particle size, the particle surface protein coverage, and the physical state of continuous and dispersed phases. Physical stability of the systems and their protection properties on β-carotene were compared. The lipid carrier type and interfacial structure were investigated as the two key factors which regulate the stability of labile lipophilic bioactive molecules in food model systems. Our results showed high β-carotene stability when O/W systems were stable (protein concentration ≥0.8% w/w.) A (partially) solid lipid carrier (HPKO) enhanced protection compared to the liquid carrier (SO) as the bioactive molecules were entrapped in isolated domains within the solid lattice and kept apart from reactive species in the surroundings. NaCas provided a better barrier than WPI due to the different amino acid composition and interface structure which significantly reduced β-carotene degradation rate.

  2. Residue level quantification of protein stability in living cells.

    PubMed

    Monteith, William B; Pielak, Gary J

    2014-08-01

    The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed. This difference has led both experimentalists and theoreticians to tackle the challenging task of understanding how the intracellular environment affects the properties of biopolymers. Despite a growing number of in-cell studies, there is a lack of quantitative, residue-level information about equilibrium thermodynamic protein stability under nonperturbing conditions. We report the use of NMR-detected hydrogen-deuterium exchange of quenched cell lysates to measure individual opening free energies of the 56-aa B1 domain of protein G (GB1) in living Escherichia coli cells without adding destabilizing cosolutes or heat. Comparisons to dilute solution data (pH 7.6 and 37 °C) show that opening free energies increase by as much as 1.14 ± 0.05 kcal/mol in cells. Importantly, we also show that homogeneous protein crowders destabilize GB1, highlighting the challenge of recreating the cellular interior. We discuss our findings in terms of hard-core excluded volume effects, charge-charge GB1-crowder interactions, and other factors. The quenched lysate method identifies the residues most important for folding GB1 in cells, and should prove useful for quantifying the stability of other globular proteins in cells to gain a more complete understanding of the effects of the intracellular environment on protein chemistry.

  3. Cellular proteomes have broad distributions of protein stability.

    PubMed

    Ghosh, Kingshuk; Dill, Ken

    2010-12-15

    Biological cells are extremely sensitive to temperature. What is the mechanism? We compute the thermal stabilities of the whole proteomes of Escherichia coli, yeast, and Caenorhabditis elegans using an analytical model and an extensive database of stabilities of individual proteins. Our results support the hypothesis that a cell's thermal sensitivities arise from the collective instability of its proteins. This model shows a denaturation catastrophe at temperatures of 49-55°C, roughly the thermal death point of mesophiles. Cells live on the edge of a proteostasis catastrophe. According to the model, it is not that the average protein is problematic; it is the tail of the distribution. About 650 of E. coli's 4300 proteins are less than 4 kcal mol(-1) stable to denaturation. And upshifting by only 4° from 37° to 41°C is estimated to destabilize an average protein by nearly 20%. This model also treats effects of denaturants, osmolytes, and other physical stressors. In addition, it predicts the dependence of cellular growth rates on temperature. This approach may be useful for studying physical forces in biological evolution and the role of climate change on biology.

  4. Sgs1, a Homologue of the Bloom's and Werner's Syndrome Genes, Is Required for Maintenance of Genome Stability in Saccharomyces Cerevisiae

    PubMed Central

    Watt, P. M.; Hickson, I. D.; Borts, R. H.; Louis, E. J.

    1996-01-01

    The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability. sgs1Δ strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1Δ mutants, although meiosis I chromosome missegregation has been shown to be elevated. sgs1Δ suppresses the slow growth of a top3Δ strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1Δ strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3Δ or a sgs1Δ strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae. PMID:8913739

  5. Redox control of iron regulatory protein 2 stability.

    PubMed

    Hausmann, Anja; Lee, Julie; Pantopoulos, Kostas

    2011-02-18

    Iron regulatory protein 2 (IRP2) is a critical switch for cellular and systemic iron homeostasis. In iron-deficient or hypoxic cells, IRP2 binds to mRNAs containing iron responsive elements (IREs) and regulates their expression. Iron promotes proteasomal degradation of IRP2 via the F-box protein FBXL5. Here, we explored the effects of oxygen and cellular redox status on IRP2 stability. We show that iron-dependent decay of tetracycline-inducible IRP2 proceeds efficiently under mild hypoxic conditions (3% oxygen) but is compromised in severe hypoxia (0.1% oxygen). A treatment of cells with exogenous H(2)O(2) protects IRP2 against iron and increases its IRE-binding activity. IRP2 is also stabilized during menadione-induced oxidative stress. These data demonstrate that the degradation of IRP2 in iron-replete cells is not only oxygen-dependent but also sensitive to redox perturbations.

  6. Structural stability of proteins in aqueous and nonpolar environments

    NASA Astrophysics Data System (ADS)

    Yasuda, Satoshi; Oshima, Hiraku; Kinoshita, Masahiro

    2012-10-01

    A protein folds into its native structure with the α-helix and/or β-sheet in aqueous solution under the physiological condition. The relative content of these secondary structures largely varies from protein to protein. However, such structural variability is not exhibited in nonaqueous environment. For example, there is a strong trend that alcohol induces a protein to form α-helices, and many of the membrane proteins within the lipid bilayer consists of α-helices. Here we investigate the structural stability of proteins in aqueous and nonpolar environments using our recently developed free-energy function F = (Λ - TS)/(kBT0) = Λ/(kBT0) - S/kB (T0 = 298 K and the absolute temperature T is set at T0) which is based on statistical thermodynamics. Λ/(kBT0) and S/kB are the energetic and entropic components, respectively, and kB is Boltzmann's constant. A smaller value of the positive quantity, -S, represents higher efficiency of the backbone and side-chain packing promoted by the entropic effect arising from the translational displacement of solvent molecules or the CH2, CH3, and CH groups which constitute nonpolar chains of lipid molecules. As for Λ, in aqueous solution, a transition to a more compact structure of a protein accompanies the break of protein-solvent hydrogen bonds: As the number of donors and acceptors buried without protein intramolecular hydrogen bonding increases, Λ becomes higher. In nonpolar solvent, lower Λ simply implies more intramolecular hydrogen bonds formed. We find the following. The α-helix and β-sheet are advantageous with respect to -S as well as Λ and to be formed as much as possible. In aqueous solution, the solvent-entropy effect on the structural stability is so strong that the close packing of side chains is dominantly important, and the α-helix and β-sheet contents are judiciously adjusted to accomplish it. In nonpolar solvent, the solvent-entropy effect is substantially weaker than in aqueous solution. Λ is

  7. Physical and oxidative stability of fish oil-in-water emulsions stabilized with fish protein hydrolysates.

    PubMed

    García-Moreno, Pedro J; Guadix, Antonio; Guadix, Emilia M; Jacobsen, Charlotte

    2016-07-15

    The emulsifying and antioxidant properties of fish protein hydrolysates (FPH) for the physical and oxidative stabilization of 5% (by weight) fish oil-in-water emulsions were investigated. Muscle proteins from sardine (Sardina pilchardus) and small-spotted catshark (Scyliorhinus canicula) were hydrolyzed to degrees of hydrolysis (DH) of 3-4-5-6% with subtilisin. Sardine hydrolysates with low DH, 3% and 4%, presented the most effective peptides to physically stabilize emulsions with smaller droplet size. This implied more protein adsorbed at the interface to act as physical barrier against prooxidants. This fact might also be responsible for the higher oxidative stability of these emulsions, as shown by their lowest peroxide value and concentration of volatiles such as 1-penten-3-one and 1-penten-3-ol. Among the hydrolysates prepared from small-spotted catshark only the hydrolysate with DH 3% yielded a physically stable emulsion with low concentration of unsaturated aldehydes. These results show the potential of FPH as alternative protein emulsifiers for the production of oxidatively stable fish oil-in-water emulsions.

  8. Bromodomain Proteins Contribute to Maintenance of Bloodstream Form Stage Identity in the African Trypanosome

    PubMed Central

    Schulz, Danae; Mugnier, Monica R.; Paulsen, Eda-Margaret; Kim, Hee-Sook; Chung, Chun-wa W.; Tough, David F.; Rioja, Inmaculada; Prinjha, Rab K.; Papavasiliou, F. Nina; Debler, Erik W.

    2015-01-01

    Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite’s surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design. PMID:26646171

  9. Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance

    SciTech Connect

    Lu, Duo; Bernstein, Douglas A.; Satyshur, Kenneth A.; Keck, James L.

    2010-09-03

    Bacterial single-stranded DNA-binding proteins (SSBs) help to recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. For all cases examined to date, these interactions are mediated by the evolutionarily conserved C terminus of SSB (SSB-Ct). The essential nature of SSB protein interactions makes inhibitors that block SSB complex formation valuable biochemical tools and attractive potential antibacterial agents. Here, we identify four small molecules that disrupt complexes formed between Escherichia coli SSB and Exonuclease I (ExoI), a well-studied SSB-interacting enzyme. Each compound disrupts ExoI/SSB-Ct peptide complexes and abrogates SSB stimulation of ExoI nuclease activity. Structural and biochemical studies support a model for three of the compounds in which they compete with SSB for binding to ExoI. The fourth appears to rely on an allosteric mechanism to disrupt ExoI/SSB complexes. Subsets of the inhibitors block SSB-Ct complex formation with two other SSB-interaction partners as well, which highlights their utility as reagents for investigating the roles of SSB/protein interactions in diverse DNA replication, recombination, and repair reactions.

  10. Intercellular chaperone transmission via exosomes contributes to maintenance of protein homeostasis at the organismal level

    PubMed Central

    Takeuchi, Toshihide; Suzuki, Mari; Fujikake, Nobuhiro; Popiel, H. Akiko; Kikuchi, Hisae; Futaki, Shiroh; Wada, Keiji; Nagai, Yoshitaka

    2015-01-01

    The heat shock response (HSR), a transcriptional response that up-regulates molecular chaperones upon heat shock, is necessary for cell survival in a stressful environment to maintain protein homeostasis (proteostasis). However, there is accumulating evidence that the HSR does not ubiquitously occur under stress conditions, but largely depends on the cell types. Despite such imbalanced HSR among different cells and tissues, molecular mechanisms by which multicellular organisms maintain their global proteostasis have remained poorly understood. Here, we report that proteostasis can be maintained by molecular chaperones not only in a cell-autonomous manner but also in a non–cell-autonomous manner. We found that elevated expression of molecular chaperones, such as Hsp40 and Hsp70, in a group of cells improves proteostasis in other groups of cells, both in cultured cells and in Drosophila expressing aggregation-prone polyglutamine proteins. We also found that Hsp40, as well as Hsp70 and Hsp90, is physiologically secreted from cells via exosomes, and that the J domain at the N terminus is responsible for its exosome-mediated secretion. Addition of Hsp40/Hsp70-containing exosomes to the culture medium of the polyglutamine-expressing cells results in efficient suppression of inclusion body formation, indicating that molecular chaperones non-cell autonomously improve the protein-folding environment via exosome-mediated transmission. Our study reveals that intercellular chaperone transmission mediated by exosomes is a novel molecular mechanism for non–cell-autonomous maintenance of organismal proteostasis that could functionally compensate for the imbalanced state of the HSR among different cells, and also provides a novel physiological role of exosomes that contributes to maintenance of organismal proteostasis. PMID:25918398

  11. Altered Dimer Interface Decreases Stability in an Amyloidogenic Protein

    SciTech Connect

    Baden, Elizabeth M.; Owen, Barbara A.L.; Peterson, Francis C.; Volkman, Brian F.; Ramirez-Alvarado, Marina; Thompson, James R.

    2008-07-21

    Amyloidoses are devastating and currently incurable diseases in which the process of amyloid formation causes fatal cellular and organ damage. The molecular mechanisms underlying amyloidoses are not well known. In this study, we address the structural basis of immunoglobulin light chain amyloidosis, which results from deposition of light chains produced by clonal plasma cells. We compare light chain amyloidosis protein AL-09 to its wild-type counterpart, the kl O18/O8 light chain germline. Crystallographic studies indicate that both proteins form dimers. However, AL-09 has an altered dimer interface that is rotated 90 degrees from the kl O18/O8 dimer interface. The three non-conservative mutations in AL-09 are located within the dimer interface, consistent with their role in the decreased stability of this amyloidogenic protein. Moreover, AL-09 forms amyloid fibrils more quickly than kl O18/O8 in vitro. These results support the notion that the increased stability of the monomer and delayed fibril formation, together with a properly formed dimer, may be protective against amyloidogenesis. This could open a new direction into rational drug design for amyloidogenic proteins.

  12. Properties and stability of oil-in-water emulsions stabilized by coconut skim milk proteins.

    PubMed

    Onsaard, Ekasit; Vittayanont, Manee; Srigam, Sukoncheun; McClements, D Julian

    2005-07-13

    Protein fractions were isolated from coconut: coconut skim milk protein isolate (CSPI) and coconut skim milk protein concentrate (CSPC). The ability of these proteins to form and stabilize oil-in-water emulsions was compared with that of whey protein isolate (WPI). The solubility of the proteins in CSPI, CSPC, and WPI was determined in aqueous solutions containing 0, 100, and 200 mM NaCl from pH 3 to 8. In the absence of salt, the minimum protein solubility occurred between pH 4 and 5 for CSPI and CSPC and around pH 5 for WPI. In the presence of salt (100 and 200 mM NaCl), all proteins had a higher solubility than in distilled water. Corn oil-in-water emulsions (10 wt %) with relatively small droplet diameters (d32 approximately 0.46, 1.0, and 0.5 mum for CSPI, CSPC, and WPI, respectively) could be produced using 0.2 wt % protein fraction. Emulsions were prepared with different pH values (3-8), salt concentrations (0-500 mM NaCl), and thermal treatments (30-90 degrees C for 30 min), and the mean particle diameter, particle size distribution, zeta-potential, and creaming stability were measured. Considerable droplet flocculation occurred in the emulsions near the isoelectric point of the proteins: CSPI, pH approximately 4.0; CSPC, pH approximately 4.5; WPI, pH approximately 4.8. Emulsions with monomodal particle size distributions, small mean droplet diameters, and good creaming stability could be produced at pH 7 for CSPI and WPI, whereas CSPC produced bimodal distributions. The CSPI and WPI emulsions remained relatively stable to droplet aggregation and creaming at NaCl concentrations of < or =50 and < or =100 mM, respectively. In the absence salt, the CSPI and WPI emulsions were also stable to thermal treatments at < or =80 and < or =90 degrees C for 30 min, respectively. These results suggest that CSPI may be suitable for use as an emulsifier in the food industry.

  13. Properties and stability of oil-in-water emulsions stabilized by coconut skim milk proteins.

    PubMed

    Onsaard, Ekasit; Vittayanont, Manee; Srigam, Sukoncheun; McClements, D Julian

    2005-07-13

    Protein fractions were isolated from coconut: coconut skim milk protein isolate (CSPI) and coconut skim milk protein concentrate (CSPC). The ability of these proteins to form and stabilize oil-in-water emulsions was compared with that of whey protein isolate (WPI). The solubility of the proteins in CSPI, CSPC, and WPI was determined in aqueous solutions containing 0, 100, and 200 mM NaCl from pH 3 to 8. In the absence of salt, the minimum protein solubility occurred between pH 4 and 5 for CSPI and CSPC and around pH 5 for WPI. In the presence of salt (100 and 200 mM NaCl), all proteins had a higher solubility than in distilled water. Corn oil-in-water emulsions (10 wt %) with relatively small droplet diameters (d32 approximately 0.46, 1.0, and 0.5 mum for CSPI, CSPC, and WPI, respectively) could be produced using 0.2 wt % protein fraction. Emulsions were prepared with different pH values (3-8), salt concentrations (0-500 mM NaCl), and thermal treatments (30-90 degrees C for 30 min), and the mean particle diameter, particle size distribution, zeta-potential, and creaming stability were measured. Considerable droplet flocculation occurred in the emulsions near the isoelectric point of the proteins: CSPI, pH approximately 4.0; CSPC, pH approximately 4.5; WPI, pH approximately 4.8. Emulsions with monomodal particle size distributions, small mean droplet diameters, and good creaming stability could be produced at pH 7 for CSPI and WPI, whereas CSPC produced bimodal distributions. The CSPI and WPI emulsions remained relatively stable to droplet aggregation and creaming at NaCl concentrations of < or =50 and < or =100 mM, respectively. In the absence salt, the CSPI and WPI emulsions were also stable to thermal treatments at < or =80 and < or =90 degrees C for 30 min, respectively. These results suggest that CSPI may be suitable for use as an emulsifier in the food industry. PMID:15998143

  14. Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR-Cas9 library.

    PubMed

    Wu, Yuanzhong; Kang, Tiebang

    2016-06-29

    The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A," recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.

  15. Fragmented proteins as food emulsion stabilizers: A theoretical study.

    PubMed

    Ettelaie, Rammile; Zengin, Adem; Lee, Hazel

    2014-09-01

    Using a model based on the primary structure of αs1 -casein, the colloid stabilizing ability of fragmented protein and that of the intact chains are compared. We perform self consistent field (SCF) calculations to obtain the induced interaction potentials between the oil droplets, resulting from the overlap of adsorbed protein layers in each case. For the intact αs1 -casein, we confirm the known result, that the mediated interparticle interaction potential develops a deep attractive energy minimum at high salt concentrations and pH values close to the isoelectric point of the protein. The same does not occur for the appropriately fragmented systems, with improved emulsion stability predicted as a result, even at pH values close to pI. It is shown that this superior performance, for the case considered, is due to the diblock-type behavior of one of the fragments. However, it is well known that αs1 -casein more closely resembles the less favorable triblock structure. However, it is also demonstrated that the presence of a "diblock" like fragment by itself may not always be enough to produce a better emulsion stabilizer. It is seen that the hydrolysis of some peptide bonds may indeed lead to a suitable polypeptide, but that this is displaced from the interface by the structurally less desirable ones, also generated by the fragmentation process. The displacement occurs due to the competitive adsorption between different fragments. The removal of the undesirable fragments from the solution is found to greatly enhance the predicted colloid stabilizing ability of the remaining polypeptide.

  16. Cleavage and polyadenylation factor, Rna14 is an essential protein required for the maintenance of genomic integrity in fission yeast Schizosaccharomyces pombe.

    PubMed

    Sonkar, Amit; Yadav, Sudhanshu; Ahmed, Shakil

    2016-02-01

    Faithful segregation of chromosomes is essential for the maintenance of genome integrity. In a genetic screen to identify genes related to checkpoint function, we have characterized the role of rna14, an essential gene in the maintenance of chromosome dynamics. We demonstrate that Rna14 localizes in the nucleus and in the absence of functional Rna14, the cells exhibit chromosomal segregation defects. The mutant allele of rna14 exhibits genetic interaction with key kinetochore components and spindle checkpoint proteins. Inactivation of rna14 leads to accumulation of Bub1-GFP foci, a protein required for spindle checkpoint activation that could be due to the defects in the attachment of mitotic spindle to the chromosome. Consistently, the double mutant of rna14-11 and bub1 knockout exhibits high degree of chromosome mis-segregation. At restrictive condition, the rna14-11 mutant cells exhibit defects in cell cycle progression with high level of septation. The orthologs of Rna14 in Saccharomyces cerevisiae (sc Rna14) and human (CstF3) contain similar domain architecture and are required for 3'-end processing of pre-mRNA. We have also demonstrated that the fission yeast Rna14 is required to prevent transcriptional read-through. These findings reveal the importance of transcription termination in the maintenance of genomic stability through the regulation of kinetochore function.

  17. Liquid drop stability for protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Owen, Robert B.; Broom, Beth H.; Snyder, Robert S.; Daniel, Ron

    1987-01-01

    It is possible to grow protein crystals for biomedical research in microgravity by deploying a protein-rich solution from a syringe, forming a drop in which crystallization can occur with the proper degree of supersaturation. Drop stability is critical to the success of this research, due to the large drop sizes which can be achieved in space. In order to determine the type of syringe tips most suitable to support these large drops, tests were performed during brief periods of weightlessness onboard the NASA KC-135 low-gravity simulation aircraft. The drops were analyzed using three simple models in which the samples were approximated by modified pendulum and spring systems. It was concluded that the higher frequency systems were the most stable, indicating that of the syringes utilized, a disk-shaped configuration provided the most stable environment of low-gravity protein crystal growth.

  18. Protein folding, stability, and solvation structure in osmolyte solutions hydrophobicity

    NASA Astrophysics Data System (ADS)

    Montgomery Pettitt, B.

    2008-03-01

    The hydrophobic effect between solutes in aqueous solutions plays a central role in our understanding of recognition and folding of proteins and self assembly of lipids. Hydrophobicity induces nonideal solution behavior which plays a role in many aspects of biophysics. Work on the use of small biochemical compounds to crowd protein solutions indicates that a quantitative description of their non-ideal behavior is possible and straightforward. Here, we will show what the structural origin of this non-ideal solution behavior is from expression derived from a semi grand ensemble approach. We discuss the consequences of these findings regarding protein folding stability and solvation in crowded solutions through a structural analysis of the m-value or the change in free energy difference of a macromolecule in solution with respect to the concentration of a third component. This effect has recently been restudied and new mechanisms proposed for its origins in terms of transfer free energies and hydrophobicity.

  19. SHA1, a novel RING finger protein, functions in shoot apical meristem maintenance in Arabidopsis.

    PubMed

    Sonoda, Yutaka; Yao, Shan-Guo; Sako, Kaori; Sato, Takeo; Kato, Wataru; Ohto, Masa-aki; Ichikawa, Takanari; Matsui, Minami; Yamaguchi, Junji; Ikeda, Akira

    2007-05-01

    Post-embryonic plant growth is dependent on a functional shoot apical meristem (SAM) that provides cells for continuous development of new aerial organs. However, how the SAM is dynamically maintained during vegetative development remains largely unclear. We report here the characterization of a new SAM maintenance mutant, sha1-1 (shoot apical meristem arrest 1-1), that shows a primary SAM-deficient phenotype at the adult stage. The SHA1 gene encodes a novel RING finger protein, and is expressed most intensely in the shoot apex. We show that, in the sha1-1 mutant, the primary SAM develops normally during the juvenile vegetative stage, but cell layer structure becomes disorganized after entering the adult vegetative stage, resulting in a dysfunctional SAM that cannot initiate floral primordia. The sha1-1 SAM terminates completely at the stage when the wild-type begins to bolt, producing adult plants with a primary inflorescence-deficient phenotype. These observations indicate that SHA1, a putative E3 ligase, is required for post-embryonic SAM maintenance by controlling proper cellular organization. PMID:17461786

  20. Chromatin protein HMGB2 regulates articular cartilage surface maintenance via β-catenin pathway

    PubMed Central

    Taniguchi, Noboru; Caramés, Beatriz; Kawakami, Yasuhiko; Amendt, Brad A.; Komiya, Setsuro; Lotz, Martin

    2009-01-01

    The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis. Mechanisms that regulate the unique phenotype of SZ chondrocytes and maintain SZ integrity are unknown. We recently demonstrated that expression of the chromatin protein high mobility group box (HMGB) protein 2 is restricted to the SZ in articular cartilage suggesting a transcriptional regulation involving HMGB2 in SZ. Here, we show that an interaction between HMGB2 and the Wnt/β-catenin pathway regulates the maintenance of the SZ. We found that the Wnt/β-catenin pathway is active specifically in the SZ in normal mouse knee joints and colocalizes with HMGB2. Both Wnt signaling and HMGB2 expression decrease with aging in mouse joints. Our molecular studies show that HMGB2 enhances the binding of Lef-1 to its target sequence and potentiates transcriptional activation of the Lef-1-β-catenin complex. The HMG domain within HMGB2 is crucial for interaction with Lef-1, suggesting that both HMGB2 and HMGB1 may be involved in this function. Furthermore, conditional deletion of β-catenin in cultured mouse chondrocytes induced apoptosis. These findings define a pathway where protein interactions of HMGB2 and Lef-1 enhance Wnt signaling and promote SZ chondrocyte survival. Loss of the HMGB2-Wnt signaling interaction is a new mechanism in aging-related cartilage pathology. PMID:19805379

  1. Effective stabilization of CLA by microencapsulation in pea protein.

    PubMed

    Costa, A M M; Nunes, J C; Lima, B N B; Pedrosa, C; Calado, V; Torres, A G; Pierucci, A P T R

    2015-02-01

    CLA was microencapsulated by spray drying in ten varied wall systems (WS) consisting of pea protein isolate or pea protein concentrate (PPC) alone at varied core:WS ratios (1:2; 1:3 and 1:4), or blended with maltodextrin (M) and carboxymethylcellulose at a pea protein:carbohydrate ratio of 3:1. The physical-chemical properties of the CLA microparticles were characterised by core retention, microencapsulation efficiency (ME), particle size and moisture. CLA:M:PPC (1:1:3) showed the most promising results, thus we evaluated the effect of M addition in the WS on other physical-chemical characteristics and oxidative stability (CLA isomer profile, quantification of CLA and volatile compounds by SPME coupled with CG-MS) during two months of storage at room temperature, CLA:PPC (1:4) was selected for comparisons. CLA:M:PPC (1:1:3) microparticles demonstrated better morphology, solubility, dispersibility and higher glass-transition temperature values. M addition did not influence the oxidative stability of CLA, however its presence improved physical-chemical characteristics necessary for food applications.

  2. Diets with High or Low Protein Content and Glycemic Index for Weight-Loss Maintenance

    PubMed Central

    Larsen, Thomas Meinert; Dalskov, Stine-Mathilde; van Baak, Marleen; Jebb, Susan A.; Papadaki, Angeliki; Pfeiffer, Andreas F.H.; Martinez, J. Alfredo; Handjieva-Darlenska, Teodora; Kunešová, Marie; Pihlsgård, Mats; Stender, Steen; Holst, Claus; Saris, Wim H.M.; Astrup, Arne

    2012-01-01

    Background Studies of weight-control diets that are high in protein or low in glycemic index have reached varied conclusions, probably owing to the fact that the studies had insufficient power. Methods We enrolled overweight adults from eight European countries who had lost at least 8% of their initial body weight with a 3.3-MJ (800-kcal) low-calorie diet. Participants were randomly assigned, in a two-by-two factorial design, to one of five ad libitum diets to prevent weight regain over a 26-week period: a low-protein and low-glycemic-index diet, a low-protein and high-glycemic-index diet, a high-protein and low-glycemic-index diet, a high-protein and high-glycemic-index diet, or a control diet. Results A total of 1209 adults were screened (mean age, 41 years; body-mass index [the weight in kilograms divided by the square of the height in meters], 34), of whom 938 entered the low-calorie-diet phase of the study. A total of 773 participants who completed that phase were randomly assigned to one of the five maintenance diets; 548 completed the intervention (71%). Fewer participants in the high-protein and the low-glycemic-index groups than in the low-protein–high-glycemic-index group dropped out of the study (26.4% and 25.6%, respectively, vs. 37.4%; P = 0.02 and P = 0.01 for the respective comparisons). The mean initial weight loss with the low-calorie diet was 11.0 kg. In the analysis of participants who completed the study, only the low-protein–high-glycemic-index diet was associated with subsequent significant weight regain (1.67 kg; 95% confidence interval [CI], 0.48 to 2.87). In an intention-to-treat analysis, the weight regain was 0.93 kg less (95% CI, 0.31 to 1.55) in the groups assigned to a high-protein diet than in those assigned to a low-protein diet (P = 0.003) and 0.95 kg less (95% CI, 0.33 to 1.57) in the groups assigned to a low-glycemic-index diet than in those assigned to a high-glycemic-index diet (P = 0.003). The analysis involving

  3. Biacore analysis with stabilized G-protein-coupled receptors.

    PubMed

    Rich, Rebecca L; Errey, James; Marshall, Fiona; Myszka, David G

    2011-02-15

    Using stabilized forms of β₁ adrenergic and A₂(A) adenosine G-protein-coupled receptors, we applied Biacore to monitor receptor activity and characterize binding constants of small-molecule antagonists spanning more than 20,000-fold in affinity. We also illustrate an improved method for tethering His-tagged receptors on NTA (carboxymethylated dextran preimmobilized with nitrilotriacetic acid) chips to yield stable, high-capacity, high-activity surfaces as well as a novel approach to regenerate receptor binding sites. Based on our success with this approach, we expect that the combination of stabilized receptors with biosensor technology will become a common method for characterizing members of this receptor family.

  4. Exploring the Metabolic Stability of Engineered Hairy Roots after 16 Years Maintenance

    PubMed Central

    Häkkinen, Suvi T.; Moyano, Elisabeth; Cusidó, Rosa M.; Oksman-Caldentey, Kirsi-Marja

    2016-01-01

    Plants remain a major source of new drugs, leads and fine chemicals. Cell cultures deriving from plants offer a fascinating tool to study plant metabolic pathways and offer large scale production systems for valuable compounds – commercial examples include compounds such as paclitaxel. The major constraint with undifferentiated cell cultures is that they are generally considered to be genetically unstable and cultured cells tend to produce low yields of secondary metabolites especially over time. Hairy roots, a tumor tissue caused by infection of Agrobacterium rhizogenes is a relevant alternative for plant secondary metabolite production for being fast growing, able to grow without phytohormones, and displaying higher stability than undifferentiated cells. Although genetic and metabolic stability has often been connected to transgenic hairy roots, there are only few reports on how a very long-term subculturing effects on the production capacity of hairy roots. In this study, hairy roots producing high tropane alkaloid levels were subjected to 16-year follow-up in relation to genetic and metabolic stability. Cryopreservation method for hairy roots of Hyoscyamus muticus was developed to replace laborious subculturing, and although the post-thaw recovery rates remained low, the expression of transgene remained unaltered in cryopreserved roots. It was shown that although displaying some fluctuation in the metabolite yields, even an exceedingly long-term subculturing was successfully applied without significant loss of metabolic activity. PMID:27746806

  5. SLIRP stabilizes LRPPRC via an RRM-PPR protein interface.

    PubMed

    Spåhr, Henrik; Rozanska, Agata; Li, Xinping; Atanassov, Ilian; Lightowlers, Robert N; Chrzanowska-Lightowlers, Zofia M A; Rackham, Oliver; Larsson, Nils-Göran

    2016-08-19

    LRPPRC is a protein that has attracted interest both for its role in post-transcriptional regulation of mitochondrial gene expression and more recently because numerous mutated variants have been characterized as causing severe infantile mitochondrial neurodegeneration. LRPPRC belongs to the pentatricopeptide repeat (PPR) protein family, originally defined by their RNA binding capacity, and forms a complex with SLIRP that harbours an RNA recognition motif (RRM) domain. We show here that LRPPRC displays a broad and strong RNA binding capacity in vitro in contrast to SLIRP that associates only weakly with RNA. The LRPPRC-SLIRP complex comprises a hetero-dimer via interactions by polar amino acids in the single RRM domain of SLIRP and three neighbouring PPR motifs in the second quarter of LRPPRC, which critically contribute to the LRPPRC-SLIRP binding interface to enhance its stability. Unexpectedly, specific amino acids at this interface are located within the PPRs of LRPPRC at positions predicted to interact with RNA and within the RNP1 motif of SLIRP's RRM domain. Our findings thus unexpectedly establish that despite the prediction that these residues in LRPPRC and SLIRP should bind RNA, they are instead used to facilitate protein-protein interactions, enabling the formation of a stable complex between these two proteins.

  6. Cockayne syndrome group B protein (CSB) plays a general role in chromatin maintenance and remodeling

    PubMed Central

    Newman, John C.; Bailey, Arnold D.; Weiner, Alan M.

    2006-01-01

    Cockayne syndrome (CS) is an inherited neurodevelopmental disorder with progeroid features. Although the genes responsible for CS have been implicated in a variety of DNA repair- and transcription-related pathways, the nature of the molecular defect in CS remains mysterious. Using expression microarrays and a unique method for comparative expression analysis called L2L, we sought to define this defect in cells lacking a functional CS group B (CSB) protein, the SWI/SNF-like ATPase responsible for most cases of CS. Remarkably, many of the genes regulated by CSB are also affected by inhibitors of histone deacetylase and DNA methylation, as well as by defects in poly(ADP-ribose)-polymerase function and RNA polymerase II elongation. Moreover, consistent with these microarray expression data, CSB-null cells are sensitive to inhibitors of histone deacetylase or poly(ADP-ribose)-polymerase. Our data indicate a general role for CSB protein in maintenance and remodeling of chromatin structure and suggest that CS is a disease of transcriptional deregulation caused by misexpression of growth-suppressive, inflammatory, and proapoptotic pathways. PMID:16772382

  7. Effect of minichromosome maintenance protein 2 deficiency on the locations of DNA replication origins

    PubMed Central

    Kunnev, Dimiter; Freeland, Amy; Qin, Maochun; Leach, Robert W.; Wang, Jianmin; Shenoy, Rajani M.

    2015-01-01

    Minichromosome maintenance (MCM) proteins are loaded onto chromatin during G1-phase and define potential locations of DNA replication initiation. MCM protein deficiency results in genome instability and high rates of cancer in mouse models. Here we develop a method of nascent strand capture and release and show that MCM2 deficiency reduces DNA replication initiation in gene-rich regions of the genome. DNA structural properties are shown to correlate with sequence motifs associated with replication origins and with locations that are preferentially affected by MCM2 deficiency. Reduced nascent strand density correlates with sites of recurrent focal CNVs in tumors arising in MCM2-deficient mice, consistent with a direct relationship between sites of reduced DNA replication initiation and genetic damage. Between 10% and 90% of human tumors, depending on type, carry heterozygous loss or mutation of one or more MCM2-7 genes, which is expected to compromise DNA replication origin licensing and result in elevated rates of genome damage at a subset of gene-rich locations. PMID:25762552

  8. Maintenance of mitochondrial genome distribution by mitochondrial AAA+ protein ClpX.

    PubMed

    Kasashima, Katsumi; Sumitani, Megumi; Endo, Hitoshi

    2012-11-01

    The segregation of mitochondrial DNA (mtDNA) is important for the maintenance and transmission of the genome between generations. Recently, we clarified that human mitochondrial transcription factor A (TFAM) is required for equal distribution and symmetric segregation of mtDNA in cultured cells; however, the molecular mechanism involved is largely unknown. ClpX is an ATPase associated with various cellular activities (AAA+) proteins that localize to the mitochondrial matrix and is suggested to associate with mtDNA. In this study, we found that RNAi-mediated knockdown of ClpX in HeLa cells resulted in enlarged mtDNA nucleoids, which is very similar to that observed in TFAM-knockdown cells in several properties. The expression of TFAM protein was not significantly reduced in ClpX-knockdown cells. However, the enlarged mtDNA nucleoids caused by ClpX-knockdown were suppressed by overexpression of recombinant TFAM and the phenotype was not observed in knockdown with ClpP, a protease subunit of ClpXP. Endogenous ClpX and TFAM exist in close vicinity, and ClpX enhanced DNA-binding activity of TFAM in vitro. These results suggest that human ClpX, a novel mtDNA regulator, maintains mtDNA nucleoid distribution through TFAM function as a chaperone rather than as a protease and its involvement in mtDNA segregation. PMID:22841477

  9. Protein structure, stability and solubility in water and other solvents.

    PubMed Central

    Pace, C Nick; Treviño, Saul; Prabhakaran, Erode; Scholtz, J Martin

    2004-01-01

    Proteins carry out the most difficult tasks in living cells. They do so by interacting specifically with other molecules. This requires that they fold to a unique, globular conformation that is only marginally more stable than the large ensemble of unfolded states. The folded state is stabilized mainly by the burial and tight packing of over 80% of the peptide groups and non-polar side chains. If life as we know it is to exist in a solvent other than water, the folded state must be stable and soluble in the new solvent. Our analysis suggests that proteins will be unstable in most polar solvents such as ethanol, extremely stable in non-polar solvents such as cyclohexane, and even more stable in a vacuum. Our solubility studies suggest that protein solubility will be markedly lower in polar solvents such as ethanol and that proteins will be essentially insoluble in non-polar solvents such as cyclohexane. For these and other reasons it seems unlikely that the life we know could exist in any solvent system other than water. PMID:15306378

  10. Protein attributes contribute to halo-stability, bioinformatics approach.

    PubMed

    Ebrahimie, Esmaeil; Ebrahimi, Mansour; Sarvestani, Narjes Rahpayma; Ebrahimi, Mahdi

    2011-01-01

    Halophile proteins can tolerate high salt concentrations. Understanding halophilicity features is the first step toward engineering halostable crops. To this end, we examined protein features contributing to the halo-toleration of halophilic organisms. We compared more than 850 features for halophilic and non-halophilic proteins with various screening, clustering, decision tree, and generalized rule induction models to search for patterns that code for halo-toleration. Up to 251 protein attributes selected by various attribute weighting algorithms as important features contribute to halo-stability; from them 14 attributes selected by 90% of models and the count of hydrogen gained the highest value (1.0) in 70% of attribute weighting models, showing the importance of this attribute in feature selection modeling. The other attributes mostly were the frequencies of di-peptides. No changes were found in the numbers of groups when K-Means and TwoStep clustering modeling were performed on datasets with or without feature selection filtering. Although the depths of induced trees were not high, the accuracies of trees were higher than 94% and the frequency of hydrophobic residues pointed as the most important feature to build trees. The performance evaluation of decision tree models had the same values and the best correctness percentage recorded with the Exhaustive CHAID and CHAID models. We did not find any significant difference in the percent of correctness, performance evaluation, and mean correctness of various decision tree models with or without feature selection. For the first time, we analyzed the performance of different screening, clustering, and decision tree algorithms for discriminating halophilic and non-halophilic proteins and the results showed that amino acid composition can be used to discriminate between halo-tolerant and halo-sensitive proteins. PMID:21592393

  11. Protein attributes contribute to halo-stability, bioinformatics approach

    PubMed Central

    2011-01-01

    Halophile proteins can tolerate high salt concentrations. Understanding halophilicity features is the first step toward engineering halostable crops. To this end, we examined protein features contributing to the halo-toleration of halophilic organisms. We compared more than 850 features for halophilic and non-halophilic proteins with various screening, clustering, decision tree, and generalized rule induction models to search for patterns that code for halo-toleration. Up to 251 protein attributes selected by various attribute weighting algorithms as important features contribute to halo-stability; from them 14 attributes selected by 90% of models and the count of hydrogen gained the highest value (1.0) in 70% of attribute weighting models, showing the importance of this attribute in feature selection modeling. The other attributes mostly were the frequencies of di-peptides. No changes were found in the numbers of groups when K-Means and TwoStep clustering modeling were performed on datasets with or without feature selection filtering. Although the depths of induced trees were not high, the accuracies of trees were higher than 94% and the frequency of hydrophobic residues pointed as the most important feature to build trees. The performance evaluation of decision tree models had the same values and the best correctness percentage recorded with the Exhaustive CHAID and CHAID models. We did not find any significant difference in the percent of correctness, performance evaluation, and mean correctness of various decision tree models with or without feature selection. For the first time, we analyzed the performance of different screening, clustering, and decision tree algorithms for discriminating halophilic and non-halophilic proteins and the results showed that amino acid composition can be used to discriminate between halo-tolerant and halo-sensitive proteins. PMID:21592393

  12. Cementing proteins provide extra mechanical stabilization to viral cages

    NASA Astrophysics Data System (ADS)

    Hernando-Pérez, M.; Lambert, S.; Nakatani-Webster, E.; Catalano, C. E.; de Pablo, P. J.

    2014-07-01

    The study of virus shell stability is key not only for gaining insights into viral biological cycles but also for using viral capsids in materials science. The strength of viral particles depends profoundly on their structural changes occurring during maturation, whose final step often requires the specific binding of ‘decoration’ proteins (such as gpD in bacteriophage lambda) to the viral shell. Here we characterize the mechanical stability of gpD-free and gpD-decorated bacteriophage lambda capsids. The incorporation of gpD into the lambda shell imparts a major mechanical reinforcement that resists punctual deformations. We further interrogate lambda particle stability with molecular fatigue experiments that resemble the sub-lethal Brownian collisions of virus shells with macromolecules in crowded environments. Decorated particles are especially robust against collisions of a few kBT (where kB is the Boltzmann’s constant and T is the temperature ~300 K), which approximate those anticipated from molecular insults in the environment.

  13. Differential stability of the bovine prion protein upon urea unfolding

    PubMed Central

    Julien, Olivier; Chatterjee, Subhrangsu; Thiessen, Angela; Graether, Steffen P; Sykes, Brian D

    2009-01-01

    Prion diseases, or transmissible spongiform encephalopathies, are a group of infectious neurological diseases associated with the structural conversion of an endogenous protein (PrP) in the central nervous system. There are two major forms of this protein: the native and noninfectious cellular form, PrPC; and the misfolded, infectious, and proteinase K-resistant form, PrPSc. The C-terminal domain of PrPC is mainly α-helical in structure, whereas PrPSc in known to aggregate into an assembly of β-sheets, forming amyloid fibrils. To identify the regions of PrPC potentially involved in the initial steps of the conversion to the infectious conformation, we have used high-resolution NMR spectroscopy to characterize the stability and structure of bovine recombinant PrPC (residues 121 to 230) during unfolding with the denaturant urea. Analysis of the 800 MHz 1H NMR spectra reveals region-specific information about the structural changes occurring upon unfolding. Our data suggest that the dissociation of the native β-sheet of PrPC is a primary step in the urea-induced unfolding process, while strong hydrophobic interactions between helices α1 and α3, and between α2 and α3, stabilize these regions even at very high concentrations of urea. PMID:19693935

  14. Effects of genes exerting growth inhibition and plasmid stability on plasmid maintenance.

    PubMed

    Boe, L; Gerdes, K; Molin, S

    1987-10-01

    Plasmid stabilization mediated by the parA+ and parB+ genes of the R1 plasmid and the ccd+ and sop+ genes of the F plasmid was tested on a mini-R1 plasmid and a pBR322 plasmid derivative. The mini-R1 plasmid is thought to be unstably inherited owing to a low copy number and to random segregation of the plasmid at cell division, whereas cells harboring the pBR322 derivative used in this work are lost through competition with plasmid-free cells, mainly as a result of the shorter generation time of cells without plasmids. The pBR322 derivative carries a fusion between part of the atp operon of Escherichia coli and the bacteriophage lambda pR promoter, and the cI857 repressor gene. The insertion of sop+ from the F plasmid or parB+ from the R1 plasmid reduced the loss frequency by a factor of 10(3) for the pBR322 derivative and by at least a factor of 10(2) for the mini-R1 plasmid. Insertion of parA+ from the R1 plasmid decreased the loss frequency of the pBR322 derivative by a factor of 10 and that of the mini-R1 plasmid by a factor of 50. When ccd+ from the F plasmid was inserted, the loss frequency of the pBR322 derivative was decreased by a factor of 10, but it had only a marginal effect on the stability of the mini-R1 plasmid. In no case was any significant structural instability of the plasmids observed.

  15. Protein stability induced by ligand binding correlates with changes in protein flexibility

    PubMed Central

    Celej, María Soledad; Montich, Guillermo G.; Fidelio, Gerardo D.

    2003-01-01

    The interaction between ligands and proteins usually induces changes in protein thermal stability with modifications in the midpoint denaturation temperature, enthalpy of unfolding, and heat capacity. These modifications are due to the coupling of unfolding with binding equilibrium. Furthermore, they can be attained by changes in protein structure and conformational flexibility induced by ligand interaction. To study these effects we have used bovine serum albumin (BSA) interacting with three different anilinonaphthalene sulfonate derivatives (ANS). These ligands have different effects on protein stability, conformation, and dynamics. Protein stability was studied by differential scanning calorimetry and fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism and infrared spectroscopy including kinetics of hydrogen/deuterium exchange. The order of calorimetric midpoint of denaturation was: 1,8-ANS-BSA > 2,6-ANS-BSA > free BSA >> (nondetected) bis-ANS-BSA. Both 1,8-ANS and 2,6-ANS did not substantially modify the secondary structure of BSA, whereas bis-ANS induced a distorted α-helix conformation with an increase of disordered structure. Protein flexibility followed the order: 1,8-ANS-BSA < 2,6-ANS-BSA < free BSA << bis-ANS-BSA, indicating a clear correlation between stability and conformational flexibility. The structure induced by an excess of bis-ANS to BSA is compatible with a molten globule-like state. Within the context of the binding landscape model, we have distinguished five conformers (identified by subscript): BSA1,8-ANS, BSA2,6-ANS, BSAfree, BSAbis-ANS, and BSAunfolded among the large number of possible states of the conformational dynamic ensemble. The relative population of each distinguishable conformer depends on the type and concentration of ligand and the temperature of the system. PMID:12824495

  16. Soy protein polymers: Enhancing the water stability property

    NASA Astrophysics Data System (ADS)

    Srinivasan, Gowrishankar

    Soy protein based plastics have been processed in the past by researchers for various short-term applications; however a common issue is the high water sensitivity of these plastics. This work concentrates on resolving this water sensitivity issue of soy protein polymers by employing chemical and mechanical interaction at the molecular level during extrusion. The primary chemical interactions employed were anhydride chemistries such as maleic anhydride (MA), phthalic anhydride (PTA), and butylated hydroxyanisole (BHA). These were respectively used in conjunction with glycerol as a plasticizer to produce relatively water stable soy protein based plastics. Formulations with varying additive levels of the chemistries were extruded and injection molded to form the samples for characterization. The additive levels of anhydrides were varied between 3-10% tw/tw (total mass). Results indicated that phthalic anhydride formulations resulted in highest water stability. Plastic formulations with concentration up to 10% phthalic anhydride were observed to have water absorption as low as 21.5% after 24 hrs of exposure to water with respect to 250% for the control formulation. Fourier transform infrared spectroscopy (FTIR) was utilized to characterize and confirm the fundamental mechanisms of water stability achieved by phthalic and maleic anhydride chemistries. In addition, the anhydride formulations were modified by inclusion of cotton fibers and pretreated cotton powder in order to improve mechanical properties. The incorporation of cotton fibers improved the dry strength by 18%, but did not significantly improve the wet state strength of the plastics. It was also observed that the butylated-hydroxy anisole (BHA) formulation exhibited high extension values in the dry state and had inferior water absorption properties in comparison with anhydride formulations.

  17. Potential Benefit of the Charge-Stabilized Nanostructure Saline RNS60 for Myelin Maintenance and Repair.

    PubMed

    Rao, Vijayaraghava T S; Khan, Damla; Jones, Russell G; Nakamura, Diane S; Kennedy, Timothy E; Cui, Qiao-Ling; Rone, Malena B; Healy, Luke M; Watson, Richard; Ghosh, Supurna; Antel, Jack P

    2016-01-01

    Myelin injury in multiple sclerosis (MS) has been attributed both to "outside-in" primary immune mediated and "inside-out" metabolic stress of oligodendrocyte (OL) related mechanisms. Subsequent remyelination is dependent on recruitment and differentiation of oligodendrocyte progenitor cells (OPCs). RNS60 is a physically-modified saline containing charge-stabilized nanobubbles generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. Administration of RNS60 has been shown to reduce the severity of EAE by dampening the immune response and myelin loss. Additionally, RNS60 has been demonstrated to enhance mitochondrial ATP synthesis in neurons. Here, we used post-natal rat derived OLs and OPCs to assess the impact of RNS60 on the response of OLs to metabolic stress in vitro (glucose-nutrient deprivation, referred to as 'NG') and on OPC differentiation capacity. Under the NG condition, our findings indicate that RNS60 decreases caspases 3/7 activation. Respirometric analyses revealed that RNS60 increased spare glycolytic capacity (SGC) under normal culture conditions. However, RNS60 enhanced OL spare respiratory capacity (SRC) when a metabolic stress was present. Furthermore, we show that RNS60 promotes OPC differentiation under physiological conditions. Our findings provide evidence for the potential therapeutic efficacy of RNS60 through the promotion of OL survival and OPC differentiation. PMID:27451946

  18. Multiple Functions of Drosophila BLM Helicase in Maintenance of Genome Stability

    PubMed Central

    McVey, Mitch; Andersen, Sabrina L.; Broze, Yuri; Sekelsky, Jeff

    2007-01-01

    Bloom Syndrome, a rare human disorder characterized by genomic instability and predisposition to cancer, is caused by mutation of BLM, which encodes a RecQ-family DNA helicase. The Drosophila melanogaster ortholog of BLM, DmBlm, is encoded by mus309. Mutations in mus309 cause hypersensitivity to DNA-damaging agents, female sterility, and defects in repairing double-strand breaks (DSBs). To better understand these phenotypes, we isolated novel mus309 alleles. Mutations that delete the N terminus of DmBlm, but not the helicase domain, have DSB repair defects as severe as those caused by null mutations. We found that female sterility is due to a requirement for DmBlm in early embryonic cell cycles; embryos lacking maternally derived DmBlm have anaphase bridges and other mitotic defects. These defects were less severe for the N-terminal deletion alleles, so we used one of these mutations to assay meiotic recombination. Crossovers were decreased to about half the normal rate, and the remaining crossovers were evenly distributed along the chromosome. We also found that spontaneous mitotic crossovers are increased by several orders of magnitude in mus309 mutants. These results demonstrate that DmBlm functions in multiple cellular contexts to promote genome stability. PMID:17507683

  19. Potential Benefit of the Charge-Stabilized Nanostructure Saline RNS60 for Myelin Maintenance and Repair

    PubMed Central

    Rao, Vijayaraghava T. S.; Khan, Damla; Jones, Russell G.; Nakamura, Diane S.; Kennedy, Timothy E.; Cui, Qiao-Ling; Rone, Malena B.; Healy, Luke M.; Watson, Richard; Ghosh, Supurna; Antel, Jack P.

    2016-01-01

    Myelin injury in multiple sclerosis (MS) has been attributed both to “outside-in” primary immune mediated and “inside-out” metabolic stress of oligodendrocyte (OL) related mechanisms. Subsequent remyelination is dependent on recruitment and differentiation of oligodendrocyte progenitor cells (OPCs). RNS60 is a physically-modified saline containing charge-stabilized nanobubbles generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. Administration of RNS60 has been shown to reduce the severity of EAE by dampening the immune response and myelin loss. Additionally, RNS60 has been demonstrated to enhance mitochondrial ATP synthesis in neurons. Here, we used post-natal rat derived OLs and OPCs to assess the impact of RNS60 on the response of OLs to metabolic stress in vitro (glucose-nutrient deprivation, referred to as ‘NG’) and on OPC differentiation capacity. Under the NG condition, our findings indicate that RNS60 decreases caspases 3/7 activation. Respirometric analyses revealed that RNS60 increased spare glycolytic capacity (SGC) under normal culture conditions. However, RNS60 enhanced OL spare respiratory capacity (SRC) when a metabolic stress was present. Furthermore, we show that RNS60 promotes OPC differentiation under physiological conditions. Our findings provide evidence for the potential therapeutic efficacy of RNS60 through the promotion of OL survival and OPC differentiation. PMID:27451946

  20. Potential Benefit of the Charge-Stabilized Nanostructure Saline RNS60 for Myelin Maintenance and Repair.

    PubMed

    Rao, Vijayaraghava T S; Khan, Damla; Jones, Russell G; Nakamura, Diane S; Kennedy, Timothy E; Cui, Qiao-Ling; Rone, Malena B; Healy, Luke M; Watson, Richard; Ghosh, Supurna; Antel, Jack P

    2016-01-01

    Myelin injury in multiple sclerosis (MS) has been attributed both to "outside-in" primary immune mediated and "inside-out" metabolic stress of oligodendrocyte (OL) related mechanisms. Subsequent remyelination is dependent on recruitment and differentiation of oligodendrocyte progenitor cells (OPCs). RNS60 is a physically-modified saline containing charge-stabilized nanobubbles generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. Administration of RNS60 has been shown to reduce the severity of EAE by dampening the immune response and myelin loss. Additionally, RNS60 has been demonstrated to enhance mitochondrial ATP synthesis in neurons. Here, we used post-natal rat derived OLs and OPCs to assess the impact of RNS60 on the response of OLs to metabolic stress in vitro (glucose-nutrient deprivation, referred to as 'NG') and on OPC differentiation capacity. Under the NG condition, our findings indicate that RNS60 decreases caspases 3/7 activation. Respirometric analyses revealed that RNS60 increased spare glycolytic capacity (SGC) under normal culture conditions. However, RNS60 enhanced OL spare respiratory capacity (SRC) when a metabolic stress was present. Furthermore, we show that RNS60 promotes OPC differentiation under physiological conditions. Our findings provide evidence for the potential therapeutic efficacy of RNS60 through the promotion of OL survival and OPC differentiation.

  1. Solubilizing and Stabilizing Proteins in Anhydrous Ionic Liquids through Formation of Protein-Polymer Surfactant Nanoconstructs.

    PubMed

    Brogan, Alex P S; Hallett, Jason P

    2016-04-01

    Nonaqueous biocatalysis is rapidly becoming a desirable tool for chemical and fuel synthesis in both the laboratory and industry. Similarly, ionic liquids are increasingly popular anhydrous reaction media for a number of industrial processes. Consequently, the use of enzymes in ionic liquids as efficient, environment-friendly, commercial biocatalysts is highly attractive. However, issues surrounding the poor solubility and low stability of enzymes in truly anhydrous media remain a significant challenge. Here, we demonstrate for the first time that engineering the surface of a protein to yield protein-polymer surfactant nanoconstructs allows for dissolution of dry protein into dry ionic liquids. Using myoglobin as a model protein, we show that this method can deliver protein molecules with near native structure into both hydrophilic and hydrophobic anhydrous ionic liquids. Remarkably, using temperature-dependent synchrotron radiation circular dichroism spectroscopy to measure half-denaturation temperatures, our results show that protein stability increases by 55 °C in the ionic liquid as compared to aqueous solution, pushing the solution thermal denaturation beyond the boiling point of water. Therefore, the work presented herein could provide a platform for the realization of biocatalysis at high temperatures or in anhydrous solvent systems. PMID:26976718

  2. Structural stability study of protein monolayers in air

    NASA Astrophysics Data System (ADS)

    Pompa, P. P.; Biasco, A.; Cingolani, R.; Rinaldi, R.; Verbeet, M. Ph.; Canters, G. W.

    2004-03-01

    The assessment of the folding and of the structural stability of a protein in air, upon immobilization in the solid state, represents a critical point from both a fundamental point of view and for the development of solid state nanobioelectronics. The recent demonstrations by Rinaldi et al. [R. Rinaldi et al., Adv. Mater. 14, 1453 (2002); Appl. Phys. Lett. 82, 472 (2003); Ann. (N.Y.) Acad. Sci. 1006, 187 (2003)] of protein-based solid state devices and transistors working in air have raised an intriguing question about the behavior of a biomolecule under nonphysiological conditions. The operation principle of the realized devices is based on the physiological electron transfer function of the metalloprotein azurin. This means that azurin should retain its shape and functionality also in the solid state when utilized in air and at room temperature. In this Brief Report, we prove this claim by analyzing the conformational state of the azurin monolayers developed for such devices by means of intrinsic fluorescence spectroscopy. We show that the immobilization of azurins in the solid state under nonliquid conditions, by means of a specific chemisorption process, does not necessarily lead to protein denaturation. This result is of great importance because it opens up interesting perspectives for the development of solid state hybrid nanodevices for electronic applications requiring nonliquid environments.

  3. The intricate relationship between microtubules and their associated motor proteins during axon growth and maintenance

    PubMed Central

    2013-01-01

    The hallmarks of neurons are their slender axons which represent the longest cellular processes of animals and which act as the cables that electrically wire the brain, and the brain to the body. Axons extend along reproducible paths during development and regeneration, and they have to be maintained for the lifetime of an organism. Both axon extension and maintenance essentially depend on the microtubule (MT) cytoskeleton. For this, MTs organize into parallel bundles that are established through extension at the leading axon tips within growth cones, and these bundles then form the architectural backbones, as well as the highways for axonal transport essential for supply and intracellular communication. Axon transport over these enormous distances takes days or even weeks and is a substantial logistical challenge. It is performed by kinesins and dynein/dynactin, which are molecular motors that form close functional links to the MTs they walk along. The intricate machinery which regulates MT dynamics, axonal transport and the motors is essential for nervous system development and function, and its investigation has huge potential to bring urgently required progress in understanding the causes of many developmental and degenerative brain disorders. During the last years new explanations for the highly specific properties of axonal MTs and for their close functional links to motor proteins have emerged, and it has become increasingly clear that motors play active roles also in regulating axonal MT networks. Here, I will provide an overview of these new developments. PMID:24010872

  4. The RNA-binding protein Puf1 functions in the maintenance of gametocytes in Plasmodium falciparum.

    PubMed

    Shrestha, Sony; Li, Xiaolian; Ning, Gang; Miao, Jun; Cui, Liwang

    2016-08-15

    Translation control plays an important role in the regulation of gene expression in the malaria parasite Plasmodium falciparum, especially in transition stages between the vertebrate host and mosquito vector. Here, we determined the function of the Puf-family member Puf1 (denoted as PfPuf1 for the P. falciparum protein) during P. falciparum sexual development. We show that PfPuf1 was expressed in all gametocyte stages and at higher levels in female gametocytes. PfPuf1 disruption did not interfere with the asexual erythrocyte cycle of the parasite but resulted in an approximately tenfold decrease of mature gametocytes. In the PfPuf1-disrupted lines, gametocytes appeared normal before stage III but subsequently exhibited a sharp decline in gametocytemia. This was accompanied by a concomitant accumulation of dead and dying late-stage gametocytes, which retained normal gross morphology. In addition, significantly more female gametocytes were lost in the PfPuf1-disrupted lines during development, resulting in a reversed male-to-female sex ratio. These results indicate that PfPuf1 is important for the differentiation and maintenance of gametocytes, especially female gametocytes. PMID:27383769

  5. The kinesin related motor protein, Eg5, is essential for maintenance of pre-implantation embryogenesis

    SciTech Connect

    Castillo, Andrew; Justice, Monica J. . E-mail: mjustice@bcm.tmc.edu

    2007-06-08

    Eg5 is a plus end directed kinesin related motor protein (KRP) previously shown to be involved in the assembly and maintenance of the mitotic spindle. KRPs are molecular motors capable of generating forces upon microtubules (MTs) in dividing cells and driving structural rearrangements necessary in the developing spindle. In vitro experiments demonstrate that loss of Eg5 results in cell cycle arrest and defective centrosome separation resulting in the development of monopolar spindles. Here we describe mice with a genetrap insertion in Eg5. Heterozygous mutant mice appear phenotypically normal. In contrast, embryos homozygous for the Eg5 null allele recovered at embryonic days 2.5-3.5 display signs of a proliferation defect as reduced cell numbers and failure of compaction and progression to the blastocyst stage was observed. These data, in conjunction with previous in vitro data, suggest that loss of Eg5 results in abnormal spindle structure, cell cycle arrest and thereby reduced cell proliferation of early cleavage pre-implantation embryos. These observations further support the conclusion that Eg5 is essential for cell division early in mouse development, and that maternal contribution may sustain the embryo through the maternal to zygotic transition at which point supplies of functional Eg5 are exhausted, preventing further cell cleavage.

  6. Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function

    SciTech Connect

    Dement, Gregory A.; Maloney, Scott C.; Reeves, Raymond . E-mail: reevesr@mail.wsu.edu

    2007-01-01

    We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential ({delta}{psi}{sub m}). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7C cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression.

  7. SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein.

    PubMed

    Vishwamitra, Deeksha; Curry, Choladda V; Shi, Ping; Alkan, Serhan; Amin, Hesham M

    2015-09-01

    Nucleophosmin-anaplastic lymphoma kinase-expressing (NPM-ALK+) T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5)(p23;q35) that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs) with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm.

  8. Crowding in extremophiles: linkage between solvation and weak protein-protein interactions, stability and dynamics, provides insight into molecular adaptation.

    PubMed

    Ebel, Christine; Zaccai, Giuseppe

    2004-01-01

    The study of the molecular adaptation of microorganisms to extreme environments (solvent, temperature, etc.) has provided tools to investigate the complex relationships between protein-solvent and protein-protein interactions, protein stability and protein dynamics, and how they are modulated by the crowded environment of the cell. We have evaluated protein-solvent and protein-protein interactions by solution experiments (analytical ultracentrifugation, small angle neutron and X-ray scattering, density) and crystallography, and protein dynamics by energy resolved neutron scattering. This review concerns work from our laboratory on (i) proteins from extreme halophilic Archaea, and (ii) psychrophile, mesophile, thermophile and hyperthermophile bacterial cells.

  9. Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

    SciTech Connect

    Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A.

    2012-08-21

    DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

  10. Lower Protein Stability Does Not Necessarily Increase Local Dynamics.

    PubMed

    McClelland, Levi J; Bowler, Bruce E

    2016-05-17

    Overall protein stability is thought to have an important impact on the millisecond time scale dynamics modulating enzyme function. In order to better understand the effects of overall stability on the substructure dynamics of mitochondrial cytochrome c, we test the effect of a destabilizing L85A mutation on the kinetics and equilibrium thermodynamics of the alkaline conformational transition. The alkaline conformational transition replaces the Met80 ligand of the heme with a lysine residue from Ω-loop D, the heme crevice loop, consisting of residues 70-85. Residues 67-87 are the most conserved portion of the sequence of mitochondrial cytochrome c, suggesting that this region is of prime importance for function. Mutations to Ω-loop D affect the stability of the heme crevice directly, modulating the pKapp of the alkaline transition. Two variants of yeast iso-1-cytochrome c, WT*/L85A and WT*/K73H/L85A, were prepared for these studies. Guanidine-HCl unfolding monitored by circular dichroism and pH titrations at 695 nm, respectively, were used to study the thermodynamics of global and local unfolding of these variants. The kinetics of the alkaline transition were measured by pH-jump stopped-flow methods. Gated electron transfer techniques using bis(2,2',2″-terpyridine)cobalt(II) as a reducing reagent were implemented to measure the heme crevice dynamics for the WT*/K73H/L85A variant. Contrary to the expectation that dynamics around the heme crevice would be faster for the less stable WT*/K73H/L85A variant, based on the behavior of psychrophilic versus mesophilic enzymes, they were similar to those for a variant without the L85A mutation. In fact, below pH 7, the dynamics of the WT*/K73H/L85A variant were slower. PMID:27104373

  11. Mathematics, Thermodynamics, and Modeling to Address Ten Common Misconceptions about Protein Structure, Folding, and Stability

    ERIC Educational Resources Information Center

    Robic, Srebrenka

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative…

  12. A meta-analysis of energy and protein requirements for maintenance and growth of Nellore cattle.

    PubMed

    Chizzotti, M L; Tedeschi, L O; Valadares Filho, S C

    2008-07-01

    A meta-analysis was conducted to determine NE and net protein requirements of growing bulls, steers, and heifers of Nellore purebred and Nellore x Bos taurus crossbreds. A database of 16 comparative slaughter studies (n = 389 animals) was gathered to provide enough information to develop equations to predict the requirements of NE(m), NE(g), and net protein for maintenance (NP(m)) and growth (NP(g)). The data were analyzed using a random coefficients model, considering studies as random effects, and sex and castrate status (bulls, steers, and heifers; n = 262, 103, and 24, respectively) and breeds as fixed effects. There were no differences in NE(m) requirements among sex and castrate status (P = 0.73) and breeds (P = 0.82). The combined data indicated a NE(m) requirement of 75 kcal/ kg(0.75) of empty BW (EBW) with a partial efficiency of use of ME for NE(m) of 0.67. The NE(g) requirement was different (P = 0.009) among sex and castrate status and tended (P = 0.06) to be different among breeds. The equation for NE(g) requirement for bulls was 0.0514 x EBW(0.75) x EWG(1.070); for steers, it was 0.0700 x EBW(0.75) x EWG(1.070); and for heifers, it was 0.0771 x EBW(0.75) x EWG(1.070), where EWG = EBW gain (kg/d). The partial efficiency of use of ME for NE(g) was not different among sex and castrate status (P = 0.33) and breeds (P = 0.20) and averaged 0.44. There were no differences in NP(m) requirement among sex and castrate status (P = 0.59) and breeds (P = 0.92); the overall NP(m) requirement was 1.74 g of NP.kg(-0.75) of EBW.d(-1). The overall MP requirement for maintenance was 2.59 g of MP.kg(-0.75) EBW.d(-1). The NP(g) requirement (g/d) was not different among sex and castrate status (P = 0.59) and breeds (P = 0.14); the overall equation was EWG x [217 - (12.8 x RE/EWG)], where RE = retained energy (Mcal/d). The percentage of RE deposited as protein (%RE(p)) decreased exponentially as the content of RE in the gain (REc, Mcal/kg of EWG) increased. Because no study

  13. Physiology, pharmacology, and rationale for colloid administration for the maintenance of effective hemodynamic stability in critically ill patients.

    PubMed

    Vercueil, Andre; Grocott, Michael P W; Mythen, Michael G

    2005-04-01

    The semisynthetic colloid solutions (gelatins, dextrans, and hydroxyethyl starches) are complex drugs. Their principal role in the care of the critically ill is as plasma volume expanders, but they may also affect hemorrheology, hemostasis, and inflammatory processes. The pattern of beneficial and detrimental effects varies between products. Understanding of the physiology of plasma volume expansion, as well as the nature and magnitude of these additional pharmacological qualities, is necessary for rational prescription of these commonly used products. The composition of the solute carrier solution can influence the clinical effects of colloid solutions. A large amount of data from laboratory and small clinical studies is available to inform this choice of colloid in a variety of situations. Significant patient outcome data from large studies has until recently been lacking, and clinicians have continued to prescribe a variety of crystalloids and colloids for the maintenance of effective hemodynamic stability in critically ill patients. The recently published Saline vs Albumin Fluid Evaluation Study demonstrates that albumin has an equivalent effectiveness and safety profile to 0.9% saline as a resuscitation fluid. The choice of clinical endpoints to guide dosage (infused volume) of colloids is probably therefore more important than the choice between individual products.

  14. More than 10% of yeast genes are related to genome stability and influence cellular senescence via rDNA maintenance

    PubMed Central

    Saka, Kimiko; Takahashi, Akihiro; Sasaki, Mariko; Kobayashi, Takehiko

    2016-01-01

    Genome instability triggers cellular senescence and is a common cause of cancer. The ribosomal RNA genes (rDNA), due to their repetitive structure, form a fragile site with frequent rearrangements. To identify eukaryotic factors that connect reduced genome stability to senescence we screened 4,876 strains of a Saccharomyces cerevisiae deletion library for aberrant rDNA and found 708 genes that contribute to its upkeep. 28 mutants caused abnormalities in non-rDNA chromosomes and among them 12 mutants have abnormalities both in rDNA and in non-rDNA chromosomes. Many mutated genes have not previously been implicated with genome maintenance nor their homologues with tumorigenesis in mammals. The link between rDNA state and senescence was broken after deletion of factors related with DNA polymerase ϵ. These mutations also suppressed the short lifespan phenotype of a sir2 mutant, suggesting a model in which molecular events at the heart of the replication fork induce abnormal rDNA recombination and are responsible for the emergence of an aging signal. PMID:26912831

  15. Reduced native state stability in crowded cellular environment due to protein-protein interactions.

    PubMed

    Harada, Ryuhei; Tochio, Naoya; Kigawa, Takanori; Sugita, Yuji; Feig, Michael

    2013-03-01

    The effect of cellular crowding environments on protein structure and stability is a key issue in molecular and cellular biology. The classical view of crowding emphasizes the volume exclusion effect that generally favors compact, native states. Here, results from molecular dynamics simulations and NMR experiments show that protein crowders may destabilize native states via protein-protein interactions. In the model system considered here, mixtures of villin head piece and protein G at high concentrations, villin structures become increasingly destabilized upon increasing crowder concentrations. The denatured states observed in the simulation involve partial unfolding as well as more subtle conformational shifts. The unfolded states remain overall compact and only partially overlap with unfolded ensembles at high temperature and in the presence of urea. NMR measurements on the same systems confirm structural changes upon crowding based on changes of chemical shifts relative to dilute conditions. An analysis of protein-protein interactions and energetic aspects suggests the importance of enthalpic and solvation contributions to the crowding free energies that challenge an entropic-centered view of crowding effects.

  16. Effect of cosolvent on protein stability: A theoretical investigation

    NASA Astrophysics Data System (ADS)

    Chalikian, Tigran V.

    2014-12-01

    We developed a statistical thermodynamic algorithm for analyzing solvent-induced folding/unfolding transitions of proteins. The energetics of protein transitions is governed by the interplay between the cavity formation contribution and the term reflecting direct solute-cosolvent interactions. The latter is viewed as an exchange reaction in which the binding of a cosolvent to a solute is accompanied by release of waters of hydration to the bulk. Our model clearly differentiates between the stoichiometric and non-stoichiometric interactions of solvent or co-solvent molecules with a solute. We analyzed the urea- and glycine betaine (GB)-induced conformational transitions of model proteins of varying size which are geometrically approximated by a sphere in their native state and a spherocylinder in their unfolded state. The free energy of cavity formation and its changes accompanying protein transitions were computed based on the concepts of scaled particle theory. The free energy of direct solute-cosolvent interactions were analyzed using empirical parameters previously determined for urea and GB interactions with low molecular weight model compounds. Our computations correctly capture the mode of action of urea and GB and yield realistic numbers for (∂ΔG°/∂a3)T,P which are related to the m-values of protein denaturation. Urea is characterized by negative values of (∂ΔG°/∂a3)T,P within the entire range of urea concentrations analyzed. At concentrations below ˜1 M, GB exhibits positive values of (∂ΔG°/∂a3)T,P which turn positive at higher GB concentrations. The balance between the thermodynamic contributions of cavity formation and direct solute-cosolvent interactions that, ultimately, defines the mode of cosolvent action is extremely subtle. A 20% increase or decrease in the equilibrium constant for solute-cosolvent binding may change the sign of (∂ΔG°/∂a3)T,P thereby altering the mode of cosolvent action (stabilizing to destabilizing or vice

  17. Effect of cosolvent on protein stability: A theoretical investigation

    SciTech Connect

    Chalikian, Tigran V.

    2014-12-14

    We developed a statistical thermodynamic algorithm for analyzing solvent-induced folding/unfolding transitions of proteins. The energetics of protein transitions is governed by the interplay between the cavity formation contribution and the term reflecting direct solute-cosolvent interactions. The latter is viewed as an exchange reaction in which the binding of a cosolvent to a solute is accompanied by release of waters of hydration to the bulk. Our model clearly differentiates between the stoichiometric and non-stoichiometric interactions of solvent or co-solvent molecules with a solute. We analyzed the urea- and glycine betaine (GB)-induced conformational transitions of model proteins of varying size which are geometrically approximated by a sphere in their native state and a spherocylinder in their unfolded state. The free energy of cavity formation and its changes accompanying protein transitions were computed based on the concepts of scaled particle theory. The free energy of direct solute-cosolvent interactions were analyzed using empirical parameters previously determined for urea and GB interactions with low molecular weight model compounds. Our computations correctly capture the mode of action of urea and GB and yield realistic numbers for (∂ΔG°/∂a{sub 3}){sub T,P} which are related to the m-values of protein denaturation. Urea is characterized by negative values of (∂ΔG°/∂a{sub 3}){sub T,P} within the entire range of urea concentrations analyzed. At concentrations below ∼1 M, GB exhibits positive values of (∂ΔG°/∂a{sub 3}){sub T,P} which turn positive at higher GB concentrations. The balance between the thermodynamic contributions of cavity formation and direct solute-cosolvent interactions that, ultimately, defines the mode of cosolvent action is extremely subtle. A 20% increase or decrease in the equilibrium constant for solute-cosolvent binding may change the sign of (∂ΔG°/∂a{sub 3}){sub T,P} thereby altering the mode of

  18. AHSP (α-haemoglobin-stabilizing protein) stabilizes apo-α-haemoglobin in a partially folded state.

    PubMed

    Krishna Kumar, Kaavya; Dickson, Claire F; Weiss, Mitchell J; Mackay, Joel P; Gell, David A

    2010-12-01

    To produce functional Hb (haemoglobin), nascent α-globin (αo) and β-globin (βo) chains must each bind a single haem molecule (to form αh and βh) and interact together to form heterodimers. The precise sequence of binding events is unknown, and it has been suggested that additional factors might enhance the efficiency of Hb folding. AHSP (α-haemoglobin-stabilizing protein) has been shown previously to bind αh and regulate redox activity of the haem iron. In the present study, we used a combination of classical and dynamic light scattering and NMR spectroscopy to demonstrate that AHSP forms a heterodimeric complex with αo that inhibits αo aggregation and promotes αo folding in the absence of haem. These findings indicate that AHSP may function as an αo-specific chaperone, and suggest an important role for αo in guiding Hb assembly by stabilizing βo and inhibiting off-pathway self-association of βh.

  19. Alpha-haemoglobin stabilizing protein (AHSP) stabilizes apo-α-haemoglobin in a partially folded state

    PubMed Central

    Krishna Kumar, Kaavya; Dickson, Claire F.; Weiss, Mitchell J.; Mackay, Joel P.; Gell, David A.

    2015-01-01

    SYNOPSIS To produce functional haemoglobin, nascent α-globin (αo) and β-globin (βo) chains must each bind a single haem molecule (to form αh and βh) and interact together to form heterodimers. The precise sequence of binding events is unknown, and it has been suggested that additional factors might enhance the efficiency of Hb folding. The α-haemoglobin stabilizing protein (AHSP) has previously been shown to bind αh and regulate redox activity of the haem iron. Here, we use a combination of classical and dynamic light scattering and NMR spectroscopy to demonstrate that AHSP forms a heterodimeric complex with αo that inhibits αo aggregation and promotes αo folding in the absence of haem. These findings indicate that AHSP may function as an αo-specific chaperone, and suggest an important role for αo in guiding Hb assembly by stabilizing βo and inhibiting off-pathway self-association of βh. PMID:20860551

  20. Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response.

    PubMed

    Lin, Yi-Fan; Schulz, Anna M; Pellegrino, Mark W; Lu, Yun; Shaham, Shai; Haynes, Cole M

    2016-05-19

    Mitochondrial genomes (mitochondrial DNA, mtDNA) encode essential oxidative phosphorylation (OXPHOS) components. Because hundreds of mtDNAs exist per cell, a deletion in a single mtDNA has little impact. However, if the deletion genome is enriched, OXPHOS declines, resulting in cellular dysfunction. For example, Kearns-Sayre syndrome is caused by a single heteroplasmic mtDNA deletion. More broadly, mtDNA deletion accumulation has been observed in individual muscle cells and dopaminergic neurons during ageing. It is unclear how mtDNA deletions are tolerated or how they are propagated in somatic cells. One mechanism by which cells respond to OXPHOS dysfunction is by activating the mitochondrial unfolded protein response (UPR(mt)), a transcriptional response mediated by the transcription factor ATFS-1 that promotes the recovery and regeneration of defective mitochondria. Here we investigate the role of ATFS-1 in the maintenance and propagation of a deleterious mtDNA in a heteroplasmic Caenorhabditis elegans strain that stably expresses wild-type mtDNA and mtDNA with a 3.1-kilobase deletion (∆mtDNA) lacking four essential genes. The heteroplasmic strain, which has 60% ∆mtDNA, displays modest mitochondrial dysfunction and constitutive UPR(mt) activation. ATFS-1 impairment reduced the ∆mtDNA nearly tenfold, decreasing the total percentage to 7%. We propose that in the context of mtDNA heteroplasmy, UPR(mt) activation caused by OXPHOS defects propagates or maintains the deleterious mtDNA in an attempt to recover OXPHOS activity by promoting mitochondrial biogenesis and dynamics. PMID:27135930

  1. Maintenance of Golgi apparatus structure in the face of continuous protein recycling to the endoplasmic reticulum: making ends meet.

    PubMed

    Storrie, Brian

    2005-01-01

    I focus here on the Golgi apparatus and the dynamic relationship between the Golgi apparatus, the central organelle of the secretory pathway, and the endoplasmic reticulum (ER). The proteins and lipids of the Golgi apparatus originate in the ER, and cargo proteins and lipids that also originate in the ER are processed and sorted within the Golgi apparatus. The Golgi apparatus is indeed the central organelle of the secretory pathway. Surprisingly, many, if not all, of the proteins and accompanying lipids of the Golgi apparatus cycle continuously between the Golgi and the ER. Neither the Cisternal Maturation nor the Vesicular Transport/Stable Compartment model of Golgi apparatus function predicts continuous cycling of Golgi resident proteins through the ER. Evidence for this cycling comes from multiple experimental approaches, including ER-exit block-revealed accumulation of recycled Golgi resident proteins in the ER, evidence for exchange of green fluorescent protein (GFP)-tagged Golgi proteins or their analogues between Golgi and ER pools, and cisternal rab overexpression-induced redistribution of Golgi resident proteins to the ER. The implications of Golgi protein cycling for the maintenance of Golgi structure in the interphase mammalian cell are discussed. The challenge for the future is to put Golgi resident protein cycling pathway(s) to protein machinery and to characterize the cumulative, weak, dynamic interactions that hold the Golgi apparatus together. In doing so, new paradigms of organelle biogenesis will emerge.

  2. Polyglutamylated Tubulin Binding Protein C1orf96/CSAP Is Involved in Microtubule Stabilization in Mitotic Spindles

    PubMed Central

    Ohta, Shinya; Hamada, Mayako; Sato, Nobuko; Toramoto, Iyo

    2015-01-01

    The centrosome-associated C1orf96/Centriole, Cilia and Spindle-Associated Protein (CSAP) targets polyglutamylated tubulin in mitotic microtubules (MTs). Loss of CSAP causes critical defects in brain development; however, it is unclear how CSAP association with MTs affects mitosis progression. In this study, we explored the molecular mechanisms of the interaction of CSAP with mitotic spindles. Loss of CSAP caused MT instability in mitotic spindles and resulted in mislocalization of Nuclear protein that associates with the Mitotic Apparatus (NuMA), with defective MT dynamics. Thus, CSAP overload in the spindles caused extensive MT stabilization and recruitment of NuMA. Moreover, MT stabilization by CSAP led to high levels of polyglutamylation on MTs. MT depolymerization by cold or nocodazole treatment was inhibited by CSAP binding. Live-cell imaging analysis suggested that CSAP-dependent MT-stabilization led to centrosome-free MT aster formation immediately upon nuclear envelope breakdown without γ-tubulin. We therefore propose that CSAP associates with MTs around centrosomes to stabilize MTs during mitosis, ensuring proper bipolar spindle formation and maintenance. PMID:26562023

  3. Stability and Immunogenicity of Hypoallergenic Peanut Protein-Polyphenol Complexes During In Vitro Pepsin Digestion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Allergenic peanut proteins are relatively resistant to digestion, and if digested, metabolized peptides tend to remain large and immunoreactive, triggering allergic reactions in sensitive individuals. In this study, the stability of hypoallergenic peanut protein-polyphenol complexes was evaluated d...

  4. Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridge in Gαo Protein.

    PubMed

    Nagibina, Galina S; Tin, Ulyana F; Glukhov, Anatoly S; Melnik, Tatiana N; Melnik, Bogdan S

    2016-01-01

    In this study, we have used an approach that allows us to determine in what region of the polypeptide chain of protein it is required to insert a disulphide bond in order to stabilize it. In our previous paper [Melnik et al., JBSD. 2012] it was proposed that to search for a "weak" site in the protein, it is possible to use programs (for example, PONDR-FIT and IsUnstruct) finding intrinsic disorder protein regions. We suggested that in structured globular proteins, such programs predict not protein regions in the polypeptide chain disordered under native conditions, but "weakened", feebly stabilized ones. Accordingly, an artificial introduction of SS-bridges using mutations in such regions would reliably result in the protein stabilization. We have taken advantage of this approach to stabilize protein Gαo from Drosophila melanogaster. The designed SS-bridge increased by 4 degrees the melting temperature of one domain of protein Gαo.

  5. Analysis of protein stability and ligand interactions by thermal shift assay.

    PubMed

    Huynh, Kathy; Partch, Carrie L

    2015-02-02

    Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye.

  6. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy.

    PubMed

    Quan, Shu; Koldewey, Philipp; Tapley, Tim; Kirsch, Nadine; Ruane, Karen M; Pfizenmaier, Jennifer; Shi, Rong; Hofmann, Stephan; Foit, Linda; Ren, Guoping; Jakob, Ursula; Xu, Zhaohui; Cygler, Miroslaw; Bardwell, James C A

    2011-03-01

    To optimize the in vivo folding of proteins, we linked protein stability to antibiotic resistance, thereby forcing bacteria to effectively fold and stabilize proteins. When we challenged Escherichia coli to stabilize a very unstable periplasmic protein, it massively overproduced a periplasmic protein called Spy, which increases the steady-state levels of a set of unstable protein mutants up to 700-fold. In vitro studies demonstrate that the Spy protein is an effective ATP-independent chaperone that suppresses protein aggregation and aids protein refolding. Our strategy opens up new routes for chaperone discovery and the custom tailoring of the in vivo folding environment. Spy forms thin, apparently flexible cradle-shaped dimers. The structure of Spy is unlike that of any previously solved chaperone, making it the prototypical member of a new class of small chaperones that facilitate protein refolding in the absence of energy cofactors.

  7. Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres.

    PubMed Central

    Nakamura, Toru M; Moser, Bettina A; Russell, Paul

    2002-01-01

    Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Delta mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres. PMID:12196391

  8. Protocols for Studying Protein Stability in an Arabidopsis Protoplast Transient Expression System.

    PubMed

    Planchais, Séverine; Camborde, Laurent; Jupin, Isabelle

    2016-01-01

    Protein stability influences many aspects of biology, and measuring their stability in vivo can provide important insights into biological systems.This chapter describes in details two methods to assess the stability of a specific protein based on its transient expression in Arabidopsis protoplasts. First, a pulse-chase assay based on radioactive metabolic labeling of cellular proteins, followed by immunoprecipitation of the protein of interest. The decrease in radioactive signal is monitored over time and can be used to determine the protein's half-life.Alternatively, we also present a nonradioactive assay based on the use of reporter proteins, whose ratio can be quantified. This assay can be used to determine the relative stability of a protein of interest under specific conditions. PMID:27424754

  9. Protocols for Studying Protein Stability in an Arabidopsis Protoplast Transient Expression System.

    PubMed

    Planchais, Séverine; Camborde, Laurent; Jupin, Isabelle

    2016-01-01

    Protein stability influences many aspects of biology, and measuring their stability in vivo can provide important insights into biological systems.This chapter describes in details two methods to assess the stability of a specific protein based on its transient expression in Arabidopsis protoplasts. First, a pulse-chase assay based on radioactive metabolic labeling of cellular proteins, followed by immunoprecipitation of the protein of interest. The decrease in radioactive signal is monitored over time and can be used to determine the protein's half-life.Alternatively, we also present a nonradioactive assay based on the use of reporter proteins, whose ratio can be quantified. This assay can be used to determine the relative stability of a protein of interest under specific conditions.

  10. Using state diagrams for predicting colloidal stability of whey protein beverages.

    PubMed

    Wagoner, Ty B; Ward, Loren; Foegeding, E Allen

    2015-05-01

    A method for evaluating aspects of colloidal stability of whey protein beverages after thermal treatment was established. Three state diagrams for beverages (pH 3-7) were developed representing protein solubility, turbidity, and macroscopic state after two ultrahigh-temperature (UHT) treatments. Key transitions of stability in the state diagrams were explored using electrophoresis and chromatography to determine aggregation propensities of β-lactoglobulin, α-lactalbumin, bovine serum albumin, and glycomacropeptide. The state diagrams present an overlapping view of high colloidal stability at pH 3 accompanied by high solubility of individual whey proteins. At pH 5, beverages were characterized by poor solubility, high turbidity, and aggregation/gelation of whey proteins with the exception of glycomacropeptide. Stability increased at pH 6, due to increased solubility of α-lactalbumin. The results indicate that combinations of state diagrams can be used to identify key regions of stability for whey protein containing beverages.

  11. Identification of Multiple Proteins Coupling Transcriptional Gene Silencing to Genome Stability in Arabidopsis thaliana

    PubMed Central

    Hale, Christopher J.; Potok, Magdalena E.; Lopez, Jennifer; Do, Truman; Liu, Ao; Michaels, Scott D.; Jacobsen, Steven E.

    2016-01-01

    Eukaryotic genomes are regulated by epigenetic marks that act to modulate transcriptional control as well as to regulate DNA replication and repair. In Arabidopsis thaliana, mutation of the ATXR5 and ATXR6 histone methyltransferases causes reduction in histone H3 lysine 27 monomethylation, transcriptional upregulation of transposons, and a genome instability defect in which there is an accumulation of excess DNA corresponding to pericentromeric heterochromatin. We designed a forward genetic screen to identify suppressors of the atxr5/6 phenotype that uncovered loss-of-function mutations in two components of the TREX-2 complex (AtTHP1, AtSAC3B), a SUMO-interacting E3 ubiquitin ligase (AtSTUbL2) and a methyl-binding domain protein (AtMBD9). Additionally, using a reverse genetic approach, we show that a mutation in a plant homolog of the tumor suppressor gene BRCA1 enhances the atxr5/6 phenotype. Through characterization of these mutations, our results suggest models for the production atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell, and suggest that the atxr5 atxr6 transcriptional defects may be the cause of the genome instability defects in the mutants. These findings highlight the critical intersection of transcriptional silencing and DNA replication in the maintenance of genome stability of heterochromatin. PMID:27253878

  12. Conjugation Strategy Strongly Impacts the Conformational Stability of a PEG-Protein Conjugate.

    PubMed

    Lawrence, Paul B; Billings, Wendy M; Miller, McKenzie B; Pandey, Brijesh K; Stephens, Andrew R; Langlois, Minnie I; Price, Joshua L

    2016-07-15

    Site-specific PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs, and has been enabled by the recent development of many chemoselective reactions for protein side-chain modification. However, the impact of these different conjugation strategies on the properties of PEG-protein conjugates is poorly understood. Here we show that the ability of PEG to enhance protein conformational stability depends strongly on the identity of the PEG-protein linker, with the most stabilizing linkers involving conjugation of PEG to planar polar groups near the peptide backbone. We also find that branched PEGs provide superior stabilization relative to their linear counterparts, suggesting additional applications for branched PEGs in protein stabilization. PMID:27191252

  13. Associations between dairy protein intake and body weight and risk markers of diabetes and CVD during weight maintenance.

    PubMed

    Bendtsen, Line Q; Lorenzen, Janne K; Larsen, Thomas M; van Baak, Marleen; Papadaki, Angeliki; Martinez, J Alfredo; Handjieva-Darlenska, Teodora; Jebb, Susan A; Kunešová, Marie; Pfeiffer, Andreas F H; Saris, Wim H M; Astrup, Arne; Raben, Anne

    2014-03-14

    Dairy products have previously been reported to be associated with beneficial effects on body weight and metabolic risk markers. Moreover, primary data from the Diet, Obesity and Genes (DiOGenes) study indicate a weight-maintaining effect of a high-protein-low-glycaemic index diet. The objective of the present study was to examine putative associations between consumption of dairy proteins and changes in body weight and metabolic risk markers after weight loss in obese and overweight adults. Results were based on secondary analyses of data obtained from overweight and obese adults who completed the DiOGenes study. The study consisted of an 8-week weight-loss phase and a 6-month weight-maintenance (WM) phase, where the subjects were given five different diets varying in protein content and glycaemic index. In the present study, data obtained from all the subjects were pooled. Dairy protein intake was estimated from 3 d dietary records at two time points (week 4 and week 26) during the WM phase. Body weight and metabolic risk markers were determined at baseline (week -9 to -11) and before and at the end of the WM phase (week 0 and week 26). Overall, no significant associations were found between consumption of dairy proteins and changes in body weight and metabolic risk markers. However, dairy protein intake tended to be negatively associated with body weight gain (P=0·08; β=-0·17), but this was not persistent when controlled for total protein intake, which indicates that dairy protein adds no additional effect to the effect of total protein. Therefore, the present study does not report that dairy proteins are more favourable than other proteins for body weight regulation.

  14. Effect of glycosylation on protein folding: A close look at thermodynamic stabilization

    PubMed Central

    Shental-Bechor, Dalit; Levy, Yaakov

    2008-01-01

    Glycosylation is one of the most common posttranslational modifications to occur in protein biosynthesis, yet its effect on the thermodynamics and kinetics of proteins is poorly understood. A minimalist model based on the native protein topology, in which each amino acid and sugar ring was represented by a single bead, was used to study the effect of glycosylation on protein folding. We studied in silico the folding of 63 engineered SH3 domain variants that had been glycosylated with different numbers of conjugated polysaccharide chains at different sites on the protein's surface. Thermal stabilization of the protein by the polysaccharide chains was observed in proportion to the number of attached chains. Consistent with recent experimental data, the degree of thermal stabilization depended on the position of the glycosylation sites, but only very weakly on the size of the glycans. A thermodynamic analysis showed that the origin of the enhanced protein stabilization by glycosylation is destabilization of the unfolded state rather than stabilization of the folded state. The higher free energy of the unfolded state is enthalpic in origin because the bulky polysaccharide chains force the unfolded ensemble to adopt more extended conformations by prohibiting formation of a residual structure. The thermodynamic stabilization induced by glycosylation is coupled with kinetic stabilization. The effects introduced by the glycans on the biophysical properties of proteins are likely to be relevant to other protein polymeric conjugate systems that regularly occur in the cell as posttranslational modifications or for biotechnological purposes. PMID:18550810

  15. Effects of Protein Stabilizing Agents on Thermal Backbone Motions: A Disulfide Trapping Study†

    PubMed Central

    Butler, Scott L.; Falke, Joseph J.

    2010-01-01

    Chemical stabilizers are widely used to enhance protein stability, both in nature and in the laboratory. Here, the molecular mechanism of chemical stabilizers is studied using a disulfide trapping assay to measure the effects of stabilizers on thermal backbone dynamics in the Escherichia coli galactose/glucose binding protein. Two types of backbone fluctuations are examined: (a) relative movements of adjacent surface α-helices within the same domain and (b) interdomain twisting motions. Both types of fluctuations are significantly reduced by all six stabilizers tested (glycerol, sucrose, trehalose, l-glucose, d-glucose, and d-galactose), and in each case larger amplitude motions are inhibited more than smaller ones. Motional inhibition does not require a high-affinity stabilizer binding site, indicating that the effects of stabilizers are nonspecific. Overall, the results support the theory that effective stabilizing agents act by favoring the most compact structure of a protein, thereby reducing local backbone fluctuations away from the fully folded state. Such inhibition of protein backbone dynamics may be a general mechanism of protein stabilization in extreme thermal or chemical environments. PMID:8718847

  16. Direct delivery of functional proteins and enzymes to the cytosol using nanoparticle-stabilized nanocapsules.

    PubMed

    Tang, Rui; Kim, Chang Soo; Solfiell, David J; Rana, Subinoy; Mout, Rubul; Velázquez-Delgado, Elih M; Chompoosor, Apiwat; Jeong, Youngdo; Yan, Bo; Zhu, Zheng-Jiang; Kim, Chaekyu; Hardy, Jeanne A; Rotello, Vincent M

    2013-08-27

    Intracellular protein delivery is an important tool for both therapeutic and fundamental applications. Effective protein delivery faces two major challenges: efficient cellular uptake and avoiding endosomal sequestration. We report here a general strategy for direct delivery of functional proteins to the cytosol using nanoparticle-stabilized capsules (NPSCs). These NPSCs are formed and stabilized through supramolecular interactions between the nanoparticle, the protein cargo, and the fatty acid capsule interior. The NPSCs are ~130 nm in diameter and feature low toxicity and excellent stability in serum. The effectiveness of these NPSCs as therapeutic protein carriers was demonstrated through the delivery of fully functional caspase-3 to HeLa cells with concomitant apoptosis. Analogous delivery of green fluorescent protein (GFP) confirmed cytosolic delivery as well as intracellular targeting of the delivered protein, demonstrating the utility of the system for both therapeutic and imaging applications.

  17. A Multi-layered Protein Network Stabilizes the Escherichia coli FtsZ-ring and Modulates Constriction Dynamics

    PubMed Central

    Buss, Jackson; Coltharp, Carla; Shtengel, Gleb; Yang, Xinxing; Hess, Harald; Xiao, Jie

    2015-01-01

    The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation. PMID:25848771

  18. The role of maintenance proteins in the preservation of epithelial cell identity during mammary gland remodeling and breast cancer initiation.

    PubMed

    Coradini, Danila; Oriana, Saro

    2014-02-01

    During normal postnatal mammary gland development and adult remodeling related to the menstrual cycle, pregnancy, and lactation, ovarian hormones and peptide growth factors contribute to the delineation of a definite epithelial cell identity. This identity is maintained during cell replication in a heritable but DNA-independent manner. The preservation of cell identity is fundamental, especially when cells must undergo changes in response to intrinsic and extrinsic signals. The maintenance proteins, which are required for cell identity preservation, act epigenetically by regulating gene expression through DNA methylation, histone modification, and chromatin remodeling. Among the maintenance proteins, the Trithorax (TrxG) and Polycomb (PcG) group proteins are the best characterized. In this review, we summarize the structures and activities of the TrxG and PcG complexes and describe their pivotal roles in nuclear estrogen receptor activity. In addition, we provide evidence that perturbations in these epigenetic regulators are involved in disrupting epithelial cell identity, mammary gland remodeling, and breast cancer initiation.

  19. The beta-isoform of the BRCA2 and CDKN1A(p21)-interacting protein (BCCIP) stabilizes nuclear RPL23/uL14.

    PubMed

    Wyler, Emanuel; Wandrey, Franziska; Badertscher, Lukas; Montellese, Christian; Alper, Daniel; Kutay, Ulrike

    2014-10-16

    BRCA2 and CDKN1A(p21,CIP1)-interacting protein (BCCIP) is an evolutionary conserved protein implicated in maintenance of genome stability and cell cycle progression. Two isoforms of BCCIP with distinct C-terminal domains exist in humans. We show that mammalian BCCIPβ, but not BCCIPα, forms a ternary complex with the ribosomal protein RPL23/uL14 and the pre-60S trans-acting factor eIF6. Complex formation is dependent on an intact C-terminal domain of BCCIPβ. Depletion of BCCIPβ reduces the pool of free RPL23, and decreases eIF6 levels in nucleoli. Overexpression of BCCIPβ leads to nucleoplasmic accumulation of extra-ribosomal RPL23 and stabilizes overexpressed RPL23, suggesting that BCCIPβ functions as nuclear chaperone for RPL23.

  20. A C alpha-H...O hydrogen bond in a membrane protein is not stabilizing.

    PubMed

    Yohannan, Sarah; Faham, Salem; Yang, Duan; Grosfeld, David; Chamberlain, Aaron K; Bowie, James U

    2004-03-01

    Hydrogen bonds involving a carbon donor are very common in protein structures, and energy calculations suggest that Calpha-H...O hydrogen bonds could be about one-half the strength of traditional hydrogen bonds. It has therefore been proposed that these nontraditional hydrogen bonds could be a significant factor in stabilizing proteins, particularly membrane proteins as there is a low dielectric and no competition from water in the bilayer core. Nevertheless, this proposition has never been tested experimentally. Here, we report an experimental test of the significance of Calpha-H...O bonds for protein stability. Thr24 in bacteriorhodopsin, which makes an interhelical Calpha-H...O hydrogen bond to the Calpha of Ala51, was changed to Ala, Val, and Ser, and the thermodynamic stability of the mutants was measured. None of the mutants had significantly reduced stability. In fact, T24A was more stable than the wild-type protein by 0.6 kcal/mol. Crystal structures were determined for each of the mutants, and, while some structural changes were seen for T24S and T24V, T24A showed essentially no apparent structural alteration that could account for the increased stability. Thus, Thr24 appears to destabilize the protein rather than stabilize. Our results suggest that Calpha-H...O bonds are not a major contributor to protein stability.

  1. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution.

    PubMed

    He, Yi-Ming; Ma, Bin-Guang

    2016-01-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions. PMID:27220911

  2. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    NASA Astrophysics Data System (ADS)

    He, Yi-Ming; Ma, Bin-Guang

    2016-05-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

  3. A functional protein retention and release multilayer with high stability

    NASA Astrophysics Data System (ADS)

    Nie, Kun; An, Qi; Zhang, Yihe

    2016-04-01

    Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by varying the number of capping layers. Furthermore, we demonstrate that the protein-loaded interfacial layers could not only be used to construct catalytic-active interfaces, but also be integrated as the power-generating unit to propel a macroscopic floating device.Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by

  4. Stability and DNA-binding properties of the omega regulator protein from the broad-host range Streptococcus pyogenes plasmid pSM19035.

    PubMed

    Misselwitz, R; de la Hoz, A B; Ayora, S; Welfle, K; Behlke, J; Murayama, K; Saenger, W; Alonso, J C; Welfle, H

    2001-09-21

    At the transcriptional level, the pSM19035-encoded omega protein coordinates the expression of proteins required for control of copy number and maintenance of plasmids. Using circular dichroism, fluorescence spectroscopy, ultracentrifugation and an electrophoretic mobility shift assay, the wild-type omega protein and a variant with a C-terminal hexa-histidine tag (omega-H(6)) were characterized. The omega protein is mainly alpha-helical (42%), occurs as homodimer in solution, unfolds thermally with half transition temperatures, T(m), between approximately 43 and approximately 78 degrees C depending on the ionic strength of the buffer, and binds PcopS-DNA with high affinity. The omega-H(6) protein has a modified conformation with lower alpha-helix content (29%), lower thermal stability, and strongly reduced affinity to PcopS-DNA.

  5. The Stability and Formation of Native Proteins from Unfolded Monomers Is Increased through Interactions with Unrelated Proteins

    PubMed Central

    Rodríguez-Almazán, Claudia; Torner, Francisco J.; Costas, Miguel; Pérez-Montfort, Ruy; de Gómez-Puyou, Marieta Tuena; Puyou, Armando Gómez

    2007-01-01

    The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins. PMID:17551578

  6. Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance

    PubMed Central

    Cohen, Laurie D.; Zuchman, Rina; Sorokina, Oksana; Müller, Anke; Dieterich, Daniela C.; Armstrong, J. Douglas; Ziv, Tamar; Ziv, Noam E.

    2013-01-01

    Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non–Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2–5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load

  7. Maintenance of immune hyporesponsiveness to melanosomal proteins by DHICA-mediated antioxidation: Possible implications for autoimmune vitiligo.

    PubMed

    Liu, Xiao-Ming; Zhou, Qiong; Xu, Shi-Zheng; Wakamatsu, Kazumasa; Lei, Tie-Chi

    2011-05-01

    Melanocyte destruction in the skin of vitiligo patients has been considered to be a consequence of an autoimmune response against melanosomal proteins. However, little is known about the molecular mechanisms by which the immune system recognizes these sequestered intracellular self-proteins, which are confined in specialized organelles termed melanosomes, and is provoked into an autoimmune response to melanocytes. Here, we utilize a sucrose density-gradient ultracentrifugation protocol to enrich melanosomal components from dopachrome tautomerase (Dct)-mutant or wild-type melanocytes exposed to a pulse of hydrogen peroxide at a noncytotoxic concentration to evaluate their immunogenicity in mice challenged with the corresponding melanosomal proteins. The results demonstrate that enhanced humoral and cellular immune responses to a challenge with late-stage melanosomal proteins, especially with those derived from Dct-mutant melanocytes, are found in the immunized mice. To elucidate whether a reduced 5,6-dihydroxyindole-2-carboxylic acid (DHICA) content in melanin might cause a loss in antioxidative protection to the proteins, we incubated these melanosomal proteins in vitro with synthetic 5,6-dihydroindole (DHI)-melanin or DHI/DHICA (1:1)-melanin and then used them to immunize mice. T cell proliferation and IgG antibody responsiveness to the challenges were significantly induced by melanosomal proteins treated with DHI-melanin, but not by those treated with DHI/DHICA (1:1)-melanin. Moreover, we observed that melanosomal proteins derived from Dct-mutant melanocytes are subject to oxidative modifications that alter their antigenic configurations to attain an enhanced immunogenicity compared with those derived from wild-type melanocytes. From these results, we conclude that DHICA-mediated antioxidation plays a critical role in the maintenance of immune hyporesponsiveness to melanosomal proteins.

  8. Role of loops connecting secondary structure elements in the stabilization of proteins isolated from thermophilic organisms

    PubMed Central

    Balasco, Nicole; Esposito, Luciana; Simone, Alfonso De; Vitagliano, Luigi

    2013-01-01

    It has been recently discovered that the connection of secondary structure elements (ββ-unit, βα- and αβ-units) in proteins follows quite stringent principles regarding the chirality and the orientation of the structural units (Koga et al., Nature 2012;491:222–227). By exploiting these rules, a number of protein scaffolds endowed with a remarkable thermal stability have been designed (Koga et al., Nature 2012;491:222–227). By using structural databases of proteins isolated from either mesophilic or thermophilic organisms, we here investigate the influence of supersecondary associations on the thermal stability of natural proteins. Our results suggest that β-hairpins of proteins from thermophilic organisms are very frequently characterized by shortenings of the loops. Interestingly, this shortening leads to states that display a very strong preference for the most common connectivity of the strands observed in native protein hairpins. The abundance of selective states in these proteins suggests that they may achieve a high stability by adopting a strategy aimed to reduce the possible conformations of the unfolded ensemble. In this scenario, our data indicate that the shortening is effective if it increases the adherence to these rules. We also show that this mechanism may operate in the stabilization of well-known protein folds (thioredoxin and RNase A). These findings suggest that future investigations aimed at defining mechanism of protein stabilization should also consider these effects. PMID:23661276

  9. Stability Curve Prediction of Homologous Proteins Using Temperature-Dependent Statistical Potentials

    PubMed Central

    Pucci, Fabrizio; Rooman, Marianne

    2014-01-01

    The unraveling and control of protein stability at different temperatures is a fundamental problem in biophysics that is substantially far from being quantitatively and accurately solved, as it requires a precise knowledge of the temperature dependence of amino acid interactions. In this paper we attempt to gain insight into the thermal stability of proteins by designing a tool to predict the full stability curve as a function of the temperature for a set of 45 proteins belonging to 11 homologous families, given their sequence and structure, as well as the melting temperature () and the change in heat capacity () of proteins belonging to the same family. Stability curves constitute a fundamental instrument to analyze in detail the thermal stability and its relation to the thermodynamic stability, and to estimate the enthalpic and entropic contributions to the folding free energy. In summary, our approach for predicting the protein stability curves relies on temperature-dependent statistical potentials derived from three datasets of protein structures with targeted thermal stability properties. Using these potentials, the folding free energies () at three different temperatures were computed for each protein. The Gibbs-Helmholtz equation was then used to predict the protein's stability curve as the curve that best fits these three points. The results are quite encouraging: the standard deviations between the experimental and predicted 's, 's and folding free energies at room temperature () are equal to 13 , 1.3 ) and 4.1 , respectively, in cross-validation. The main sources of error and some further improvements and perspectives are briefly discussed. PMID:25032839

  10. Folding and Stabilization of Native-Sequence-Reversed Proteins.

    PubMed

    Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

    2016-04-26

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols.

  11. Folding and Stabilization of Native-Sequence-Reversed Proteins

    PubMed Central

    Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

    2016-01-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844

  12. RINT1 functions as a multitasking protein at the crossroads between genomic stability, ER homeostasis, and autophagy.

    PubMed

    Grigaravicius, Paulius; von Deimling, Andreas; Frappart, Pierre-Olivier

    2016-08-01

    RINT1 was first identified as an RAD50-interacting protein and its function was therefore linked to the maintenance of genomic stability. It was also shown that RINT1 was a key player in ER-Golgi trafficking as a member of an ER tethering complex interacting with STX18. However, due to early embryonic lethality of rint1-null mice, the in vivo functions of RINT1 remained for the most part elusive. We recently described the consequences of Rint1 inactivation in various neuronal cells of the central nervous system. We observed that lack of RINT1 in vivo triggers genomic instability and ER stress leading to depletion of the neural progenitor pool and neurodegeneration. Surprisingly, we also observed inhibition of autophagy in RINT1-deficient neurons, indicating an involvement of RINT1 in the regulation of neuronal autophagy. Here, we summarize our main RINT1 findings and discuss its putative roles in autophagy.

  13. RINT1 functions as a multitasking protein at the crossroads between genomic stability, ER homeostasis, and autophagy.

    PubMed

    Grigaravicius, Paulius; von Deimling, Andreas; Frappart, Pierre-Olivier

    2016-08-01

    RINT1 was first identified as an RAD50-interacting protein and its function was therefore linked to the maintenance of genomic stability. It was also shown that RINT1 was a key player in ER-Golgi trafficking as a member of an ER tethering complex interacting with STX18. However, due to early embryonic lethality of rint1-null mice, the in vivo functions of RINT1 remained for the most part elusive. We recently described the consequences of Rint1 inactivation in various neuronal cells of the central nervous system. We observed that lack of RINT1 in vivo triggers genomic instability and ER stress leading to depletion of the neural progenitor pool and neurodegeneration. Surprisingly, we also observed inhibition of autophagy in RINT1-deficient neurons, indicating an involvement of RINT1 in the regulation of neuronal autophagy. Here, we summarize our main RINT1 findings and discuss its putative roles in autophagy. PMID:27367497

  14. Zwitterionic gel encapsulation promotes protein stability, enhances pharmacokinetics, and reduces immunogenicity

    PubMed Central

    Zhang, Peng; Sun, Fang; Tsao, Caroline; Liu, Sijun; Jain, Priyesh; Sinclair, Andrew; Hung, Hsiang-Chieh; Bai, Tao; Wu, Kan; Jiang, Shaoyi

    2015-01-01

    Advances in protein therapy are hindered by the poor stability, inadequate pharmacokinetic (PK) profiles, and immunogenicity of many therapeutic proteins. Polyethylene glycol conjugation (PEGylation) is the most successful strategy to date to overcome these shortcomings, and more than 10 PEGylated proteins have been brought to market. However, anti-PEG antibodies induced by treatment raise serious concerns about the future of PEGylated therapeutics. Here, we demonstrate a zwitterionic polymer network encapsulation technology that effectively enhances protein stability and PK while mitigating the immune response. Uricase modified with a comprehensive zwitterionic polycarboxybetaine (PCB) network exhibited exceptional stability and a greatly prolonged circulation half-life. More importantly, the PK behavior was unchanged, and neither anti-uricase nor anti-PCB antibodies were detected after three weekly injections in a rat model. This technology is applicable to a variety of proteins and unlocks the possibility of adopting highly immunogenic proteins for therapeutic or protective applications. PMID:26371311

  15. Cooperative hydration effect causes thermal unfolding of proteins and water activity plays a key role in protein stability in solutions.

    PubMed

    Miyawaki, Osato; Dozen, Michiko; Hirota, Kaede

    2016-08-01

    The protein unfolding process observed in a narrow temperature range was clearly explained by evaluating the small difference in the enthalpy of hydrogen-bonding between amino acid residues and the hydration of amino acid residue separately. In aqueous solutions, the effect of cosolute on the protein stability is primarily dependent on water activity, aw, the role of which has been long neglected in the literature. The effect of aw on protein stability works as a power law so that a small change in aw is amplified substantially through the cooperative hydration effect. In the present approach, the role of hydrophobic interaction stands behind. This affects protein stability indirectly through the change in solution structure caused by the existence of cosolute.

  16. Cooperative hydration effect causes thermal unfolding of proteins and water activity plays a key role in protein stability in solutions.

    PubMed

    Miyawaki, Osato; Dozen, Michiko; Hirota, Kaede

    2016-08-01

    The protein unfolding process observed in a narrow temperature range was clearly explained by evaluating the small difference in the enthalpy of hydrogen-bonding between amino acid residues and the hydration of amino acid residue separately. In aqueous solutions, the effect of cosolute on the protein stability is primarily dependent on water activity, aw, the role of which has been long neglected in the literature. The effect of aw on protein stability works as a power law so that a small change in aw is amplified substantially through the cooperative hydration effect. In the present approach, the role of hydrophobic interaction stands behind. This affects protein stability indirectly through the change in solution structure caused by the existence of cosolute. PMID:26896315

  17. Protein Structure and Stability in Neat Ionic Liquid

    NASA Astrophysics Data System (ADS)

    Bihari, Malvika; Russell, Thomas P.; Hoagland, David A.

    2010-03-01

    Ionic liquid (IL) as a medium for room temperature preservation of biomacromolecules has been proposed, and to investigate the possibility, we studied physicochemical and enzymatic properties of several proteins in the neat hydrophilic IL, ethylmethyl imidazolium ethyl sulfate [EMIM][EtSO4]. Molecular dissolution of α-chymotypsin, cytochrome-c and other proteins could be achieved with moderate heating (60C). Dynamic light scattering and dilute solution viscometry typically reveal protein size slightly larger than in buffer, suggesting different solvation or protein unfolding. Spectroscopic methods (UV-Vis, fluorescence, FTIR, CD) show largely unchanged secondary structure but significantly changed tertiary structure. IL-dissolved cytochrome-c has heightened peroxidase activity, supporting the same conclusions. Transfer of dissolved protein from IL to buffer and ensuing alterations to protein conformation/activity will be discussed.

  18. Fast Dynamics and Stabilization of Proteins: Binary Glasses of Trehalose and Glycerol

    PubMed Central

    Cicerone, Marcus T.; Soles, Christopher L.

    2004-01-01

    We present elastic and inelastic incoherent neutron scattering data from a series of trehalose glasses diluted with glycerol. A strong correlation with recently published protein stability data in the same series of glasses illustrates that the dynamics at Q ≥ 0.71 Å−1 and ω > 200 MHz are important to stabilization of horseradish peroxidase and yeast alcohol dehydrogenase in these glasses. To the best of our knowledge, this is the first direct evidence that enzyme stability in a room temperature glass depends upon suppressing these short-length scale, high-frequency dynamics within the glass. We briefly discuss the coupling of protein motions to the local dynamics of the glass. Also, we show that Tg alone is not a good indicator for the protein stability in this series of glasses; the glass that confers the maximum room-temperature stability does not have the highest Tg. PMID:15189880

  19. A chloroplast-targeted DnaJ protein contributes to maintenance of photosystem II under chilling stress.

    PubMed

    Kong, Fanying; Deng, Yongsheng; Zhou, Bin; Wang, Guodong; Wang, Yu; Meng, Qingwei

    2014-01-01

    DnaJ proteins act as essential molecular chaperones in protein homeostasis and protein complex stabilization under stress conditions. The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1), whose expression was upregulated by treatment at 4 and 42 °C, and with high light, NaCl, polyethylene glycol, and H2O2, were investigated here using sense and antisense transgenic tomatoes. The sense plants exhibited not only higher chlorophyll content, fresh weight and net photosynthetic rate, but also lower accumulation of reactive oxygen species and membrane damage under chilling stress. Moreover, the maximal photochemistry efficiency of photosystem II (PSII) (F v/F m) and D1 protein content were higher in the sense plants and lower in the antisense plants, and the photoinhibitory quenching was lower in the sense plants and higher in the antisense plants, suggesting that the inhibition of PSII was less severe in the sense plants and more severe in the antisense plants compared with the wild type. Furthermore, the PSII protein complexes were also more stable in the sense plants. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher F v/F m, D1 protein content and PSII stability than the SM-untreated antisense plants. This finding suggested that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, chloroplast heat-shock protein 70 was identified as the partner of LeCDJ1. These results indicate that LeCDJ1 has essential functions in maintaining PSII under chilling stress. PMID:24227338

  20. Multiple interaction partners for Cockayne syndrome proteins: implications for genome and transcriptome maintenance

    PubMed Central

    Aamann, Maria D.; Muftuoglu, Meltem; Bohr, Vilhelm A.; Stevnsner, Tinna

    2013-01-01

    Cockayne syndrome (CS) is characterized by progressive multisystem degeneration and is classified as a segmental premature aging syndrome. The majority of CS cases are caused by defects in the CS complementation group B (CSB) protein and the rest are mainly caused by defects in the CS complementation group A (CSA) protein. Cells from CS patients are sensitive to UV light and a number of other DNA damaging agents including various types of oxidative stress. The cells also display transcription deficiencies, abnormal apoptotic response to DNA damage, and DNA repair deficiencies. Herein we have critically reviewed the current knowledge about known protein interactions of the CS proteins. The review focuses on the participation of the CSB and CSA proteins in many different protein interactions and complexes, and how these interactions inform us about pathways that are defective in the disease. PMID:23583689

  1. The UHRF1 Protein Stimulates the Activity and Specificity of the Maintenance DNA Methyltransferase DNMT1 by an Allosteric Mechanism*

    PubMed Central

    Bashtrykov, Pavel; Jankevicius, Gytis; Jurkowska, Renata Z.; Ragozin, Sergey; Jeltsch, Albert

    2014-01-01

    The ubiquitin-like, containing PHD and RING finger domains protein 1 (UHRF1) is essential for maintenance DNA methylation by DNA methyltransferase 1 (DNMT1). UHRF1 has been shown to recruit DNMT1 to replicated DNA by the ability of its SET and RING-associated (SRA) domain to bind to hemimethylated DNA. Here, we demonstrate that UHRF1 also increases the activity of DNMT1 by almost 5-fold. This stimulation is mediated by a direct interaction of both proteins through the SRA domain of UHRF1 and the replication focus targeting sequence domain of DNMT1, and it does not require DNA binding by the SRA domain. Disruption of the interaction between DNMT1 and UHRF1 by replacement of key residues in the replication focus targeting sequence domain led to a strong reduction of DNMT1 stimulation. Additionally, the interaction with UHRF1 increased the specificity of DNMT1 for methylation of hemimethylated CpG sites. These findings show that apart from the targeting of DNMT1 to the replicated DNA UHRF1 increases the activity and specificity of DNMT1, thus exerting a multifaceted influence on the maintenance of DNA methylation. PMID:24368767

  2. Membrane protein stability analyses by means of protein energy profiles in case of nephrogenic diabetes insipidus.

    PubMed

    Heinke, Florian; Labudde, Dirk

    2012-01-01

    Diabetes insipidus (DI) is a rare endocrine, inheritable disorder with low incidences in an estimated one per 25,000-30,000 live births. This disease is characterized by polyuria and compensatory polydypsia. The diverse underlying causes of DI can be central defects, in which no functional arginine vasopressin (AVP) is released from the pituitary or can be a result of defects in the kidney (nephrogenic DI, NDI). NDI is a disorder in which patients are unable to concentrate their urine despite the presence of AVP. This antidiuretic hormone regulates the process of water reabsorption from the prourine that is formed in the kidney. It binds to its type-2 receptor (V2R) in the kidney induces a cAMP-driven cascade, which leads to the insertion of aquaporin-2 water channels into the apical membrane. Mutations in the genes of V2R and aquaporin-2 often lead to NDI. We investigated a structure model of V2R in its bound and unbound state regarding protein stability using a novel protein energy profile approach. Furthermore, these techniques were applied to the wild-type and selected mutations of aquaporin-2. We show that our results correspond well to experimental water ux analysis, which confirms the applicability of our theoretical approach to equivalent problems.

  3. The multifaceted role of Lon proteolysis in seedling establishment and maintenance of plant organelle function: living from protein destruction.

    PubMed

    Rigas, Stamatis; Daras, Gerasimos; Tsitsekian, Dikran; Hatzopoulos, Polydefkis

    2012-05-01

    Intracellular selective proteolysis is an important post-translational regulatory mechanism maintaining protein quality control by removing defective, damaged or even deleterious protein aggregates. The ATP-dependent Lon protease is a key component of protein quality control that is highly conserved across the kingdoms of living organisms. Major advancements have been made in bacteria and in non-plant organisms to understand the role of Lon in protection against protein oxidation, ageing and neurodegenerative diseases. This review presents the progress currently made in plants. The Lon gene family in Arabidopsis consists of four members that produce distinct protein isoforms localized in several organelles. Lon1 and Lon4 that potentially originate from a recent gene duplication event are dual-targeted to mitochondria and chloroplasts through distinct mechanisms revealing divergent evolution. Arabidopsis mutant analysis showed that mitochondria and peroxisomes biogenesis or maintenance of function is modulated by Lon1 and Lon2, respectively. Consequently, the lack of Lon selective proteolysis leading to growth retardation and impaired seedling establishment can be attributed to defects in the oil reserve mobilization pathway. The current progress in Arabidopsis research uncovers the role of Lon in the proteome homeostasis of plant organelles and stimulates biotechnology scenarios of plant tolerance against harsh abiotic conditions because of climate instability.

  4. Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein

    PubMed Central

    Ravindranathan, Ramya; Dereli, Ihsan; Stanzione, Marcello; Tóth, Attila

    2016-01-01

    Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during

  5. Proteins from hyperthermophiles: stability and enzymatic catalysis close to the boiling point of water.

    PubMed

    Ladenstein, R; Antranikian, G

    1998-01-01

    It has become clear since about a decade ago, that the biosphere contains a variety of microorganisms that can live and grow in extreme environments. Hyperthermophilic microorganisms, present among Archaea and Bacteria, proliferate at temperatures of around 80-100 degrees C. The majority of the genera known to date are of marine origin, however, some of them have been found in continental hot springs and solfataric fields. Metabolic processes and specific biological functions of these organisms are mediated by enzymes and proteins that function optimally under these extreme conditions. We are now only starting to understand the structural, thermodynamic and kinetic basis for function and stability under conditions of high temperature, salt and extremes of pH. Insights gained from the study of such macromolecules help to extend our understanding of protein biochemistry and -biophysics and are becoming increasingly important for the investigation of fundamental problems in structure biology such as protein stability and protein folding. Extreme conditions in the biosphere require either the adaptation of the amino acid sequence of a protein by mutations, the optimization of weak interactions within the protein and at the protein-solvent boundary, the influence of extrinsic factors such as metabolites, cofactors, compatible solutes. Furthermore folding catalysts, known as chaperones, that assist the folding of proteins may be involved or increased protein protein synthesis in order to compensate for destruction by extreme conditions. The comparison of structure and stability of homologous proteins from mesophiles and hyperthermophiles has revealed important determinants of thermal stability of proteins. Rather than being the consequence of one dominant type of interactions or of a general stabilization strategy, it appears that the adaptation to high temperatures reflects a number of subtle interactions, often characteristic for each protein species, that minimize the

  6. Nucleic acid aptamers stabilize proteins against different types of stress conditions.

    PubMed

    Jetani, Hardik C; Bhadra, Ankan Kumar; Jain, Nishant Kumar; Roy, Ipsita

    2014-01-01

    It has been observed that the same osmolyte cannot provide protection to a protein exposed to more than one stress condition. We wanted to study the effect of nucleic acid aptamers on the stabilization of proteins against a variety of stress conditions. Adjuvanted tetanus toxoid was exposed to thermal, freeze-thawing, and agitation stress. The stability and antigenicity of the toxoid were measured. Using nucleic acid aptamers selected against tetanus toxoid, we show that these specific RNA sequences were able to stabilize alumina-adsorbed tetanus toxoid against thermal-, agitation-, and freeze-thawing-induced stress. Binding affinity of the aptamer-protein complex did not show any significant change at elevated temperature as compared with that at room temperature, indicating that the aptamer protected the protein by remaining bound to it under stress conditions and did not allow either the protein to unfold or to promote protein-protein interaction. Thus, we show that by changing the stabilization strategy from a solvent-centric to a protein-centric approach, the same molecule can be employed as a stabilizer against more than one stress condition and thus probably reduce the cost of the product during its formulation.

  7. Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability

    PubMed Central

    Tong, X; Kono, T; Evans-Molina, C

    2015-01-01

    The sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl l-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-d,l-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca2+ imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca2+ and decreased ER Ca2+ levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein

  8. Long-term manure amendments reduced soil aggregate stability via redistribution of the glomalin-related soil protein in macroaggregates.

    PubMed

    Xie, Hongtu; Li, Jianwei; Zhang, Bin; Wang, Lianfeng; Wang, Jingkuan; He, Hongbo; Zhang, Xudong

    2015-01-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm; 2000-250 μm; 250-53 μm; and <53 μm). The treatments include no fertilization (CK), low and high manure amendment (M1, M2), chemical nitrogen, phosphorus and potassium fertilizers (NPK), and combined manure and chemical fertilizers (NPKM1, NPKM2). Though SOC, GRSPe and GRSPt in soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000-250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture. PMID:26423355

  9. Long-term manure amendments reduced soil aggregate stability via redistribution of the glomalin-related soil protein in macroaggregates

    NASA Astrophysics Data System (ADS)

    Xie, Hongtu; Li, Jianwei; Zhang, Bin; Wang, Lianfeng; Wang, Jingkuan; He, Hongbo; Zhang, Xudong

    2015-10-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm 2000-250 μm 250-53 μm and <53 μm). The treatments include no fertilization (CK), low and high manure amendment (M1, M2), chemical nitrogen, phosphorus and potassium fertilizers (NPK), and combined manure and chemical fertilizers (NPKM1, NPKM2). Though SOC, GRSPe and GRSPt in soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000-250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture.

  10. Long-term manure amendments reduced soil aggregate stability via redistribution of the glomalin-related soil protein in macroaggregates

    PubMed Central

    Xie, Hongtu; Li, Jianwei; Zhang, Bin; Wang, Lianfeng; Wang, Jingkuan; He, Hongbo; Zhang, Xudong

    2015-01-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm; 2000–250 μm; 250–53 μm; and <53 μm). The treatments include no fertilization (CK), low and high manure amendment (M1, M2), chemical nitrogen, phosphorus and potassium fertilizers (NPK), and combined manure and chemical fertilizers (NPKM1, NPKM2). Though SOC, GRSPe and GRSPt in soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000–250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture. PMID:26423355

  11. Clinical application for the preservation of phospho-proteins through in-situ tissue stabilization

    PubMed Central

    2010-01-01

    Background Protein biomarkers will play a pivotal role in the future of personalized medicine for both diagnosis and treatment decision-making. While the results of several pre-clinical and small-scale clinical studies have demonstrated the value of protein biomarkers, there have been significant challenges to translating these findings into routine clinical care. Challenges to the use of protein biomarkers include inter-sample variability introduced by differences in post-collection handling and ex vivo degradation of proteins and protein modifications. Results In this report, we re-create laboratory and clinical scenarios for sample collection and test the utility of a new tissue stabilization technique in preserving proteins and protein modifications. In the laboratory setting, tissue stabilization with the Denator Stabilizor T1 resulted in a significantly higher yield of phospho-protein when compared to standard snap freeze preservation. Furthermore, in a clinical scenario, tissue stabilization at collection resulted in a higher yield of total phospho-protein, total phospho-tyrosine, pErkT202/Y204 and pAktS473 when compared to standard methods. Tissue stabilization did not have a significant effect on other post-translational modifications such as acetylation and glycosylation, which are more stable ex-vivo. Tissue stabilization did decrease total RNA quantity and quality. Conclusion Stabilization at the time of collection offers the potential to better preserve tissue protein and protein modification levels, as well as reduce the variability related to tissue processing delays that are often associated with clinical samples. PMID:21092202

  12. Protein lipid interaction in bile: effects of biliary proteins on the stability of cholesterol-lecithin vesicles.

    PubMed

    Luk, A S; Kaler, E W; Lee, S P

    1998-02-23

    The nucleation of cholesterol crystals is an obligatory precursor to cholesterol gallstone formation. Nucleation, in turn, is believed to be preceded by aggregation and fusion of cholesterol-rich vesicles. We have investigated the effects of two putative pro-nucleating proteins, a concanavalin A-binding protein fraction and a calcium-binding protein, on the stability of sonicated small unilamellar cholesterol-lecithin vesicles. Vesicle aggregation is followed by monitoring absorbance, and upon addition of the concanavalin A-binding protein fraction the absorbance of a vesicle dispersion increases continuously with time. Vesicle fusion is probed by a fluorescence contents-mixing assay. Vesicles apparently fuse slowly after the addition of the concanavalin A-binding protein, although inner filter effects confound the quantitative measurement of fusion rates. The rates of change of absorbance and fluorescence increase with the concentration of the protein, and the second-order dimerization rate constant increases with both the protein concentration and the cholesterol content of the vesicles. On the other hand, the calcium-binding protein has no effect on the stability of the vesicle dispersion. This protein may therefore affect cholesterol crystal formation not by promoting the nucleation process, but by enhancing crystal growth and packaging. Our results demonstrate that biliary proteins can destabilize lipid vesicles and that different proteins play different roles in the mechanism of cholesterol gallstone formation.

  13. A single disulfide bond restores thermodynamic and proteolytic stability to an extensively mutated protein.

    PubMed Central

    Roesler, K. R.; Rao, A. G.

    2000-01-01

    The potential for engineering stable proteins with multiple amino acid substitutions was explored. Eleven lysine, five methionine, two tryptophan, one glycine, and three threonine substitutions were simultaneously made in barley chymotrypsin inhibitor-2 (CI-2) to substantially improve the essential amino acid content of the protein. These substitutions were chosen based on the three-dimensional structure of CI-2 and an alignment of homologous sequences. The initial engineered protein folded into a wild-type-like structure, but had a free energy of unfolding of only 2.2 kcal/mol, considerably less than the wild-type value of 7.5 kcal/mol. Restoration of the lysine mutation at position 67 to the wild-type arginine increased the free energy of unfolding to 3.1 kcal/mol. Subsequent cysteine substitutions at positions 22 and 82 resulted in disulfide bond formation and a protein with nearly wild-type thermodynamic stability (7.0 kcal/mol). None of the engineered proteins retained inhibitory activity against chymotrypsin or elastase, and all had substantially reduced inhibitory activity against subtilisin. The proteolytic stabilities of the proteins correlated with their thermodynamic stabilities. Reduction of the disulfide bond resulted in substantial loss of both thermodynamic and proteolytic stabilities, confirming that the disulfide bond, and not merely the cysteine substitutions, was responsible for the increased stability. We conclude that it is possible to replace over a third of the residues in CI-2 with minimal disruption of stability and structural integrity. PMID:11045611

  14. Effect of pasteurization on the protein composition and oxidative stability of beer during storage.

    PubMed

    Lund, Marianne N; Hoff, Signe; Berner, Torben S; Lametsch, René; Andersen, Mogens L

    2012-12-19

    The impacts of pasteurization of a lager beer on protein composition and the oxidative stability were studied during storage at 22 °C for 426 days in the dark. Pasteurization clearly improved the oxidative stability of beer determined by ESR spectroscopy, whereas it had a minor negative effect on the volatile profile by increasing volatile compounds that is generally associated with heat treatment and a loss of fruity ester aroma. A faster rate of radical formation in unpasteurized beer was consistent with a faster consumption of sulfite. Beer proteins in the unpasteurized beer were more degraded, most likely due to proteolytic enzyme activity of yeast remnants and more precipitation of proteins was also observed. The differences in soluble protein content and composition are suggested to result in differences in the contents of prooxidative metals as a consequence of the proteins ability to bind metals. This also contributes to the differences in oxidative stabilities of the beers.

  15. Evolutionary perspectives on protein structure, stability, and functionality

    NASA Astrophysics Data System (ADS)

    Goldstein, Richard A.

    Proteins are the result of a long process of evolution. It is due to this process that they have developed properties rather different from those of random strings of amino acids. If we wish to understand the properties of proteins, we need to understand the underlying process of Darwinian evolution, and how its stochastic nature interacts with the underlying fitness landscape. In this review, I describe some of the underlying theory of evolution. I then discuss how these theories can help us understand the structure, thermodynamics, and functioning of naturally-occurring proteins.

  16. Accelerated Disease Onset with Stabilized Familial Amyotrophic Lateral Sclerosis (ALS)-linked Mutant TDP-43 Proteins*

    PubMed Central

    Watanabe, Shoji; Kaneko, Kumi; Yamanaka, Koji

    2013-01-01

    Abnormal protein accumulation is a pathological hallmark of neurodegenerative diseases, including accumulation of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS). Dominant mutations in the TDP-43 gene are causative for familial ALS; however, the relationship between mutant protein biochemical phenotypes and disease course and their significance to disease pathomechanism are not known. Here, we found that longer half-lives of mutant proteins correlated with accelerated disease onset. Based on our findings, we established a cell model in which chronic stabilization of wild-type TDP-43 protein provoked cytotoxicity and recapitulated pathogenic protein cleavage and insolubility to the detergent Sarkosyl, TDP-43 properties that have been observed in sporadic ALS lesions. Furthermore, these cells showed proteasomal impairment and dysregulation of their own mRNA levels. These results suggest that chronically increased stability of mutant or wild-type TDP-43 proteins results in a gain of toxicity through abnormal proteostasis. PMID:23235148

  17. Two-Photon Fluorescence Anisotropy Imaging to Elucidate the Dynamics and the Stability of Immobilized Proteins.

    PubMed

    Orrego, Alejandro H; García, Carolina; Mancheño, José M; Guisán, Jose M; Lillo, M Pilar; López-Gallego, Fernando

    2016-01-28

    Time/spatial-resolved fluorescence determines anisotropy values of supported-fluorescent proteins through different immobilization chemistries, evidencing some of the molecular mechanisms that drive the stabilization of proteins at the interfaces with solid surfaces. Fluorescence anisotropy imaging provides a normalized protein mobility parameter that serves as a guide to study the effect of different immobilization parameters (length and flexibility of the spacer arm and multivalency of the protein-support interaction) on the final stability of the supported proteins. Proteins in a more constrained environment correspond to the most thermostable ones, as was shown by thermal inactivation studies. This work contributes to explain the experimental evidence found with conventional methods based on observable measurements; thus this advanced characterization technique provides reliable molecular information about the immobilized proteins with sub-micrometer spatial resolution. Such information has been very useful for fabricating highly stable heterogeneous biocatalysts with high interest in industrial developments.

  18. Molecular basis for polyol-induced protein stability revealed by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Liu, Fu-Feng; Ji, Luo; Zhang, Lin; Dong, Xiao-Yan; Sun, Yan

    2010-06-01

    Molecular dynamics simulations of chymotrypsin inhibitor 2 in different polyols (glycerol, xylitol, sorbitol, trehalose, and sucrose) at 363 K were performed to probe the molecular basis of the stabilizing effect, and the data in water, ethanol, and glycol were compared. It is found that protein protection by polyols is positively correlated with both the molecular volume and the fractional polar surface area, and the former contributes more significantly to the protein's stability. Polyol molecules have only a few direct hydrogen bonds with the protein, and the number of hydrogen bonds between a polyol and the protein is similar for different polyols. Thus, it is concluded that the direct interactions contribute little to the stabilizing effect. It is clarified that the preferential exclusion of the polyols is the origin of their protective effects, and it increases with increasing polyol size. Namely, there is preferential hydration on the protein surface (2 Å), and polyol molecules cluster around the protein at a distance of about 4 Å. The preferential exclusion of polyols leads to indirect interactions that prevent the protein from thermal unfolding. The water structure becomes more ordered with increasing the polyol size. So, the entropy of water in the first hydration shell decreases, and a larger extent of decrease is observed with increasing polyol size, leading to larger transfer free energy. The findings suggest that polyols protect the protein from thermal unfolding via indirect interactions. The work has thus elucidated the molecular mechanism of structural stability of the protein in polyol solutions.

  19. Principles and equations for measuring and interpreting protein stability: From monomer to tetramer.

    PubMed

    Bedouelle, Hugues

    2016-02-01

    The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins.

  20. Principles and equations for measuring and interpreting protein stability: From monomer to tetramer.

    PubMed

    Bedouelle, Hugues

    2016-02-01

    The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins. PMID:26607240

  1. Organization and maintenance of Drosophila telomeres: the roles of terminin and non-terminin proteins.

    PubMed

    Raffa, G D; Cenci, G; Ciapponi, L; Gatti, M

    2013-01-01

    Drosophila telomeres are elongated by occasional transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the sequence of the DNA termini. Drosophila telomeres are capped by terminin, a complex formed by the HOAP, Moi, Ver and HipHop proteins that localize exclusively at telomeres and protect them from fusion events. Other proteins required to prevent end-to-end fusion include HP 1 Eff/UbcD 1, ATM, the components of the Mrel 1-Rad50-Nbs (MRN) complex, and the Woc transcription factor. The terminin proteins are encoded by fast-evolving genes and are not evolutionarily conserved outside the Drosophila species. In contrast, the non-terminin telomere capping proteins are not fast-evolving, do not localize only at telomeres and are conserved from yeasts to mammals. We propose that following telomerase loss, Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent manner, and that non-terminin proteins did not evolve as rapidly as terminin because of the functional constraints imposed by their involvement in diverse cellular processes. This hypothesis suggests that the Drosophila non-terminin proteins might correspond to ancestral telomere-associated proteins with homologues in other organisms including humans. PMID:23795467

  2. Organization and maintenance of Drosophila telomeres: the roles of terminin and non-terminin proteins.

    PubMed

    Raffa, G D; Cenci, G; Ciapponi, L; Gatti, M

    2013-01-01

    Drosophila telomeres are elongated by occasional transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the sequence of the DNA termini. Drosophila telomeres are capped by terminin, a complex formed by the HOAP, Moi, Ver and HipHop proteins that localize exclusively at telomeres and protect them from fusion events. Other proteins required to prevent end-to-end fusion include HP 1 Eff/UbcD 1, ATM, the components of the Mrel 1-Rad50-Nbs (MRN) complex, and the Woc transcription factor. The terminin proteins are encoded by fast-evolving genes and are not evolutionarily conserved outside the Drosophila species. In contrast, the non-terminin telomere capping proteins are not fast-evolving, do not localize only at telomeres and are conserved from yeasts to mammals. We propose that following telomerase loss, Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent manner, and that non-terminin proteins did not evolve as rapidly as terminin because of the functional constraints imposed by their involvement in diverse cellular processes. This hypothesis suggests that the Drosophila non-terminin proteins might correspond to ancestral telomere-associated proteins with homologues in other organisms including humans.

  3. Volume exclusion and soft interaction effects on protein stability under crowded conditions.

    PubMed

    Miklos, Andrew C; Li, Conggang; Sharaf, Naima G; Pielak, Gary J

    2010-08-24

    Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams of macromolecules per liter often interfere with the observation of the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test protein. Stability was quantified with NMR-detected amide (1)H exchange. We analyzed the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a stabilizing effect. The exceptions are residues for which stabilities are unchanged. At a PVP concentration of 100 g/L, the data are consistent with theories of hard particle exclusion. At higher concentrations, the data are more consistent with confinement. The data show that the crowder also stabilizes the test protein by weakly binding its native state. We conclude that the role of native-state binding and other soft interactions needs to be seriously considered when applying both theory and experiment to studies of macromolecular crowding.

  4. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    PubMed

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

  5. Stabilization of protein crystals by electrostatic interactions as revealed by a numerical approach.

    PubMed

    Takahashi, T; Endo, S; Nagayama, K

    1993-11-20

    We developed a novel algorithm to solve numerically the Poisson-Boltzmann equations under a periodic boundary condition. By employing this algorithm to calculate the electrostatic potentials in two different types of protein crystals, a bovine pancreatic trypsin inhibitor (BPTI) orthorhombic crystal and a pig-insulin cubic crystal, the energy contributions of the electrostatic interactions to the crystals' stability were evaluated. At a high ionic strength, the condensed state of proteins in the crystal was stabilized electrostatically compared with that isolated in dilute solution because the attractive electrostatic interactions between neighboring protein molecules overcame the repulsive forces that originated from the same net charges of the equivalent protein molecules. On the other hand, at a low ionic strength the electrostatic interactions destabilized the crystalline state of both proteins, although a different dependence on the ionic strength was found between them. Here, the insulin crystal was more stable than the BPTI one because of the higher charge density in the BPTI crystal. In all of the solvent ionic strengths investigated, the attractive electrostatic interactions between charge pairs separated by less than 5 A on the respective protein molecules prominently stabilize the protein crystals. Therefore, two protein molecules in the crystals are oriented to compensate each other for their opposite charges on the surfaces. We also found a specific role for bound phosphate ions in the stabilization of the BPTI crystal, based on comparison of the electrostatic energies of the two crystals with and without the ions. By determining the contribution of each atomic charge in the crystals to the electrostatic energy, it was revealed that several electrostatic pairs specifically contributed to the crystal's stability. On the basis of our numerical calculation results, we propose a new method to design protein molecules that adopt stable crystals by replacing

  6. ngs (notochord granular surface) gene encodes a novel type of intermediate filament family protein essential for notochord maintenance in zebrafish.

    PubMed

    Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

    2013-01-25

    The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins.

  7. A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation.

    PubMed

    Kanno, Tatsuo; Bucher, Etienne; Daxinger, Lucia; Huettel, Bruno; Böhmdorfer, Gudrun; Gregor, Wolfgang; Kreil, David P; Matzke, Marjori; Matzke, Antonius J M

    2008-05-01

    RNA-directed DNA methylation (RdDM) is a process in which dicer-generated small RNAs guide de novo cytosine methylation at the homologous DNA region. To identify components of the RdDM machinery important for Arabidopsis thaliana development, we targeted an enhancer active in meristems for methylation, which resulted in silencing of a downstream GFP reporter gene. This silencing system also features secondary siRNAs, which trigger methylation that spreads beyond the targeted enhancer region. A screen for mutants defective in meristem silencing and enhancer methylation retrieved six dms complementation groups, which included the known factors DRD1 (ref. 3; a SNF2-like chromatin-remodeling protein) and Pol IVb subunits. Additionally, we identified a previously unknown gene DMS3 (At3g49250), encoding a protein similar to the hinge-domain region of structural maintenance of chromosomes (SMC) proteins. This finding implicates a putative chromosome architectural protein that can potentially link nucleic acids in facilitating an RNAi-mediated epigenetic modification involving secondary siRNAs and spreading of DNA methylation.

  8. A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

    PubMed Central

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering. PMID:22792305

  9. Rigidity versus flexibility: the dilemma of understanding protein thermal stability.

    PubMed

    Karshikoff, Andrey; Nilsson, Lennart; Ladenstein, Rudolf

    2015-10-01

    The role of fluctuations in protein thermostability has recently received considerable attention. In the current literature a dualistic picture can be found: thermostability seems to be associated with enhanced rigidity of the protein scaffold in parallel with the reduction of flexible parts of the structure. In contradiction to such arguments it has been shown by experimental studies and computer simulation that thermal tolerance of a protein is not necessarily correlated with the suppression of internal fluctuations and mobility. Both concepts, rigidity and flexibility, are derived from mechanical engineering and represent temporally insensitive features describing static properties, neglecting that relative motion at certain time scales is possible in structurally stable regions of a protein. This suggests that a strict separation of rigid and flexible parts of a protein molecule does not describe the reality correctly. In this work the concepts of mobility/flexibility versus rigidity will be critically reconsidered by taking into account molecular dynamics calculations of heat capacity and conformational entropy, salt bridge networks, electrostatic interactions in folded and unfolded states, and the emerging picture of protein thermostability in view of recently developed network theories. Last, but not least, the influence of high temperature on the active site and activity of enzymes will be considered. PMID:26074325

  10. Size and molecular flexibility of sugars determine the storage stability of freeze-dried proteins.

    PubMed

    Tonnis, W F; Mensink, M A; de Jager, A; van der Voort Maarschalk, K; Frijlink, H W; Hinrichs, W L J

    2015-03-01

    Protein-based biopharmaceuticals are generally produced as aqueous solutions and stored refrigerated to obtain sufficient shelf life. Alternatively, proteins may be freeze-dried in the presence of sugars to allow storage stability at ambient conditions for prolonged periods. However, to act as a stabilizer, these sugars should remain in the glassy state during storage. This requires a sufficiently high glass transition temperature (Tg). Furthermore, the sugars should be able to replace the hydrogen bonds between the protein and water during drying. Frequently used disaccharides are characterized by a relatively low Tg, rendering them sensitive to plasticizing effects of residual water, which strongly reduces the Tg values of the formulation. Larger sugars generally have higher Tgs, but it is assumed that these sugars are limited in their ability to interact with the protein due to steric hindrance. In this paper, the size and molecular flexibility of sugars was related to their ability to stabilize proteins. Four diverse proteins varying in size from 6 kDa to 540 kDa were freeze-dried in the presence of different sugars varying in size and molecular flexibility. Subsequently, the different samples were subjected to an accelerated stability test. Using protein specific assays and intrinsic fluorescence, stability of the proteins was monitored. It was found that the smallest sugar (disaccharide trehalose) best preserved the proteins, but also that the Tg of the formulations was only just high enough to maintain sufficient vitrification. When trehalose-based formulations are exposed to high relative humidities, water uptake by the product reduces the Tgs too much. In that respect, sugars with higher Tgs are desired. Addition of polysaccharide dextran 70 kDa to trehalose greatly increased the Tg of the formulation. Moreover, this combination also improved the stability of the proteins compared to dextran only formulations. The molecularly flexible oligosaccharide

  11. Interaction of HTLV-1 Tax with minichromosome maintenance proteins accelerates the replication timing program.

    PubMed

    Boxus, Mathieu; Twizere, Jean-Claude; Legros, Sébastien; Kettmann, Richard; Willems, Luc

    2012-01-01

    The Tax oncoprotein encoded by the human T-cell leukemia virus type 1 plays a pivotal role in viral persistence and pathogenesis. Human T-cell leukemia virus type 1-infected cells proliferate faster than normal lymphocytes, expand through mitotic division, and accumulate genomic lesions. Here, we show that Tax associates with the minichromosome maintenance MCM2-7 helicase complex and localizes to origins of replication. Tax modulates the spatiotemporal program of origin activation and fires supplementary origins at the onset of S phase. Thereby, Tax increases the DNA replication rate, accelerates S phase progression, but also generates a replicative stress characterized by the presence of genomic lesions. Mechanistically, Tax favors p300 recruitment and histone hyperacetylation at late replication domains, advancing their replication timing in early S phase. PMID:22058115

  12. Effect of Oxygen-containing Functional Groups on Protein Stability in Ionic Liquid Solutions

    NASA Technical Reports Server (NTRS)

    Turner, Megan B.; Holbrey, John D.; Spear, Scott K.; Pusey, Marc L.; Rogers, Robin D.

    2004-01-01

    The ability of functionalized ionic liquids (ILs) to provide an environment of increased stability for biomolecules has been studied. Serum albumin is an inexpensive, widely available protein that contributes to the overall colloid osmotic blood pressure within the vascular system. Albumin is used in the present study as a marker of biomolecular stability in the presence of various ILs in a range of concentrations. The incorporation of hydroxyl functionality into the methylimidazolium-based cation leads to increased protein stability detected by fluorescence spectroscopy and circular dichroic (CD) spectrometry.

  13. Stability of whey-protein-stabilized oil-in-water emulsions during chilled storage and temperature cycling.

    PubMed

    Kiokias, Sotirios; Reiffers-Magnani, Christel K; Bot, Arjen

    2004-06-16

    The stability of heat-treated and/or acidified, partly-crystalline-fat-based, whey-protein-stabilized oil-in-water (o/w) emulsions against partial coalescence was investigated during chilled storage (at 5 degrees C) and repeated temperature cycling (three times between 5 and 25 degrees C). Experiments focused on the evolution of firmness and droplet size (using pulsed field gradient NMR and scanning electron microscopy). Besides the effects of denaturation and/or acidification, the influence of the droplet size of the dispersed phase on emulsion stability was investigated also. It was found that heat treatment or acidification before emulsification led to unstable emulsions during temperature cycling, whereas heat treatment after acidification resulted in stable emulsions. PMID:15186103

  14. Protein modification by acrolein: Formation and stability of cysteine adducts

    PubMed Central

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2010-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intra-molecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues. PMID:19231900

  15. Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings.

    PubMed

    Alexander, Crispin G; Wanner, Randy; Johnson, Christopher M; Breitsprecher, Dennis; Winter, Gerhard; Duhr, Stefan; Baaske, Philipp; Ferguson, Neil

    2014-12-01

    Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein-ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide-PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes.

  16. On the Potential Origins of the High Stability of Reconstructed Ancestral Proteins.

    PubMed

    Trudeau, Devin L; Kaltenbach, Miriam; Tawfik, Dan S

    2016-10-01

    Ancestral reconstruction provides instrumental insights regarding the biochemical and biophysical characteristics of past proteins. A striking observation relates to the remarkably high thermostability of reconstructed ancestors. The latter has been linked to high environmental temperatures in the Precambrian era, the era relating to most reconstructed proteins. We found that inferred ancestors of the serum paraoxonase (PON) enzyme family, including the mammalian ancestor, exhibit dramatically increased thermostabilities compared with the extant, human enzyme (up to 30 °C higher melting temperature). However, the environmental temperature at the time of emergence of mammals is presumed to be similar to the present one. Additionally, the mammalian PON ancestor has superior folding properties (kinetic stability)-unlike the extant mammalian PONs, it expresses in E. coli in a soluble and functional form, and at a high yield. We discuss two potential origins of this unexpectedly high stability. First, ancestral stability may be overestimated by a "consensus effect," whereby replacing amino acids that are rare in contemporary sequences with the amino acid most common in the family increases protein stability. Comparison to other reconstructed ancestors indicates that the consensus effect may bias some but not all reconstructions. Second, we note that high stability may relate to factors other than high environmental temperature such as oxidative stress or high radiation levels. Foremost, intrinsic factors such as high rates of genetic mutations and/or of transcriptional and translational errors, and less efficient protein quality control systems, may underlie the high kinetic and thermodynamic stability of past proteins.

  17. On the Potential Origins of the High Stability of Reconstructed Ancestral Proteins.

    PubMed

    Trudeau, Devin L; Kaltenbach, Miriam; Tawfik, Dan S

    2016-10-01

    Ancestral reconstruction provides instrumental insights regarding the biochemical and biophysical characteristics of past proteins. A striking observation relates to the remarkably high thermostability of reconstructed ancestors. The latter has been linked to high environmental temperatures in the Precambrian era, the era relating to most reconstructed proteins. We found that inferred ancestors of the serum paraoxonase (PON) enzyme family, including the mammalian ancestor, exhibit dramatically increased thermostabilities compared with the extant, human enzyme (up to 30 °C higher melting temperature). However, the environmental temperature at the time of emergence of mammals is presumed to be similar to the present one. Additionally, the mammalian PON ancestor has superior folding properties (kinetic stability)-unlike the extant mammalian PONs, it expresses in E. coli in a soluble and functional form, and at a high yield. We discuss two potential origins of this unexpectedly high stability. First, ancestral stability may be overestimated by a "consensus effect," whereby replacing amino acids that are rare in contemporary sequences with the amino acid most common in the family increases protein stability. Comparison to other reconstructed ancestors indicates that the consensus effect may bias some but not all reconstructions. Second, we note that high stability may relate to factors other than high environmental temperature such as oxidative stress or high radiation levels. Foremost, intrinsic factors such as high rates of genetic mutations and/or of transcriptional and translational errors, and less efficient protein quality control systems, may underlie the high kinetic and thermodynamic stability of past proteins. PMID:27413048

  18. Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO).

    PubMed

    Ma, Jianqiang; Pazos, Ileana M; Gai, Feng

    2014-06-10

    Although it is widely known that trimethylamine N-oxide (TMAO), an osmolyte used by nature, stabilizes the folded state of proteins, the underlying mechanism of action is not entirely understood. To gain further insight into this important biological phenomenon, we use the C≡N stretching vibration of an unnatural amino acid, p-cyano-phenylalanine, to directly probe how TMAO affects the hydration and conformational dynamics of a model peptide and a small protein. By assessing how the lineshape and spectral diffusion properties of this vibration change with cosolvent conditions, we are able to show that TMAO achieves its protein-stabilizing ability through the combination of (at least) two mechanisms: (i) It decreases the hydrogen bonding ability of water and hence the stability of the unfolded state, and (ii) it acts as a molecular crowder, as suggested by a recent computational study, that can increase the stability of the folded state via the excluded volume effect. PMID:24912147

  19. Structure and stability of designed TPR protein superhelices: unusual crystal packing and implications for natural TPR proteins.

    PubMed

    Kajander, Tommi; Cortajarena, Aitziber L; Mochrie, Simon; Regan, Lynne

    2007-07-01

    The structure and stability of repeat proteins has been little studied in comparison to the properties of the more familiar globular proteins. Here, the structure and stability of designed tetratricopeptide-repeat (TPR) proteins is described. The TPR is a 34-amino-acid motif which adopts a helix-turn-helix structure and occurs as tandem repeats. The design of a consensus TPR motif (CTPR) has previously been described. Here, the crystal structures and stabilities of proteins that contain eight or 20 identical tandem repeats of the CTPR motif (CTPR8 and CTPR20) are presented. Both CTPR8 and CTPR20 adopt a superhelical overall structure. The structures of the different-length CTPR proteins are compared with each other and with the structures of natural TPR domains. Also, the unusual and perhaps unique crystal-packing interactions resulting in pseudo-infinite crystalline superhelices observed in the different crystal forms of CTPR8 and CTPR20 are discussed. Finally, it is shown that the thermodynamic behavior of CTPR8 and CTPR20 can be predicted from the behavior of other TPRs in this series using an Ising model-based analysis. The designed protein series CTPR2-CTPR20 covers the natural size repertoire of TPR domains and as such is an excellent model system for natural TPR proteins.

  20. Mathematics, thermodynamics, and modeling to address ten common misconceptions about protein structure, folding, and stability.

    PubMed

    Robic, Srebrenka

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative phenomena in undergraduate classes. In the process of learning about these topics, students often form incorrect ideas. For example, by learning about protein folding in the context of protein synthesis, students may come to an incorrect conclusion that once synthesized on the ribosome, a protein spends its entire cellular life time in its fully folded native confirmation. This is clearly not true; proteins are dynamic structures that undergo both local fluctuations and global unfolding events. To prevent and address such misconceptions, basic concepts of protein science can be introduced in the context of simple mathematical models and hands-on explorations of publicly available data sets. Ten common misconceptions about proteins are presented, along with suggestions for using equations, models, sequence, structure, and thermodynamic data to help students gain a deeper understanding of basic concepts relating to protein structure, folding, and stability.

  1. Mtr4-like protein coordinates nuclear RNA processing for heterochromatin assembly and for telomere maintenance

    PubMed Central

    Lee, Nathan N.; Chalamcharla, Venkata R.; Reyes-Turcu, Francisca; Mehta, Sameet; Zofall, Martin; Balachandran, Vanivilasini; Dhakshnamoorthy, Jothy; Taneja, Nitika; Yamanaka, Soichiro; Zhou, Ming; Grewal, Shiv I. S.

    2013-01-01

    SUMMARY The regulation of protein-coding and noncoding RNAs is linked to nuclear processes including chromatin modifications and gene silencing. However, the mechanisms that distinguish RNAs and mediate their functions are poorly understood. We describe a nuclear RNA processing network in fission yeast with a core module comprising the Mtr4-like protein, Mtl1, and the zinc finger protein, Red1. The Mtl1-Red1 core promotes degradation of mRNAs and noncoding RNAs, and associates with different proteins to assemble heterochromatin via distinct mechanisms. Mtl1 also forms Red1-independent interactions with evolutionarily conserved proteins named Nrl1 and Ctr1, which associate with splicing factors. Whereas Nrl1 targets transcripts with cryptic introns to form heterochromatin at developmental genes and retrotransposons, Ctr1 functions in processing intron-containing telomerase RNA. Together with our discovery of widespread cryptic introns, including in noncoding RNAs, these findings reveal unique cellular strategies for recognizing regulatory RNAs and coordinating their functions in response to developmental and environmental cues. PMID:24210919

  2. G protein-coupled receptors in stem cell maintenance and somatic reprogramming to pluripotent or cancer stem cells.

    PubMed

    Choi, Hye Yeon; Saha, Subbroto Kumar; Kim, Kyeongseok; Kim, Sangsu; Yang, Gwang-Mo; Kim, BongWoo; Kim, Jin-hoi; Cho, Ssang-Goo

    2015-02-01

    G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of selfrenewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs.

  3. G protein-coupled receptors in stem cell maintenance and somatic reprogramming to pluripotent or cancer stem cells

    PubMed Central

    Choi, Hye Yeon; Saha, Subbroto Kumar; Kim, Kyeongseok; Kim, Sangsu; Yang, Gwang-Mo; Kim, BongWoo; Kim, Jin-hoi; Cho, Ssang-Goo

    2015-01-01

    G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of selfrenewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs. [BMB Reports 2015; 48(2): 68-80] PMID:25413305

  4. Protein requirements of man: comparative nitrogen balance response within the submaintenance-to-maintenance range of intakes of wheat and beef proteins.

    PubMed

    Young, V R; Fajardo, L; Murray, E; Rand, W M; Scrimshaw, N S

    1975-05-01

    Sixteen young male students participated in two studies designed to determine the nitrogen balance response to beef or stone-ground, whole wheat protein over the submaintenance-to-maintenance range of protein intake. The objective of the studies was to evaluate the relative capacities of these proteins to meet the minimum protein needs in young adult males. A modified Latin-square design was used to allocate subjects to the four 15-day metabolic balance diet periods in each study. The last 10 days were used for fecal nitrogen determination and the last 5 days for evaluation of urinary nitrogen excretion. The diet periods were separated by a 4-day break period and began with 1 day on a "protein-free" diet. The levels of protien (N times 6.25) intake tested were 0.2, 0.3, 0.4, and 0.5 g/kg body weight/day in the beef study. The results have been compared with those obtained in a previous similar study with egg protein. The regression of estimated "true" nitrogen balance (Y) (mg N/kg/day) on nitrogen intake (X) (mg N/kg/day) was: Y equals 0.51(plus or minus 0.08)X minus 41.9(plus or minus 4.6) for beef and Y equals 0.27(plus or minus 0.06)X minus 33.6(plus or minus 5.0) for whole wheat protein. The amounts of beef and wheat proteins estimated to be requried to support body nitrogen balance in 97.5% of the population supplied 96 and 178 mg N/kg/day, respectively. The relative protein value of beef and wheat proteins, in comparison with egg protein, was 78 plus or minus 12 and 41 plus or minus 10, respectively. It is concluded that the variations in dietary protein quality should be taken into account in assessing the protein adequacy of diets for individuals and population groups.

  5. Characterization of milk proteins-lutein complexes and the impact on lutein chemical stability.

    PubMed

    Yi, Jiang; Fan, Yuting; Yokoyama, Wallace; Zhang, Yuzhu; Zhao, Liqing

    2016-06-01

    In this study, the interaction of WPI (whey protein isolate) and SC (sodium caseinate) with hydrophobic lutein was investigated through UV-vis spectroscopy and circular dichroism (CD) as well as fluorescence. The effects on lutein's chemical stability were also examined. The decrease of turbidity of lutein suggested that lutein's aqueous solubility was improved after binding with milk proteins. CD analysis indicated lutein had little impact on the secondary structures of both proteins. Different preparation methods have significant impacts on the binding constant. Fluorescence results indicated that WPI and SC interact with lutein by hydrophobic contacts. Milk proteins have protective effects on lutein against oxidation and decomposition, and SC showed better capability in protecting lutein from oxidation than WPI during 16 days storage. The lutein's chemical stability was increased with increasing of proteins concentration. The results indicated that milk proteins may act as effective carriers for lipophilic nutraceuticals.

  6. SWEF Proteins Distinctly Control Maintenance and Differentiation of Hematopoietic Stem Cells.

    PubMed

    Ripich, Tatsiana; Chacón-Martínez, Carlos Andrés; Fischer, Luise; Pernis, Alessandra; Kiessling, Nadine; Garbe, Annette I; Jessberger, Rolf

    2016-01-01

    SWAP-70 and DEF6, two proteins that feature similar domain and motif arrangements, are mainly known for their functions in differentiated hematopoietic cells. Both proteins interact with and regulate RhoGTPases and F-actin dynamics, yet their role in hematopoietic stem and precursor cells (HSPCs) remained unexplored. Here, the role of the SWEF proteins SWAP-70 and DEF6 in HSPCs was examined. Both SWEF proteins are expressed in HSCs. HSCs and different precursor populations were analyzed in mice deficient for SWAP-70, DEF6, SWAP-70 and DEF6 (double knockout, DKO), and wild-type controls. HSPCs isolated from these strains were used for competitive adoptive transfer into irradiated wild-type mice. Reconstitution of the myeloid and lymphoid lineages in the recipient mice was determined. The numbers of HSPCs in the bone marrow of Swap-70-/- and Swap-70-/-Def6-/- mice were >3-fold increased. When transplanted into lethally irradiated wild-type recipients, the reconstitution potential of Swap-70-/- HSPCs was intrinsically impaired in competing with wild-type HSPCs for contribution to hematopoiesis. Def6-/- HSPCs show wild type-like reconstitution potential under the same transplantation conditions. DKO HSPCs reconstituted to only 25% of wild-type levels, indicating a partial rescue by DEF6 deficiency in the Swap-70-/- background. Our study reveals the two SWEF proteins as important contributors to HSPC biology. Despite their similarity these two proteins regulate HSC/progenitor homeostasis, self-renewal, lineage contributions and repopulation in a distinct and mostly antagonistic manner. PMID:27561029

  7. SWEF Proteins Distinctly Control Maintenance and Differentiation of Hematopoietic Stem Cells

    PubMed Central

    Ripich, Tatsiana; Chacón-Martínez, Carlos Andrés; Fischer, Luise; Pernis, Alessandra; Kiessling, Nadine; Garbe, Annette I.; Jessberger, Rolf

    2016-01-01

    SWAP-70 and DEF6, two proteins that feature similar domain and motif arrangements, are mainly known for their functions in differentiated hematopoietic cells. Both proteins interact with and regulate RhoGTPases and F-actin dynamics, yet their role in hematopoietic stem and precursor cells (HSPCs) remained unexplored. Here, the role of the SWEF proteins SWAP-70 and DEF6 in HSPCs was examined. Both SWEF proteins are expressed in HSCs. HSCs and different precursor populations were analyzed in mice deficient for SWAP-70, DEF6, SWAP-70 and DEF6 (double knockout, DKO), and wild-type controls. HSPCs isolated from these strains were used for competitive adoptive transfer into irradiated wild-type mice. Reconstitution of the myeloid and lymphoid lineages in the recipient mice was determined. The numbers of HSPCs in the bone marrow of Swap-70-/- and Swap-70-/-Def6-/- mice were >3-fold increased. When transplanted into lethally irradiated wild-type recipients, the reconstitution potential of Swap-70-/- HSPCs was intrinsically impaired in competing with wild-type HSPCs for contribution to hematopoiesis. Def6-/- HSPCs show wild type-like reconstitution potential under the same transplantation conditions. DKO HSPCs reconstituted to only 25% of wild-type levels, indicating a partial rescue by DEF6 deficiency in the Swap-70-/- background. Our study reveals the two SWEF proteins as important contributors to HSPC biology. Despite their similarity these two proteins regulate HSC/progenitor homeostasis, self-renewal, lineage contributions and repopulation in a distinct and mostly antagonistic manner. PMID:27561029

  8. Molecular insights into the stabilization of protein-protein interactions with small molecule: The FKBP12-rapamycin-FRB case study

    NASA Astrophysics Data System (ADS)

    Chaurasia, Shilpi; Pieraccini, Stefano; De Gonda, Riccardo; Conti, Simone; Sironi, Maurizio

    2013-11-01

    Targetting protein-protein interactions is a challenging task in drug discovery process. Despite the challenges, several studies provided evidences for the development of small molecules modulating protein-protein interactions. Here we consider a typical case of protein-protein interaction stabilization: the complex between FKBP12 and FRB with rapamycin. We have analyzed the stability of the complex and characterized its interactions at the atomic level by performing free energy calculations and computational alanine scanning. It is shown that rapamycin stabilizes the complex by acting as a bridge between the two proteins; and the complex is stable only in the presence of rapamycin.

  9. New insights into structural determinants of prion protein folding and stability.

    PubMed

    Benetti, Federico; Legname, Giuseppe

    2015-01-01

    Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

  10. Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings

    PubMed Central

    Alexander, Crispin G.; Wanner, Randy; Johnson, Christopher M.; Breitsprecher, Dennis; Winter, Gerhard; Duhr, Stefan; Baaske, Philipp; Ferguson, Neil

    2014-01-01

    Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein–ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide–PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes. PMID:25262836

  11. Energy and protein requirements for growth and maintenance of F1 Nellore x Red Angus bulls, steers, and heifers.

    PubMed

    Chizzotti, M L; Valadares Filho, S C; Tedeschi, L O; Chizzotti, F H M; Carstens, G E

    2007-08-01

    A comparative slaughter trial was conducted with 36 F1 Nellore x Red Angus calves (12 steers, 12 bulls, and 12 heifers), averaging 274 kg of BW, to assess the net requirements of protein and energy for growth and maintenance. Three cattle from each group (i.e., steers, bulls, and heifers) were slaughtered at the beginning of the trial to determine the initial body composition. The remaining calves were randomly assigned to 1 of 3 treatments: maintenance (diet containing 70% of DM as corn silage fed at 1.2% of BW daily) or concentrate at 0.75 or 1.5% of BW daily with corn silage available for ad libitum consumption. The diets were isonitrogenous (2% N, DM basis). The experimental design provided ranges in ME intake, BW, and ADG for the development of regression equations to predict the maintenance requirements for NE and net protein (MRNE and MRNP, respectively) and the growth requirement for NE and net protein (GRNE and GRNP, respectively). After 84 d of growth, the cattle were slaughtered. The cleaned gastrointestinal tracts, organs, carcasses, heads, hides, tails, feet, blood, and tissues were weighed to measure empty BW (EBW). These parts were ground separately and subsampled for chemical analyses. For each animal within a period, DMI was measured daily and samples of feces were collected to determine diet digestibility. There were no differences in MRNE (P = 0.06) among groups. The combined data indicated a MRNE of 71.2 kcal x kg(-0.75) of EBW x d(-1), with a partial efficiency of use of ME to NE(m) of 0.71. The partial efficiency of use of ME to NE for growth was 0.54 for bulls, 0.47 for steers, and 0.54 for heifers. The GRNE for steers and heifers were similar (P = 0.15) but were 18.7% greater (P = 0.03) for steers and heifers than for bulls. The MRNP did not differ among groups and averaged 2.53 g of CP x kg(-0.75) of EBW x d(-1). Likewise, GRNP was not different among groups. The percentage of retained energy deposited as protein (RE(p)) increased as the

  12. Improving Protein Stability and Controlling Protein Release by Adding Poly (Cyclohexane-1, 4-Diyl Acetone Dimethylene Ketal) to PLGA Microspheres.

    PubMed

    Wang, Chenhui; Yu, Changhui; Yu, Kongtong; Teng, Lesheng; Liu, Jiaxin; Wang, Xuesong; Sun, Fengying; Li, Youxin

    2015-01-01

    The use of biodegradable polymers such as PLGA to encapsulate therapeutic proteins for their controlled release has received tremendous interest. However, an acidic environment caused by PLGA degradation productions leads to protein incomplete release and chemical degradation. The aim of this study was to develop novel PCADK/PLGA microspheres to improve protein stability and release behavior. Bovine serum albumin (BSA) incubated in PCADK and PLGA degradation products was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC-HPLC), circular dichroism (CD) and fluorescence spectroscopy. Blended microspheres of PCADK/PLGA were prepared in different ratios and the release behaviors of the microspheres and the protein stability were then measured. The degradation properties of the microspheres and the pH inside the microspheres were systematically investigated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to examine the mechanism of autocatalytic degradation and protein stability. BSA was more stable in the presence of PCADK monomers than it was in the presence of PLGA monomers, revealing that PCADK is highly compatible with this protein. PCADK/PLGA microspheres were successfully prepared, and 2/8 was determined to be the optimal ratio. Further, 43% of the BSA formed water-insoluble aggregates in the presence of PCADK/PLGA microspheres, compared with 57% for the PLGA microspheres, demonstrating that the BSA encapsulated in PCADK/PLGA blended microspheres was more stable than in PLGA microspheres. The PCADK/PLGA blended microspheres improved protein stability and release behavior, providing a promising protein drug delivery system.

  13. Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins.

    PubMed

    Lea, Wendy A; O'Neil, Pierce T; Machen, Alexandra J; Naik, Subhashchandra; Chaudhri, Tapan; McGinn-Straub, Wesley; Tischer, Alexander; Auton, Matthew T; Burns, Joshua R; Baldwin, Michael R; Khar, Karen R; Karanicolas, John; Fisher, Mark T

    2016-09-01

    Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy for decreasing disease pathologies caused by protein folding defects or deleterious kinetic transitions. Current methods of examining binding of a ligand to these marginally stable native states are limited because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, and multidomain proteins) and metastable proteins (e.g., low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein, immobilized on a BLI biosensor, to increasing denaturant concentrations (urea or GuHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remains is detected by an increased level of GroEL binding. Because this kinetic denaturant pulse is brief, the amplitude of binding of GroEL to the immobilized protein depends on the duration of the exposure to the denaturant, the concentration of the denaturant, wash times, and the underlying protein unfolding-refolding kinetics; fixing all other parameters and plotting the GroEL binding amplitude versus denaturant pulse concentration result in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein manifests as a decreased level of GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant

  14. Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years

    PubMed Central

    Nicosia, Aldo; Maggio, Teresa; Costa, Salvatore; Salamone, Monica; Tagliavia, Marcello; Mazzola, Salvatore; Gianguzza, Fabrizio; Cuttitta, Angela

    2016-01-01

    Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations. PMID:26957029

  15. Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years.

    PubMed

    Nicosia, Aldo; Maggio, Teresa; Costa, Salvatore; Salamone, Monica; Tagliavia, Marcello; Mazzola, Salvatore; Gianguzza, Fabrizio; Cuttitta, Angela

    2016-01-01

    Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations. PMID:26957029

  16. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    SciTech Connect

    Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.

    2015-04-15

    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.

  17. Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins.

    PubMed Central

    Gómez, Eliana B; Catlett, Michael G; Forsburg, Susan L

    2002-01-01

    The six conserved MCM proteins are essential for normal DNA replication. They share a central core of homology that contains sequences related to DNA-dependent and AAA(+) ATPases. It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity. To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast. Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex. Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences. Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin. We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits. PMID:11973289

  18. Key challenges for the creation and maintenance of specialist protein resources.

    PubMed

    Holliday, Gemma L; Bairoch, Amos; Bagos, Pantelis G; Chatonnet, Arnaud; Craik, David J; Finn, Robert D; Henrissat, Bernard; Landsman, David; Manning, Gerard; Nagano, Nozomi; O'Donovan, Claire; Pruitt, Kim D; Rawlings, Neil D; Saier, Milton; Sowdhamini, Ramanathan; Spedding, Michael; Srinivasan, Narayanaswamy; Vriend, Gert; Babbitt, Patricia C; Bateman, Alex

    2015-06-01

    As the volume of data relating to proteins increases, researchers rely more and more on the analysis of published data, thus increasing the importance of good access to these data that vary from the supplemental material of individual articles, all the way to major reference databases with professional staff and long-term funding. Specialist protein resources fill an important middle ground, providing interactive web interfaces to their databases for a focused topic or family of proteins, using specialized approaches that are not feasible in the major reference databases. Many are labors of love, run by a single lab with little or no dedicated funding and there are many challenges to building and maintaining them. This perspective arose from a meeting of several specialist protein resources and major reference databases held at the Wellcome Trust Genome Campus (Cambridge, UK) on August 11 and 12, 2014. During this meeting some common key challenges involved in creating and maintaining such resources were discussed, along with various approaches to address them. In laying out these challenges, we aim to inform users about how these issues impact our resources and illustrate ways in which our working together could enhance their accuracy, currency, and overall value.

  19. Terminin: a protein complex that mediates epigenetic maintenance of Drosophila telomeres.

    PubMed

    Raffa, Grazia D; Ciapponi, Laura; Cenci, Giovanni; Gatti, Maurizio

    2011-01-01

    In most organisms, telomeres are extended by telomerase and contain GC-rich repeats. Drosophila telomeres are elongated by occasional transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the sequence of the DNA termini. Recent work has shown that Drosophila telomeres are capped by a complex, we call terminin, which includes HOAP, HipHop, Moi and Ver; these are fast-evolving proteins that prevent telomere fusion, directly interact with each other, and appear to localize and function only at telomeres. With the possible exception of Ver that contains an OB fold domain structurally similar to the Stn1 OB fold, none of the terminin proteins is evolutionarily conserved outside the Drosophila species. Human telomeres are protected by the shelterin complex, which comprises six proteins that bind chromosome ends in a sequence-dependent manner. Shelterin subunits are not fast-evolving proteins and are not conserved in flies, but localize and function only at telomeres like the terminin components. Based on these findings, we propose that concomitant with telomerase loss Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent fashion, and that terminin is functionally analogous to shelterin. PMID:21989238

  20. Terminin: a protein complex that mediates epigenetic maintenance of Drosophila telomeres.

    PubMed

    Raffa, Grazia D; Ciapponi, Laura; Cenci, Giovanni; Gatti, Maurizio

    2011-01-01

    In most organisms, telomeres are extended by telomerase and contain GC-rich repeats. Drosophila telomeres are elongated by occasional transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the sequence of the DNA termini. Recent work has shown that Drosophila telomeres are capped by a complex, we call terminin, which includes HOAP, HipHop, Moi and Ver; these are fast-evolving proteins that prevent telomere fusion, directly interact with each other, and appear to localize and function only at telomeres. With the possible exception of Ver that contains an OB fold domain structurally similar to the Stn1 OB fold, none of the terminin proteins is evolutionarily conserved outside the Drosophila species. Human telomeres are protected by the shelterin complex, which comprises six proteins that bind chromosome ends in a sequence-dependent manner. Shelterin subunits are not fast-evolving proteins and are not conserved in flies, but localize and function only at telomeres like the terminin components. Based on these findings, we propose that concomitant with telomerase loss Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent fashion, and that terminin is functionally analogous to shelterin.

  1. Antiapoptotic protein Lifeguard is required for survival and maintenance of Purkinje and granular cells.

    PubMed

    Hurtado de Mendoza, Tatiana; Perez-Garcia, Carlos G; Kroll, Todd T; Hoong, Nien H; O'Leary, Dennis D M; Verma, Inder M

    2011-10-11

    Lifeguard (LFG) is an inhibitor of Fas-mediated cell death and is highly expressed in the cerebellum. We investigated the biological role of LFG in the cerebellum in vivo, using mice with reduced LFG expression generated by shRNA lentiviral transgenesis (shLFG mice) as well as LFG null mice. We found that LFG plays a role in cerebellar development by affecting cerebellar size, internal granular layer (IGL) thickness, and Purkinje cell (PC) development. All these features are more severe in early developmental stages and show substantial recovery overtime, providing a remarkable example of cerebellar plasticity. In adult mice, LFG plays a role in PC maintenance shown by reduced cellular density and abnormal morphology with increased active caspase 8 and caspase 3 immunostaining in shLFG and knockout (KO) PCs. We studied the mechanism of action of LFG as an inhibitor of the Fas pathway and provided evidence of the neuroprotective role of LFG in cerebellar granule neurons (CGNs) and PCs in an organotypic cerebellar culture system. Biochemical analysis of the Fas pathway revealed that LFG inhibits Fas-mediated cell death by interfering with caspase 8 activation. This result is supported by the increased number of active caspase 8-positive PCs in adult mice lacking LFG. These data demonstrate that LFG is required for proper development and survival of granular and Purkinje cells and suggest LFG may play a role in cerebellar disorders.

  2. Glycosylation May Reduce Protein Thermodynamic Stability by Inducing a Conformational Distortion.

    PubMed

    Gavrilov, Yulian; Shental-Bechor, Dalit; Greenblatt, Harry M; Levy, Yaakov

    2015-09-17

    Glycosylation plays not only a functional role but can also modify the biophysical properties of the modified protein. Usually, natural glycosylation results in protein stabilization; however, in vitro and in silico studies showed that sometimes glycosylation results in thermodynamic destabilization. Here, we applied coarse-grained and all-atom molecular dynamics simulations to understand the mechanism underlying the loss of stability of the MM1 protein by glycosylation. We show that the origin of the destabilization is a conformational distortion of the protein caused by the interaction of the monosaccharide with the protein surface. Though glycosylation creates new short-range glycan-protein interactions that stabilize the conjugated protein, it breaks long-range protein-protein interactions. This has a destabilizing effect because the probability of long- and short-range interactions forming differs between the folded and unfolded states. The destabilization originates not from simple loss of interactions but due to a trade-off between the short- and long-range interactions. PMID:26722726

  3. Documentation of an Imperative To Improve Methods for Predicting Membrane Protein Stability.

    PubMed

    Kroncke, Brett M; Duran, Amanda M; Mendenhall, Jeffrey L; Meiler, Jens; Blume, Jeffrey D; Sanders, Charles R

    2016-09-13

    There is a compelling and growing need to accurately predict the impact of amino acid mutations on protein stability for problems in personalized medicine and other applications. Here the ability of 10 computational tools to accurately predict mutation-induced perturbation of folding stability (ΔΔG) for membrane proteins of known structure was assessed. All methods for predicting ΔΔG values performed significantly worse when applied to membrane proteins than when applied to soluble proteins, yielding estimated concordance, Pearson, and Spearman correlation coefficients of <0.4 for membrane proteins. Rosetta and PROVEAN showed a modest ability to classify mutations as destabilizing (ΔΔG < -0.5 kcal/mol), with a 7 in 10 chance of correctly discriminating a randomly chosen destabilizing variant from a randomly chosen stabilizing variant. However, even this performance is significantly worse than for soluble proteins. This study highlights the need for further development of reliable and reproducible methods for predicting thermodynamic folding stability in membrane proteins. PMID:27564391

  4. Prediction of salt effects on protein phase behavior by HIC retention and thermal stability.

    PubMed

    Baumgartner, Kai; Großhans, Steffen; Schütz, Juliane; Suhm, Susanna; Hubbuch, Jürgen

    2016-09-01

    In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step. Unwanted protein precipitation or crystallization can lead to column, pipe or filter blocking. In formulation, the formation of aggregates can even be lethal when injected into the patient. The typical methodology to illustrate protein phase states is the generation of protein phase diagrams. Commonly, protein phase behavior is shown in dependence of protein and precipitant concentration. Despite using high-throughput methods for the generation of phase diagrams, the time necessary to reach equilibrium is the bottleneck. Faster methods to predict protein phase behavior are desirable. In this study, hydrophobic interaction chromatography retention times were correlated to crystal size and form. High-throughput thermal stability measurements (melting and aggregation temperatures), using an Optim(®)2 system, were successfully correlated to glucose isomerase stability. By using hydrophobic interaction chromatography and thermal stability determinations, glucose isomerase conformational and colloidal stability were successfully predicted for different salts in a specific pH range. PMID:27268946

  5. Protein Stability and Dynamics Modulation: The Case of Human Frataxin

    PubMed Central

    Gallo, Mariana; Salvay, Andres G.; Ferreiro, Diego U.; Santos, Javier

    2012-01-01

    Frataxin (FXN) is an α/β protein that plays an essential role in iron homeostasis. Apparently, the function of human FXN (hFXN) depends on the cooperative formation of crucial interactions between helix α1, helix α2, and the C-terminal region (CTR) of the protein. In this work we quantitatively explore these relationships using a purified recombinant fragment hFXN90–195. This variant shows the hydrodynamic behavior expected for a monomeric globular domain. Circular dichroism, fluorescence, and NMR spectroscopies show that hFXN90–195 presents native-like secondary and tertiary structure. However, chemical and temperature induced denaturation show that CTR truncation significantly destabilizes the overall hFXN fold. Accordingly, limited proteolysis experiments suggest that the native-state dynamics of hFXN90–195 and hFXN90–210 are indeed different, being the former form much more sensitive to the protease at specific sites. The overall folding dynamics of hFXN fold was further explored with structure-based protein folding simulations. These suggest that the native ensemble of hFXN can be decomposed in at least two substates, one with consolidation of the CTR and the other without consolidation of the CTR. Explicit-solvent all atom simulations identify some of the proteolytic target sites as flexible regions of the protein. We propose that the local unfolding of CTR may be a critical step for the global unfolding of hFXN, and that modulation of the CTR interactions may strongly affect hFXN physiological function. PMID:23049850

  6. Identification of salivary proteins at oil-water interfaces stabilized by lysozyme and beta-lactoglobulin.

    PubMed

    Silletti, Erika; Vitorino, Rui M P; Schipper, Raymond; Amado, Francisco M L; Vingerhoeds, Monique H

    2010-04-01

    In this research, we investigated the interaction occurring between oil-in-water emulsion droplets, stabilized by different emulsifiers, i.e. lysozyme and beta-lactoglobulin (beta-lg), and salivary proteins (SPs) with a molecular mass (M(r)) above about 10kDa. Different techniques, i.e. infrared spectroscopy, Western blotting, PAS staining and SDS-PAGE coupled to MS, were employed for this purpose. This study demonstrated the interaction between several salivary proteins and the emulsifiers at the oil-water interfaces. In particular, results show that the high M(r) mucin MUC5B was strongly bound to lysozyme stabilized emulsions, whereas beta-lg stabilized emulsions associated with MUC7 and, moderately, with MUC5B. Furthermore, we observed that salivary proteins in the range M(r) 10-100kDa associated differently with emulsion droplets. A large majority of SPs was found to interact with lysozyme stabilized emulsion droplets whilst in case of beta-lg stabilized emulsions, the SPs distribute more evenly between the fraction associated and non-associated with the droplets. A clear example is alpha-amylase (M(r) approximately 55kDa) which predominantly associates with lysozyme stabilized emulsion droplets, but not with beta-lg emulsion droplets. To conclude, our findings indicate that adsorption/association of salivary protein components onto the emulsion droplets is related to the type of emulsifying proteins at the oil-water interfaces and it is probably driven by the overall net charge at the droplet's oil-water interfaces, i.e. positive for lysozyme stabilized emulsions and negative for beta-lactoglobulin stabilized emulsion at neutral pH.

  7. Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability.

    PubMed

    Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P; Takeda, Makoto

    2016-01-01

    Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. PMID:27490564

  8. Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability

    PubMed Central

    Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P.; Takeda, Makoto

    2016-01-01

    Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. PMID:27490564

  9. Role of solvation in the energy stabilization inside the hydrophobic core of the protein rubredoxin.

    PubMed

    Riley, Kevin E; Merz, Kenneth M

    2006-08-17

    There are many forces that contribute to the stability of a protein; among these are dispersion interactions, hydrogen bonding, and solvation effects. In a recent work, Vondrasek et al. estimated the in vacuo stabilization energy of the hydrophobic core of the protein rubredoxin using high level ab initio methods (Vondrasek, J.; et al. J. Am. Chem. Soc. 2005, 127, 2615). In this work, we evaluate the effects of solvation on the stability of the hydrophobic core of this protein. Solvation calculations are made using the polarizable continuum method at the MP2/aug-cc-pVDZ level of theory. It is found that, in a protein-like environment (mimicked by a continuum solvent with a dielectric constant of approximately 4), the stability of rubredoxin's hydrophobic core is decreased by 40-50%. We also observed that the stabilization energy of the hydrophobic core is only slightly lower in a protein-like medium than in an aqueous one (DeltaGether-DeltaGwater approximately 1.0-3.5 kcal/mol).

  10. Biophysical stability of hyFc fusion protein with regards to buffers and various excipients.

    PubMed

    Lim, Jun Yeul; Kim, Nam Ah; Lim, Dae Gon; Eun, Chang-yong; Choi, Donghoon; Jeong, Seong Hoon

    2016-05-01

    A novel non-cytolytic hybrid Fc (hyFc) with an intact Ig structure without any mutation in the hyFc region, was developed to construct a long-acting agonistic protein. The stability of interleukin-7 (IL-7) fused with the hyFc (GXN-04) was evaluated to develop early formulations. Various biophysical methods were utilized and three different buffer systems with various pH ranges were investigated including histidine-acetate, sodium citrate, and tris buffers. Various excipients were incorporated into the systems to obtain optimum protein stability. Two evident thermal transitions were observed with the unfolding of IL-7 and hyFc. The Tm and ΔH increased with pH, suggesting increased conformational stability. Increased Z-average size with PDI and decreased zeta potential with pH increase, with the exception of tris buffer, showed aggregation issues. Moreover, tris buffer at higher pH showed aggregation peaks from DLS. Non-ionic surfactants were effective against agitation by outcompeting protein molecules for hydrophobic surfaces. Sucrose and sorbitol accelerated protein aggregation during agitation, but exhibited a protective effect against oxidation, with preferential exclusion favoring the compact states of GXN-04. The stability of GXN-04 was varied by basal buffers and excipients, hence the buffers and excipients need to be evaluated carefully to achieve the maximum stability of proteins. PMID:26851357

  11. CEP152 is a genome maintenance protein disrupted in Seckel syndrome.

    PubMed

    Kalay, Ersan; Yigit, Gökhan; Aslan, Yakup; Brown, Karen E; Pohl, Esther; Bicknell, Louise S; Kayserili, Hülya; Li, Yun; Tüysüz, Beyhan; Nürnberg, Gudrun; Kiess, Wieland; Koegl, Manfred; Baessmann, Ingelore; Buruk, Kurtulus; Toraman, Bayram; Kayipmaz, Saadettin; Kul, Sibel; Ikbal, Mevlit; Turner, Daniel J; Taylor, Martin S; Aerts, Jan; Scott, Carol; Milstein, Karen; Dollfus, Helene; Wieczorek, Dagmar; Brunner, Han G; Hurles, Matthew; Jackson, Andrew P; Rauch, Anita; Nürnberg, Peter; Karagüzel, Ahmet; Wollnik, Bernd

    2011-01-01

    Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation. PMID:21131973

  12. The influence of the maintenance of terraced areas on slope stability during the November 2014 flood event in Liguria (northwestern Italy)

    NASA Astrophysics Data System (ADS)

    Giordan, Daniele; Poggi, Flavio; Baldo, Marco; Cignetti, Martina

    2016-04-01

    Terraced environments are a widespread feature of the coastal settlement of eastern Liguria (northwestern Italy) and they constitute a well-known favorable role in slope stability. In this region, starting from the twentieth century, the progressive abandonment of agriculture determines a progressively increasing lack of maintenance of the terraces, consequently raising the level of slope instability. Moreover, it should be taken into account not only the level of terraces maintenance, but also their interaction with several factors as i) geological and geomorphological conditions, ii) soil properties, iii) hydrological and hydrogeological conditions, and iv) land use, causing an increase in landslides occurrence. The definition of managed terraces effects on slope stability and their response to external stress like a flood event is rather complicated; a possible approach is a statistical analysis of the effects of a flood event over a large terraced area, distinguishing the maintained sectors from the abandoned ones. After the November 2014 flood event, which affected several sectors of the Liguria region, where a high number of shallow landslides were triggered, an airborne LiDAR survey of the damaged area was carried out. In particular, a high resolution Digital Terrain Model (DTM) resampled to a lower density (1 square meter grid spacing) and a photogrammetric coverage of the area was performed, in order to create a landslide map of the flood event. The surveyed area covered more than 380 square kilometers, and over 1600 shallow landslides triggered by the flood event were identified and inventoried. The high resolution DTM allowed the identification of terraced areas also in wooded sectors and a sharp mapping of the extension of terraced slopes in this portion of Liguria region. By considering: i) the terraced areas recognized through DTM analysis, ii) the mapped landslides, and iii) the land use classification, a correlation between the presence of terraces

  13. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker

    NASA Astrophysics Data System (ADS)

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-03-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a `fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures.

  14. Stabilization of charges on isolated ionic groups sequestered in proteins by polarized peptide units.

    PubMed

    Quiocho, F A; Sack, J S; Vyas, N K

    Electrostatic interactions are of considerable importance in protein structure and function, and in a variety of cellular and biochemical processes. Here we report three similar findings from highly refined atomic structures of periplasmic binding proteins. Hydrogen bonds, acting primarily through backbone peptide units, are mainly responsible for the involvement of the positively charged arginine 151 residue in the ligand site of the arabinose-binding protein, for the association between teh sulphate-binding protein and the completely buried sulphate dianion, and for the formation of the complex of the leucine/isoleucine/valine-binding protein with the leucine zwitterion. We propose a general mechanism in which the isolated charges on the various buried, desolvated ionic groups are stabilized by the polarized peptide units. This mechanism also has broad application to processes requiring binding of uncompensated ions and charged ligands and stabilization of enzyme reaction charged intermediates, as well as activation of catalytic residues.

  15. Preparation of iron bound succinylated milk protein concentrate and evaluation of its stability.

    PubMed

    Shilpashree, B G; Arora, Sumit; Sharma, Vivek; Bajaj, Rajesh Kumar; Tomar, S K

    2016-04-01

    Major problems associated with the fortification of soluble iron salts include chemical reactivity and incompatibility with other components. Milk protein concentrate (MPC) are able to bind significant amount of iron due to the presence of both casein and whey protein. MPC in its native state possess very poor solubility, therefore, succinylated derivatives of MPC (succ. MPC) were also used for the preparation of protein-iron complex. Preparation of the complex involved centrifugation (to remove insoluble iron), ultrafiltration (to remove unbound iron) and lyophilisation (to attain in dry form). Iron binding ability of MPC enhanced significantly (P<0.05) upon succinylation. Stability of bound iron from both varieties of complexes was monitored under different conditions encountered during processing. Higher stability (P<0.05) of bound iron was observed in succ. MPC-iron complex than native protein complex. This method could be adopted for the production of stable iron enriched protein, an organic iron source. PMID:26593557

  16. Preparation of iron bound succinylated milk protein concentrate and evaluation of its stability.

    PubMed

    Shilpashree, B G; Arora, Sumit; Sharma, Vivek; Bajaj, Rajesh Kumar; Tomar, S K

    2016-04-01

    Major problems associated with the fortification of soluble iron salts include chemical reactivity and incompatibility with other components. Milk protein concentrate (MPC) are able to bind significant amount of iron due to the presence of both casein and whey protein. MPC in its native state possess very poor solubility, therefore, succinylated derivatives of MPC (succ. MPC) were also used for the preparation of protein-iron complex. Preparation of the complex involved centrifugation (to remove insoluble iron), ultrafiltration (to remove unbound iron) and lyophilisation (to attain in dry form). Iron binding ability of MPC enhanced significantly (P<0.05) upon succinylation. Stability of bound iron from both varieties of complexes was monitored under different conditions encountered during processing. Higher stability (P<0.05) of bound iron was observed in succ. MPC-iron complex than native protein complex. This method could be adopted for the production of stable iron enriched protein, an organic iron source.

  17. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker

    PubMed Central

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-01-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a ‘fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures. PMID:26980593

  18. TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

    NASA Astrophysics Data System (ADS)

    Feller, Georges

    2010-08-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  19. SIRT6 stabilizes DNA-dependent Protein Kinase at chromatin for DNA double-strand break repair

    PubMed Central

    McCord, Ronald A.; Michishita, Eriko; Hong, Tao; Berber, Elisabeth; Boxer, Lisa D.; Kusumoto, Rika; Guan, Shenheng; Shi, Xiaobing; Gozani, Or; Burlingame, Alma L.; Bohr, Vilhelm A.; Chua, Katrin F.

    2009-01-01

    The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor with chromatin impacts on the efficiency of repair, and establish a link between chromatin regulation, DNA repair, and a mammalian Sir2 factor. PMID:20157594

  20. Synthesis and characterization of dendro-PLGA nanoconjugate for protein stabilization.

    PubMed

    Tiwari, Amit; Kesharwani, Prashant; Gajbhiye, Virendra; Jain, Narendra K

    2015-10-01

    The present investigation was aimed to develop the dendro-PLGA nanoconjugate (DPNC) for protection of insulin from degradation as well as its sustained release from nano-formulation. DPNC formulation was synthesized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) coupling reaction and therapeutic efficacy of encapsulated protein (insulin) was measured by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared spectroscopy (FTIR) and circular dichorism (CD) spectroscopy. We have also demonstrated the ability of DPNC formulation to prevent the native conformation of insulin within the system by comparing the amount of free protein with the protein extracted from this system. Stability study further revealed that as compared to free protein, DPNC formulation was more efficient to stabilize the protein. Additionally, in vivo data of protein loaded system in rats showed that DPNC formulation can able to maintain the native structure of insulin and hence retain therapeutic efficacy of protein. The novel dendro-PLGA nanoconjugate not only stabilize the insulin but also work as sustained release reservoir for insulin which reduces the frequency of dosage and side effect associate with denatured protein. PMID:26209778

  1. Probing Bio-Nano Interactions between Blood Proteins and Monolayer-Stabilized Graphene Sheets.

    PubMed

    Gan, Shiyu; Zhong, Lijie; Han, Dongxue; Niu, Li; Chi, Qijin

    2015-11-18

    Meeting proteins is regarded as the starting event for nanostructures to enter biological systems. Understanding their interactions is thus essential for a newly emerging field, nanomedicine. Chemically converted graphene (CCG) is a wonderful two-dimensional (2D) material for nanomedicine, but its stability in biological environments is limited. Systematic probing on the binding of proteins to CCG is currently lacking. Herein, we report a comprehensive study on the interactions between blood proteins and stabilized CCG (sCCG). CCG nanosheets are functionalized by monolayers of perylene leading to significant improvement in their resistance to electrolyte salts and long-term stability, but retain their core structural characteristics. Five types of model human blood proteins including human fibrinogen, γ-globulin, bovine serum albumin (BSA), insulin, and histone are tested. The main driving forces for blood protein binding involve the π-π interacations between the π-plane of sCCG and surface aromatic amonic acid (sAA) residues of proteins. Several key binding parameters including the binding amount, Hill coefficient, and binding constant are determined. Through a detailed analysis of key controlling factors, we conclude that the protein binding to sCCG is determined mainly by the protein size, the number, and the density of the sAA.

  2. Nutritional Status of Maintenance Dialysis Patients: Low Lean Body Mass Index and Obesity Are Common, Protein-Energy Wasting Is Uncommon

    PubMed Central

    Koefoed, Mette; Kromann, Charles Boy; Juliussen, Sophie Ryberg; Hvidtfeldt, Danni; Ekelund, Bo; Frandsen, Niels Erik; Marckmann, Peter

    2016-01-01

    Background and Aims Maintenance dialysis patients are at increased risk of abnormal nutritional status due to numerous causative factors, both nutritional and non-nutritional. The present study assessed the current prevalence of protein-energy wasting, low lean body mass index and obesity in maintenance dialysis patients, and compared different methods of nutritional assessment. Methods In a cross-sectional study conducted in 2014 at Roskilde Hospital, Denmark, we performed anthropometry (body weight, skinfolds, mid-arm, waist, and hip circumferences), and determined plasma albumin and normalized protein catabolic rate in order to assess the prevalence of protein-energy wasting, low lean body mass index and obesity in these patients. Results Seventy-nine eligible maintenance dialysis patients participated. The prevalence of protein-energy wasted patients was 4% (95% CI: 2–12) as assessed by the coexistence of low lean body mass index and low fat mass index. Low lean body mass index was seen in 32% (95% CI: 22–44). Obesity prevalence as assessed from fat mass index was 43% (95% CI: 32–55). Coexistence of low lean body mass index and obesity was seen in 10% (95% CI: 5–19). The prevalence of protein-energy wasting and obesity varied considerably, depending on nutritional assessment methodology. Conclusions Our data indicate that protein-energy wasting is uncommon, whereas low lean body mass index and obesity are frequent conditions among patients in maintenance dialysis. A focus on how to increase and preserve lean body mass in dialysis patients is suggested in the future. In order to clearly distinguish between shortage, sufficiency and abundance of protein and/or fat deposits in maintenance dialysis patients, we suggest the simple measurements of lean body mass index and fat mass index. PMID:26919440

  3. Applications of differential scanning calorimetry for thermal stability analysis of proteins: qualification of DSC.

    PubMed

    Wen, Jie; Arthur, Kelly; Chemmalil, Letha; Muzammil, Salman; Gabrielson, John; Jiang, Yijia

    2012-03-01

    Differential scanning calorimetry (DSC) has been used to characterize protein thermal stability, overall conformation, and domain folding integrity by the biopharmaceutical industry. Recently, there have been increased requests from regulatory agencies for the qualification of characterization methods including DSC. Understanding the method precision can help determine what differences between samples are significant and also establish the acceptance criteria for comparability and other characterization studies. In this study, we identify the parameters for the qualification of DSC for thermal stability analysis of proteins. We use these parameters to assess the precision and sensitivity of DSC and demonstrate that DSC is suitable for protein thermal stability analysis for these purposes. Several molecules from different structural families were studied. The experiments and data analyses were performed by different analysts using different instruments at different sites. The results show that the (apparent) thermal transition midpoint (T(m)) values obtained for the same protein by same and different instruments and/or analysts are quite reproducible, and the profile similarity values obtained for the same protein from the same instrument are also high. DSC is an appropriate method for assessing protein thermal stability and conformational changes.

  4. Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm

    PubMed Central

    Moon, C. Preston; Zaccai, Nathan R.; Fleming, Patrick J.; Gessmann, Dennis; Fleming, Karen G.

    2013-01-01

    Thermodynamic stabilities are pivotal for understanding structure–function relationships of proteins, and yet such determinations are rare for membrane proteins. Moreover, the few measurements that are available have been conducted under very different experimental conditions, which compromises a straightforward extraction of physical principles underlying stability differences. Here, we have overcome this obstacle and provided structure–stability comparisons for multiple membrane proteins. This was enabled by measurements of the free energies of folding and the m values for the transmembrane proteins PhoP/PhoQ-activated gene product (PagP) and outer membrane protein W (OmpW) from Escherichia coli. Our data were collected in the same lipid bilayer and buffer system we previously used to determine those parameters for E. coli outer membrane phospholipase A (OmpLA). Biophysically, our results suggest that the stabilities of these proteins are strongly correlated to the water-to-bilayer transfer free energy of the lipid-facing residues in their transmembrane regions. We further discovered that the sensitivities of these membrane proteins to chemical denaturation, as judged by their m values, was consistent with that previously observed for water-soluble proteins having comparable differences in solvent exposure between their folded and unfolded states. From a biological perspective, our findings suggest that the folding free energies for these membrane proteins may be the thermodynamic sink that establishes an energy gradient across the periplasm, thus driving their sorting by chaperones to the outer membranes in living bacteria. Binding free energies of these outer membrane proteins with periplasmic chaperones support this energy sink hypothesis. PMID:23440211

  5. Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm.

    PubMed

    Moon, C Preston; Zaccai, Nathan R; Fleming, Patrick J; Gessmann, Dennis; Fleming, Karen G

    2013-03-12

    Thermodynamic stabilities are pivotal for understanding structure-function relationships of proteins, and yet such determinations are rare for membrane proteins. Moreover, the few measurements that are available have been conducted under very different experimental conditions, which compromises a straightforward extraction of physical principles underlying stability differences. Here, we have overcome this obstacle and provided structure-stability comparisons for multiple membrane proteins. This was enabled by measurements of the free energies of folding and the m values for the transmembrane proteins PhoP/PhoQ-activated gene product (PagP) and outer membrane protein W (OmpW) from Escherichia coli. Our data were collected in the same lipid bilayer and buffer system we previously used to determine those parameters for E. coli outer membrane phospholipase A (OmpLA). Biophysically, our results suggest that the stabilities of these proteins are strongly correlated to the water-to-bilayer transfer free energy of the lipid-facing residues in their transmembrane regions. We further discovered that the sensitivities of these membrane proteins to chemical denaturation, as judged by their m values, was consistent with that previously observed for water-soluble proteins having comparable differences in solvent exposure between their folded and unfolded states. From a biological perspective, our findings suggest that the folding free energies for these membrane proteins may be the thermodynamic sink that establishes an energy gradient across the periplasm, thus driving their sorting by chaperones to the outer membranes in living bacteria. Binding free energies of these outer membrane proteins with periplasmic chaperones support this energy sink hypothesis.

  6. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    NASA Technical Reports Server (NTRS)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  7. Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein.

    PubMed

    Tych, Katarzyna M; Batchelor, Matthew; Hoffmann, Toni; Wilson, Michael C; Hughes, Megan L; Paci, Emanuele; Brockwell, David J; Dougan, Lorna

    2016-07-26

    Proteins from organisms that have adapted to environmental extremes provide attractive systems to explore and determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single-molecule force spectroscopy (SMFS), and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal β-strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (ΔGD-N) of the mutated proteins and show that there is a more significant reduction for TmCSP L40A (ΔΔG = 63%) than TmCSP V62A (ΔΔG = 47%), as might be expected on the basis of the relative reduction in the size of the side chain. By contrast, SMFS measures the mechanical stability (ΔG*) and shows a greater reduction for TmCSP V62A (ΔΔG* = 8.4%) than TmCSP L40A (ΔΔG* = 2.5%). While the impact on the mechanical stability is subtle, the results demonstrate the power of tuning noncovalent interactions to modulate both the thermodynamic and mechanical stability of a protein. Such understanding and control provide the opportunity to design proteins with optimized thermodynamic and mechanical properties. PMID:27338140

  8. The protein hSnm1B is stabilized when bound to the telomere-binding protein TRF2.

    PubMed

    Freibaum, Brian D; Counter, Christopher M

    2008-08-29

    hSnm1B is member of the SNM family of exonucleases involved in DNA processing and is known to be localized to telomeres via binding to the telomere-binding protein TRF2. Here we demonstrate that the C terminus of hSnm1B facilitates the concentration of hSnm1B on telomeres by promoting ubiquitin-mediated degradation of hSnm1B that is not localized to telomeres, as well as by blocking protein degradation and fostering localization to telomeres via binding of TRF2. Finally, a mutant of hSnm1B stabilized independently of exogenous TRF2-induced cell death. Taken together, we speculate that sequestering hSnm1B at telomeres by a combination of stabilizing the protein when bound to telomeres and degrading it when not bound to telomeres may be a means to prevent potentially lethal effects of unregulated hSnm1B activity.

  9. Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

    PubMed Central

    Keefe, Andrew J.; Jiang, Shaoyi

    2013-01-01

    Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein–substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity. PMID:22169873

  10. High-throughput thermal scanning for protein stability: making a good technique more robust.

    PubMed

    Seabrook, Shane A; Newman, Janet

    2013-08-12

    We present a high-throughput approach to help define experimental formulations that enhance protein stability, which is based on differential scanning fluorimetry (DSF). The method involves defining the thermal stability of a protein against a screen of 13 buffer systems, systematically sampling pH from 5.0 to 9.0 at high and low salt concentrations, using both redundancy and extensive controls to make the method robust. The screen allows rapid determination of a suitable base formulation for protein samples, and is particularly useful for difficult samples: those that are rapidly degraded or cannot be sufficiently concentrated for downstream analyses. Data obtained from three samples in this assay illustrate the vastly different values for thermal stability that can be obtained from different formulations. This approach is simple to interpret and reliable enough that it has been implemented as a service through the Collaborative Crystallisation Centre (C3). PMID:23710551

  11. Effects of selective exercise for the deep abdominal muscles and lumbar stabilization exercise on the thickness of the transversus abdominis and postural maintenance

    PubMed Central

    Lee, Jung-seok; Kim, Tae-ho; Kim, Da-yeon; Shim, Jae-ho; Lim, Jin-yong

    2015-01-01

    [Purpose] The purpose of this study was to examine the effects of selective exercise for the deep abdominal muscles (SEDA) and lumbar stabilization exercise (LSE) on the thickness of the transversus abdominis and postural maintenance on an unstable base of support. [Subjects and Methods] The subjects of this study were 20 male and 10 female adults in their 20s without lumbar pain. They were equally and randomly assigned to a SEDA group and a LSE group. The thickness of the transversus abdominis was measured using ultrasound imaging during rest and drawing-in. The thickness of the transversus abdominis was measured when subjects raised their right and left legs while lying on a Swiss ball. [Results] Initially, there were no differences between the two groups. After the intervention, significant differences were observed in all parameters. A significant interaction between group and period was not found for any parameters. [Conclusion] In conclusion, both SEDA and LSE thickened the transversus abdominis, which is a deep abdominal muscle, thereby adjusting posture, and stabilizing the trunk. These exercises increased the thickness of the deep abdominal muscles. They are important exercises for improving the stability of athletes or patients who need postural adjustment. PMID:25729169

  12. Effects of selective exercise for the deep abdominal muscles and lumbar stabilization exercise on the thickness of the transversus abdominis and postural maintenance.

    PubMed

    Lee, Jung-Seok; Kim, Tae-Ho; Kim, Da-Yeon; Shim, Jae-Ho; Lim, Jin-Yong

    2015-02-01

    [Purpose] The purpose of this study was to examine the effects of selective exercise for the deep abdominal muscles (SEDA) and lumbar stabilization exercise (LSE) on the thickness of the transversus abdominis and postural maintenance on an unstable base of support. [Subjects and Methods] The subjects of this study were 20 male and 10 female adults in their 20s without lumbar pain. They were equally and randomly assigned to a SEDA group and a LSE group. The thickness of the transversus abdominis was measured using ultrasound imaging during rest and drawing-in. The thickness of the transversus abdominis was measured when subjects raised their right and left legs while lying on a Swiss ball. [Results] Initially, there were no differences between the two groups. After the intervention, significant differences were observed in all parameters. A significant interaction between group and period was not found for any parameters. [Conclusion] In conclusion, both SEDA and LSE thickened the transversus abdominis, which is a deep abdominal muscle, thereby adjusting posture, and stabilizing the trunk. These exercises increased the thickness of the deep abdominal muscles. They are important exercises for improving the stability of athletes or patients who need postural adjustment.

  13. Importance of surrounding residues for protein stability of partially buried mutations.

    PubMed

    Gromiha, M M; Oobatake, M; Kono, H; Uedaira, H; Sarai, A

    2000-10-01

    For understanding the factors influencing protein stability, we have analyzed the relationship between changes in protein stability caused by partially buried mutations and changes in 48 physico-chemical, energetic and conformational properties of amino acid residues. Multiple regression equations were derived to predict the stability of protein mutants and the efficiency of the method has been verified with both back-check and jack-knife tests. We observed a good agreement between experimental and computed stabilities. Further, we have analyzed the effect of sequence window length from 1 to 12 residues on each side of the mutated residue to include the sequence information for predicting protein stability and we found that the preferred window length for obtaining the highest correlation is different for each secondary structure; the preferred window length for helical, strand and coil mutations are, respectively, 0, 9 and 4 residues on both sides of the mutant residues. However, all the secondary structures have significant correlation for a window length of one residue on each side of the mutant position, implying the role of short-range interactions. Extraction of surrounding residue information for various distances (3 to 20A) around the mutant position showed the highest correlation at 8A, 6A and 7A, respectively, for mutations in helical, strand and coil segments. Overall, the information about the surrounding residues within the sphere of 7 to 8A, may explain better the stability in all subsets of partially buried mutations implying that this distance is sufficient to accommodate the residues influenced by major intramolecular interactions for the stability of protein structures. PMID:11089649

  14. Npl3, a new link between RNA-binding proteins and the maintenance of genome integrity

    PubMed Central

    Santos-Pereira, José M; Herrero, Ana B; Moreno, Sergio; Aguilera, Andrés

    2014-01-01

    The mRNA is co-transcriptionally bound by a number of RNA-binding proteins (RBPs) that contribute to its processing and formation of an export-competent messenger ribonucleoprotein particle (mRNP). In the last few years, increasing evidence suggests that RBPs play a key role in preventing transcription-associated genome instability. Part of this instability is mediated by the accumulation of co-transcriptional R loops, which may impair replication fork (RF) progression due to collisions between transcription and replication machineries. In addition, some RBPs have been implicated in DNA repair and/or the DNA damage response (DDR). Recently, the Npl3 protein, one of the most abundant heterogeneous nuclear ribonucleoproteins (hnRNPs) in yeast, has been shown to prevent transcription-associated genome instability and accumulation of RF obstacles, partially associated with R-loop formation. Interestingly, Npl3 seems to have additional functions in DNA repair, and npl3∆ mutants are highly sensitive to genotoxic agents, such as the antitumor drug trabectedin. Here we discuss the role of Npl3 in particular, and RBPs in general, in the connection of transcription with replication and genome instability, and its effect on the DDR. PMID:24694687

  15. Nonlinear Model of the Specificity of DNA-Protein Interactions and Its Stability

    NASA Astrophysics Data System (ADS)

    Dwiputra, D.; Hidayat, W.; Khairani, R.; Zen, F. P.

    2016-08-01

    Specific DNA-protein interactions are fundamental processes of living cells. We propose a new model of DNA-protein interactions to explain the site specificity of the interactions. The hydrogen bonds between DNA base pairs and between DNA-protein peptide groups play a significant role in determination of the specific binding site. We adopt the Morse potential with coupling terms to construct the Hamiltonian of coupled oscillators representing the hydrogen bonds in which the depth of the potentials vary in the DNA chain. In this paper we investigate the stability of the model to determine the conditions satisfying the biological circumstances of the DNA-protein interactions.

  16. Coassembly of Tobacco Mosaic Virus Coat Proteins into Nanotubes with Uniform Length and Improved Physical Stability.

    PubMed

    Zhou, Kun; Eiben, Sabine; Wang, Qiangbin

    2016-06-01

    Using tobacco mosaic virus coat proteins (TMVcp) from both sources of the plant and bacterial expression systems as building blocks, we demonstrate here a coassembly strategy of TMV nanotubes in the presence of RNA. Specifically, plant-expressed cp (cpp) efficiently dominates the genomic RNA encapsidation to determine the length of assembled TMV nanotubes, whereas the incorporated Escherichia coli-expressed cp (cpec) improves the physical stability of TMV nanotubes by introducing disulfide bonds between the interfaces of subunits. We expect this coassembly strategy can be expanded to other virus nanomaterials to obtain desired properties based on rationally designed protein-RNA and protein-protein interfacial interactions. PMID:27188634

  17. Stability of Silk and Collagen Protein Materials in Space

    PubMed Central

    Hu, Xiao; Raja, Waseem K.; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L.

    2013-01-01

    Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered. PMID:24305951

  18. Beta-Barrel Scaffold of Fluorescent Proteins: Folding, Stability and Role in Chromophore Formation

    PubMed Central

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K.

    2013-01-01

    This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal helix. The chromophore originates from the polypeptide chain and its properties are influenced by the surrounding protein matrix of the β-barrel. On the other hand, it appears that a chromophore tightens the β-barrel scaffold and plays a crucial role in its stability. Furthermore, the presence of a mature chromophore causes hysteresis of protein unfolding and refolding. We survey studies measuring protein unfolding and refolding using traditional methods as well as new approaches, such as mechanical unfolding and reassembly of truncated fluorescent proteins. We also analyze models of fluorescent protein unfolding and refolding obtained through different approaches, and compare the results of protein folding in vitro to co-translational folding of a newly synthesized polypeptide chain. PMID:23351712

  19. Natural constraints, folding, motion, and structural stability in transmembrane helical proteins

    NASA Astrophysics Data System (ADS)

    Harrington, Susan E.; Ben-Tal, Nir

    Transmembrane (TM) helical proteins are of fundamental importance in many diverse biological processes. To understand these proteins functionally, it is necessary to characterize the forces that stabilize them. What are these forces (both within the protein itself and between the protein and membrane) and how do they give rise to the multiple conformational states and complex activity of TM helical proteins? How do they act in concert to fold TM helical proteins, create their low-energy stable states, and guide their motion? These central questions have led to the description of critical natural constraints and partial answers, which we will review. We will then describe how these constraints can be tracked through homologs and proteins of similar folds in order to better understand how amino acid sequence can specify structure and guide motion. Our emphasis throughout will be on structural features of TM helix bundles themselves, but we will also sketch the membrane-related aspects of these questions.

  20. Surface forces in model oil-in-water emulsions stabilized by proteins.

    PubMed

    Dimitrova, Tatiana D; Leal-Calderon, Fernando; Gurkov, Theodor D; Campbell, Bruce

    2004-05-20

    We have employed two complementary techniques, namely, the magnetic chaining technique (MCT) and a variant of the Mysels cell to obtain data concerning the repulsive interaction profiles between protein layers formed at liquid-liquid interfaces. For BSA-stabilized systems, a long-ranged repulsion is operative. It is not of an electrostatic origin, but originates most probably from the formation of multiple protein layers at the interface. The interactions between beta-casein layers formed at the water/oil interface are governed by electrostatic repulsion. Due to the relatively large final thickness of approximately 20 nm, the van der Waals contribution to the total disjoining pressure is inferior. The oscillatory component is also negligible for the studied protein concentration of 0.1 wt.%. For both proteins, the extracted information describes the situation where the protein-covered surfaces are approached/manipulated in a quasi-static manner. We observe a very good agreement between the data obtained from MCT and Mysels cell. The comparison of our results with literature data from surface force apparatus (SFA) experiments reveals a substantial difference in the force laws existing between protein-stabilized liquid droplets and mica surfaces covered by proteins. We explain this discrepancy in terms of the different protein absorption on solid and liquid interfaces. We also measured the threshold force necessary to induce irreversible flocculation in beta-casein and beta-lactoglobulin (BLG) stabilized emulsions. Under similar conditions, the threshold flocculation force is higher for beta-casein than for BLG stabilized droplets. The flocs formed from BLG covered droplets are tight and remain without visible change for at least 48 h. We speculate that the flocculation is due to formation of protein aggregates between the approaching droplets. PMID:15072930

  1. Salt Effects on the Conformational Stability of the Visual G-Protein-Coupled Receptor Rhodopsin

    PubMed Central

    Reyes-Alcaraz, Arfaxad; Martínez-Archundia, Marlet; Ramon, Eva; Garriga, Pere

    2011-01-01

    Membrane protein stability is a key parameter with important physiological and practical implications. Inorganic salts affect protein stability, but the mechanisms of their interactions with membrane proteins are not completely understood. We have undertaken the study of a prototypical G-protein-coupled receptor, the α-helical membrane protein rhodopsin from vertebrate retina, and explored the effects of inorganic salts on the thermal decay properties of both its inactive and photoactivated states. Under high salt concentrations, rhodopsin significantly increased its activation enthalpy change for thermal bleaching, whereas acid denaturation affected the formation of a denatured loose-bundle state for both the active and inactive conformations. This behavior seems to correlate with changes in protonated Schiff-base hydrolysis. However, chromophore regeneration with the 11-cis-retinal chromophore and MetarhodopsinII decay kinetics were slower only in the presence of sodium chloride, suggesting that in this case, the underlying phenomenon may be linked to the activation of rhodopsin and the retinal release processes. Furthermore, the melting temperature, determined by means of circular dichroism and differential scanning calorimetry measurements, was increased in the presence of high salt concentrations. The observed effects on rhodopsin could indicate that salts favor electrostatic interactions in the retinal binding pocket and indirectly favor hydrophobic interactions at the membrane protein receptor core. These effects can be exploited in applications where the stability of membrane proteins in solution is highly desirable. PMID:22261069

  2. Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

    NASA Technical Reports Server (NTRS)

    Muday, G. K.

    2000-01-01

    In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

  3. Restoring E-cadherin-mediated cell-cell adhesion increases PTEN protein level and stability in human breast carcinoma cells

    SciTech Connect

    Li Zengxia; Wang Liying; Zhang Wen; Fu Yi; Zhao Hongbo; Hu Yali; Prins, Bram Peter; Zha Xiliang

    2007-11-09

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a well-characterized tumor suppressor that negatively regulates cell growth and survival. Despite the critical role of PTEN in cell signaling, the mechanisms of its regulation are still under investigation. We reported here that PTEN expression could be controlled by overexpression or knock-down of E-cadherin in several mammary carcinoma cell lines. Furthermore, we showed that the accumulation of PTEN protein in E-cadherin overexpressing cells was due to increased PTEN protein stability rather than the regulation of its transcription. The proteasome-dependent PTEN degradation pathway was impaired after restoring E-cadherin expression. Moreover, maintenance of E-cadherin mediated cell-cell adhesion was necessary for its regulating PTEN. Altogether, our results suggested that E-cadherin mediated cell-cell adhesion was essential for preventing the proteasome degradation of PTEN, which might explain how breast carcinoma cells which lost cell-cell contact proliferate rapidly and are prone to metastasis.

  4. On the maintenance of hepatocyte intracellular pH 7.0 in the in-vitro metabolic stability assay.

    PubMed

    Berezhkovskiy, Leonid M; Wong, Susan; Halladay, Jason S

    2013-12-01

    The account of pH difference between hepatocytes (intracellular pH 7.0) and extracellular water (pH 7.4) leads to the novel equation for hepatic clearance (Berezhkovskiy, J Pharma Sci 100:1167-1683, 2011). The metabolic stability assay using hepatocytes is commonly performed in the incubation buffer of pH 7.4. If hepatocytes retain their physiological pH 7.0 in these conditions, then the assay would mimic the in vivo condition, that is pH 7.4 for plasma and extracellular water, and pH 7.0 in hepatocytes. In this case the rate of drug elimination, taken as proportional to unbound drug concentration in buffer, would correspond to the in vivo rate of drug elimination as proportional to the unbound drug concentration in the extracellular water. Consequently the commonly used PBPK equation for the rate of hepatic elimination, and the equation for hepatic clearance would be valid. However, the experiment designed to determine hepatocyte internal pH indicated that it was not maintained in the in vitro stability assay, so that hepatocytes acquire the same pH as the incubation buffer. Thus, the novel equations for hepatic clearance (that include an ionization factor) should be applied regardless if the intrinsic clearance was obtained either from microsomal or hepatocyte stability assay.

  5. StAR Protein Stability in Y1 and Kin-8 Mouse Adrenocortical Cells.

    PubMed

    Clark, Barbara J; Hudson, Elizabeth A

    2015-03-04

    The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition.

  6. In vivo architectonic stability of fully de novo designed protein-only nanoparticles.

    PubMed

    Céspedes, María Virtudes; Unzueta, Ugutz; Tatkiewicz, Witold; Sánchez-Chardi, Alejandro; Conchillo-Solé, Oscar; Álamo, Patricia; Xu, Zhikun; Casanova, Isolda; Corchero, José Luis; Pesarrodona, Mireia; Cedano, Juan; Daura, Xavier; Ratera, Imma; Veciana, Jaume; Ferrer-Miralles, Neus; Vazquez, Esther; Villaverde, Antonio; Mangues, Ramón

    2014-05-27

    The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids. PMID:24708510

  7. Sample Stability and Protein Composition of Saliva: Implications for Its Use as a Diagnostic Fluid.

    PubMed

    Esser, Diederik; Alvarez-Llamas, Gloria; de Vries, Marcel P; Weening, Desiree; Vonk, Roel J; Roelofsen, Han

    2008-02-01

    Saliva is an easy accessible plasma ultra-filtrate. Therefore, saliva can be an attractive alternative to blood for measurement of diagnostic protein markers. Our aim was to determine stability and protein composition of saliva. Protein stability at room temperature was examined by incubating fresh whole saliva with and without inhibitors of proteases and bacterial metabolism followed by Surface Enhanced Laser Desorption/Ionization (SELDI) analyses. Protein composition was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) fractionation of saliva proteins followed by digestion of excised bands and identification by liquid chromatography tandem mass spectrometry (LC-MS/MS). Results show that rapid protein degradation occurs within 30 minutes after sample collection. Degradation starts already during collection. Protease inhibitors partly prevented degradation while inhibition of bacterial metabolism did not affect degradation. Three stable degradation products of 2937 Da, 3370 Da and 4132 Da were discovered which can be used as markers to monitor sample quality. Saliva proteome analyses revealed 218 proteins of which 84 can also be found in blood plasma. Based on a comparison with seven other proteomics studies on whole saliva we identified 83 new saliva proteins. We conclude that saliva is a promising diagnostic fluid when precautions are taken towards protein breakdown.

  8. Soy protein nanoparticle aggregates as pickering stabilizers for oil-in-water emulsions.

    PubMed

    Liu, Fu; Tang, Chuan-He

    2013-09-18

    In recent years, there have been increasing interests in developing food-grade Pickering stabilizers, due to their potential applications in formulations of novel functional foods. The present work was to investigate the potential of soy proteins to be developed into a kind of Pickering-like stabilizer for oil-in-water emulsions. The nanoparticle aggregates of soy protein isolate (SPI) were formed by sequential treatments of heating at 95 °C for 15 min and then electrostatic screening with NaCl addition. The particle size and microstructure of these aggregates were characterized using dynamic light scattering and atomic force microscopy, indicating that the fabricated nanoparticle aggregates were ∼100 nm in size with more surface hydrophobic nature (relative to unheated SPI). The influence of particle concentration (c; 0.5-6.0%, w/w) and increasing oil fraction (ϕ; in the range 0.2-0.6) on the droplet size and coalescence and/or creaming stability of the emulsions stabilized by these nanoparticle aggregates was investigated. The results showed that, at ϕ = 0.2, increasing the c resulted in a progressive but slight decrease in droplet size, and improved the stability against coalescence and creaming; at a specific c, the creaming stability was progressively increased by increasing the ϕ, with better improvement observed at a higher c (e.g., 6.0% vs 2.0%). The improvement of creaming stability was largely associated with the formation of a gel-like network that could entrap the oil droplets within the network. The observations are generally consistent with those observed for the conventional Pickering emulsions, confirming that soy proteins could be applied as a kind of effective Pickering-like stabilizer. The finding may have important implications for the design and fabrication of protein-based emulsion formulations, and even for the development of soy protein products with some unique functions. To the authors' knowledge, this is the first work to report

  9. Beyond anchoring: the expanding role of the hendra virus fusion protein transmembrane domain in protein folding, stability, and function.

    PubMed

    Smith, Everett Clinton; Culler, Megan R; Hellman, Lance M; Fried, Michael G; Creamer, Trevor P; Dutch, Rebecca Ellis

    2012-03-01

    While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion.

  10. Structural determinants of protein stabilization by solutes. The important of the hairpin loop in rubredoxins.

    PubMed

    Pais, Tiago M; Lamosa, Pedro; dos Santos, Wagner; Legall, Jean; Turner, David L; Santos, Helena

    2005-02-01

    Despite their high sequence homology, rubredoxins from Desulfovibrio gigas and D. desulfuricans are stabilized to very different extents by compatible solutes such as diglycerol phosphate, the major osmolyte in the hyperthermophilic archaeon Archaeoglobus fulgidus[Lamosa P, Burke A, Peist R, Huber R, Liu M Y, Silva G, Rodrigues-Pousada C, LeGall J, Maycock C and Santos H (2000) Appl Environ Microbiol66, 1974-1979]. The principal structural difference between these two proteins is the absence of the hairpin loop in the rubredoxin from D. desulfuricans. Therefore, mutants of D. gigas rubredoxin bearing deletions in the loop region were constructed to investigate the importance of this structural feature on protein intrinsic stability, as well as on its capacity to undergo stabilization by compatible solutes. The three-dimensional structure of the mutant bearing the largest deletion, Delta17/29, was determined by 1H-NMR, demonstrating that, despite the drastic deletion, the main structural features were preserved. The dependence of the NH chemical shifts on temperature and solute concentration (diglycerol phosphate or mannosylglycerate) provide evidence of subtle conformational changes induced by the solute. The kinetic stability (as assessed from the absorption decay at 494 nm) of six mutant rubredoxins was determined at 90 degrees C and the stabilizing effect exerted by both solutes was assessed. The extent of protection conferred by each solute was highly dependent on the specific mutant examined: while the half-life for iron release in the wild-type D. gigas rubredoxin increased threefold in the presence of 0.1 M diglycerol phosphate, mutant Delta23/29 was destabilized. This study provides evidence for solute-induced compaction of the protein structure and occurrence of weak, specific interactions with the protein surface. The relevance of these findings to our understanding of the molecular basis for protein stabilization is discussed.

  11. Examining Ligand-Based Stabilization of Proteins in Cells with MEK1 Kinase Inhibitors.

    PubMed

    Auld, Douglas S; Davis, Christopher A; Jimenez, Marta; Knight, Sinead; Orme, Jonathon P

    2015-06-01

    In this study, we describe the evaluation of a cell-based protein stability assay using β-galactosidase fragment complementation technology performed in two independent laboratories. The assay is based on the ability of certain ligands to bind to a protein leading to a ligand-protein complex that has a different stability than the free protein. The assay employed a prolabeled-tagged MEK1 kinase stably expressed in A549 cells and this was used to evaluate focused sets of compounds containing known MEK1inhibitors as well as a random set of compounds. An assay using a prolabeled-tagged lysine methyltransferase known as G9a expressed in A549 cells was used as a counterscreen. In one study, it was found that the majority of MEK1 inhibitors were either found as inactive (52%) or showed a selective inhibitory response (18%) in the cell-based MEK1 assay; however, eight compounds showed a specific activation response consistent with stabilization of MEK1 in cells. Examination of these stabilizing compounds showed that three of these were analogs of hypothemycin, a known covalent allosteric MEK1 inhibitor, while the remaining compounds covered one structural class. Both laboratories were able to confirm activity in the cell-based MEK1 assay for known MEK1 inhibitors and found that this activity was highly selective over the G9a counterscreen assay. Screening of a mechanism of action library containing compounds with bioactivity annotations against the cell-based MEK1 assay did not reveal any mechanisms leading to an increase in signal other than inhibitors of MEK1. This study supports that the MEK1 cellular protein stability assay is sensitive to certain MEK1 inhibitors, often noncompetitive inhibitors with respect to ATP. The cellular stability assay format could be useful to rapidly filter kinase inhibitor hit lists for allosteric kinase inhibitors and support target engagement in cells.

  12. Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins

    NASA Astrophysics Data System (ADS)

    Faure, Guilhem; Koonin, Eugene V.

    2015-05-01

    Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins.

  13. Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins.

    PubMed

    Faure, Guilhem; Koonin, Eugene V

    2015-04-30

    Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins.

  14. Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins.

    PubMed

    Song, Albert S; Poor, Taylor A; Abriata, Luciano A; Jardetzky, Theodore S; Dal Peraro, Matteo; Lamb, Robert A

    2016-07-01

    Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design. PMID:27335462

  15. The role of zinc in the stability of the marginally stable IscU scaffold protein.

    PubMed

    Iannuzzi, Clara; Adrover, Miquel; Puglisi, Rita; Yan, Robert; Temussi, Piero Andrea; Pastore, Annalisa

    2014-09-01

    Understanding the factors that determine protein stability is interesting because it directly reflects the evolutionary pressure coming from function and environment. Here, we have combined experimental and computational methods to study the stability of IscU, a bacterial scaffold protein highly conserved in most organisms and an essential component of the iron-sulfur cluster biogenesis pathway. We demonstrate that the effect of zinc and its consequence strongly depend on the sample history. IscU is a marginally stable protein at low ionic strength to the point that undergoes cold denaturation at around -8°C with a corresponding dramatic decrease of enthalpy, which is consistent with the fluxional nature of the protein. Presence of constitutively bound zinc appreciably stabilizes the IscU fold, whereas it may cause protein aggregation when zinc is added back posthumously. We discuss how zinc coordination can be achieved by different side chains spatially available and all competent for tetrahedral coordination. The individual absence of some of these residues can be largely compensated by small local rearrangements of the others. We discuss the potential importance of our findings in vitro for the function in vivo of the protein.

  16. Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

    PubMed Central

    Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

    2012-01-01

    Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. PMID:22619179

  17. The role of zinc in the stability of the marginally stable IscU scaffold protein

    PubMed Central

    Iannuzzi, Clara; Adrover, Miquel; Puglisi, Rita; Yan, Robert; Temussi, Piero Andrea; Pastore, Annalisa

    2014-01-01

    Understanding the factors that determine protein stability is interesting because it directly reflects the evolutionary pressure coming from function and environment. Here, we have combined experimental and computational methods to study the stability of IscU, a bacterial scaffold protein highly conserved in most organisms and an essential component of the iron–sulfur cluster biogenesis pathway. We demonstrate that the effect of zinc and its consequence strongly depend on the sample history. IscU is a marginally stable protein at low ionic strength to the point that undergoes cold denaturation at around −8°C with a corresponding dramatic decrease of enthalpy, which is consistent with the fluxional nature of the protein. Presence of constitutively bound zinc appreciably stabilizes the IscU fold, whereas it may cause protein aggregation when zinc is added back posthumously. We discuss how zinc coordination can be achieved by different side chains spatially available and all competent for tetrahedral coordination. The individual absence of some of these residues can be largely compensated by small local rearrangements of the others. We discuss the potential importance of our findings in vitro for the function in vivo of the protein. PMID:24917298

  18. Protein conformational stability in the hydrofluoroalkane propellants tetrafluoroethane and heptafluoropropane analysed by Fourier transform Raman spectroscopy.

    PubMed

    Quinn, E A; Forbes, R T; Williams, A C; Oliver, M J; McKenzie, L; Purewal, T S

    1999-09-10

    Due to the inherent structural instability of proteins, development of chlorofluorocarbon (CFC) free metered dose inhalers (MDIs) containing these biomolecules is beset with numerous challenges. In assessing the conformation of proteins in any medium, both secondary and tertiary structures need to be elucidated. This study uses Fourier transform (FT-) Raman spectroscopy to probe protein conformational stability in hydrofluoroalkane (HFA) propellants. Assignments of molecular modes for lysozyme as a solid and in aqueous solution, and for the first time, HFAs tetrafluoroethane (HFA 134a) and heptafluoropropane (HFA 227) are given. The Raman spectra provided molecular structural information on the peptide backbone, disulfide bonds and C-C stretching vibrations of hen egg lysozyme, enabling the secondary conformation of protein in HFA propellants to be determined; structural integrity of this robust model protein was maintained in both propellants. These results demonstrate that FT-Raman may be a useful tool for the optimisation of protein MDI formulations.

  19. Protein extraction from heat-stabilized defatted rice bran. 1. Physical processing and enzyme treatments.

    PubMed

    Tang, Shanhu; Hettiarachchy, Navam S; Shellhammer, Thomas H

    2002-12-01

    Physical processing with or without enzyme treatments on protein extraction from heat-stabilized defatted rice bran (HDRB) was evaluated. Freeze-thaw, sonication, high-speed blending, and high-pressure methods extracted 12%, 15%, 16%, and 11% protein, respectively. Sonication (0-100%, 750 W), followed by amylase and combined amylase and protease treatments, extracted 25.6-33.9% and 54.0-57.8% protein, respectively. Blending followed by amylase and protease treatment extracted 5.0% more protein than the nonblended enzymatic treatments. High-pressure treatments, 0-800 MPa, with water or amylase-protease combinations, extracted 10.5-11.1% or 61.8-66.6% protein, respectively. These results suggest that physical processing in combination with enzyme treatments can be effective in extracting protein from HDRB.

  20. Physicochemical effects of the lipid phase and protein level on meat emulsion stability, texture, and microstructure.

    PubMed

    Youssef, M K; Barbut, S

    2010-03-01

    The effects of beef fat (25%) substitution with rendered beef fat, canola oil, palm oil, or hydrogenated palm oil at varying meat protein levels (8%, 11%, and 14%) were studied in emulsified beef meat batters. There was no significant difference in fat loss among meat batters made with beef fat, rendered beef fat, or palm oil. Hydrogenated palm oil provided the most stable batters at all protein levels. Increasing meat protein to 14% resulted in high fat loss in batters prepared with canola oil, which did not occur in the other formulations. This indicates that the physicochemical characteristics of fat/oil affect emulsion stability. Cooked batter hardness was higher (P < 0.05) when protein level was raised; highest in hydrogenated palm oil batters when compared at similar protein levels. As protein level was raised springiness values were increased in all the meat treatments. Springiness was higher in the canola oil treatments. Light microscopy revealed fat globule coalescence in canola oil meat batters prepared with 14% protein, as well as the development of fat channels and more protein aggregation; both seem to result in lower emulsion stability. Hydrogenated palm oil batters showed fat particles with sharp edges as opposed to the round ones seen in all other treatments.

  1. Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

    SciTech Connect

    Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Brozik, Susan Marie; Cox, Jason M.; Lopez, Gabriel P.; Barrick, Todd A.; Flores, Adrean

    2007-03-01

    The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

  2. Phosphoprotein Stability in Clinical Tissue and Its Relevance for Reverse Phase Protein Microarray Technology

    PubMed Central

    Espina, Virginia; Mueller, Claudius; Liotta, Lance A.

    2013-01-01

    Phosphorylated proteins reflect the activity of specific cell signaling nodes in biological kinase protein networks. Cell signaling pathways can be either activated or deactivated depending on the phosphorylation state of the constituent proteins. The state of these kinase pathways reflects the in vivo activity of the cells and tissue at any given point in time. As such, cell signaling pathway information can be extrapolated to infer which phosphorylated proteins/pathways are driving an individual tumor’s growth. Reverse Phase Protein Microarrays (RPMA) are a sensitive and precise platform that can be applied to the quantitative measurement of hundreds of phosphorylated signal proteins from a small sample of tissue. Pre-analytical variability originating from tissue procurement and preservation may cause significant variability and bias in downstream molecular analysis. Depending on the ex vivo delay time in tissue processing, and the manner of tissue handling, protein biomarkers such as signal pathway phosphoproteins will be elevated or suppressed in a manner that does not represent the biomarker levels at the time of excision. Consequently, assessment of the state of these kinase networks requires stabilization, or preservation, of the phosphoproteins immediately post tissue procurement. We have employed reverse phase protein microarray analysis of phosphoproteins to study the factors influencing stability of phosphoproteins in tissue following procurement. Based on this analysis we have established tissue procurement guidelines for clinical research with an emphasis on quantifying phosphoproteins by RPMA. PMID:21901591

  3. Lysine acetylation stabilizes SP2 protein in the silkworm Bombyx mori.

    PubMed

    Zhou, Yong; Wu, Chengcheng; Sheng, Qing; Jiang, Caiying; Chen, Qin; Lv, Zhengbing; Yao, Juming; Nie, Zuoming

    2016-01-01

    Lysine acetylation (Kac) is a vital post-translational modification that plays an important role in many cellular processes in organisms. In the present study, the nutrient storage proteins in hemolymph were first found to be highly acetylated-particularly SP2 protein, which contains 20 potential Kac sites. Further results confirmed that lysine acetylation could stabilize and up-regulate the protein level of anti-apoptosis protein SP2, thereby improving the survival of H2O2-treated BmN cells and suppressing the apoptosis induced by H2O2. The potential mechanism involved in the inhibition of ubiquitin-mediated proteasomal degradation by crosstalk between lysine acetylation and ubiquitination. Our results showed that the increase in the acetylation level by TSA could decrease the ubiquitination and improve the protein level of SP2, indicating that lysine acetylation could influence the SP2 protein level through competition between ubiquitination and the suppression of ubiquitin-mediated proteasomal degradation, thereby stabilizing the protein. SP2 is a major nutrient storage protein from hemolymph for amino acid storage and utilization. The crosstalk between lysine acetylation and ubiquitination of SP2 might imply an important role of lysine acetylation for nutrient storage and utilization in silkworm. PMID:27374983

  4. Long-lasting stability of Vaccinia virus strains in murine feces: implications for virus circulation and environmental maintenance.

    PubMed

    Abrahão, Jônatas S; Trindade, Giliane de Souza; Ferreira, Jaqueline M Siqueira; Campos, Rafael K; Bonjardim, Cláudio A; Ferreira, Paulo C Peregrino; Kroon, Erna Geessien

    2009-01-01

    Vaccinia virus (VACV) has been associated with several bovine vaccinia outbreaks in Brazil, causing exanthematic lesions in dairy cattle and humans. The way that VACV circulates in the environment is unknown, as is the way that this virus is transferred from wildlife to farms. Rodents are hypothetical VACV reservoirs, and murine feces has been identified as a potential source of viral shedding and transmission. In this work, we analyzed the stability of VACV infectious particles and DNA in feces from intranasally infected mice, exposed to environmental temperature and humidity, by titration assays and PCR, respectively. The results showed that VACV infectious particles were still detected at 20 days post-environmental-exposure (d.p.e.), while viral DNA was detected until 60 d.p.e. A gradual decrease in fecal viral load could be detected in all analyzed VACV strains. This work indicates long-lasting stability of VACV in murine feces and reinforces the idea that fecal matter may represent a potential source of circulating virus among rodents.

  5. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization.

    PubMed

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability.

  6. Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate*

    PubMed Central

    Mollan, Todd L.; Khandros, Eugene; Weiss, Mitchell J.; Olson, John S.

    2012-01-01

    Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k′AHSP) and dissociation (kAHSP) rate constants of ≈10 μm−1 s−1 and 0.2 s−1, respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp29-Pro30 peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe3+)-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro30 conformer. Both wild-type and Pro30-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe2+)-α. The dissociation rate of the met-α·AHSP complex (kAHSP ≈ 0.002 s−1) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers. PMID:22298770

  7. Characteristics of sugar surfactants in stabilizing proteins during freeze-thawing and freeze-drying.

    PubMed

    Imamura, Koreyoshi; Murai, Katsuyuki; Korehisa, Tamayo; Shimizu, Noriyuki; Yamahira, Ryo; Matsuura, Tsutashi; Tada, Hiroko; Imanaka, Hiroyuki; Ishida, Naoyuki; Nakanishi, Kazuhiro

    2014-06-01

    Sugar surfactants with different alkyl chain lengths and sugar head groups were compared for their protein-stabilizing effect during freeze-thawing and freeze-drying. Six enzymes, different in terms of tolerance against inactivation because of freeze-thawing and freeze-drying, were used as model proteins. The enzyme activities that remained after freeze-thawing and freeze-drying in the presence of a sugar surfactant were measured for different types and concentrations of sugar surfactants. Sugar surfactants stabilized all of the tested enzymes both during freeze-thawing and freeze-drying, and a one or two order higher amount of added sugar surfactant was required for achieving protein stabilization during freeze-drying than for the cryoprotection. The comprehensive comparison showed that the C10-C12 esters of sucrose or trehalose were the most effective through the freeze-drying process: the remaining enzyme activities after freeze-thawing and freeze-drying increased at the sugar ester concentrations of 1-10 and 10-100 μM, respectively, and increased to a greater extent than for the other surfactants at higher concentrations. Results also indicate that, when a decent amount of sugar was also added, the protein-stabilizing effect of a small amount of sugar ester through the freeze-drying process could be enhanced.

  8. Semiautomated Sample Preparation for Protein Stability and Formulation Screening via Buffer Exchange.

    PubMed

    Ying, William; Levons, Jaquan K; Carney, Andrea; Gandhi, Rajesh; Vydra, Vicky; Rubin, A Erik

    2016-06-01

    A novel semiautomated buffer exchange process workflow was developed to enable efficient early protein formulation screening. An antibody fragment protein, BMSdab, was used to demonstrate the workflow. The process afforded 60% to 80% cycle time and scientist time savings and significant material efficiencies. These efficiencies ultimately facilitated execution of this stability work earlier in the drug development process, allowing this tool to inform the developability of potential candidates for development from a formulation perspective. To overcome the key technical challenges, the protein solution was buffer-exchanged by centrifuge filtration into formulations for stability screening in a 96-well plate with an ultrafiltration membrane, leveraging automated liquid handling and acoustic volume measurements to allow several cycles of exchanges. The formulations were transferred into a vacuum manifold and sterile filtered into a rack holding 96 glass vials. The vials were sealed with a capmat of individual caps and placed in stability stations. Stability of the samples prepared by this process and by the standard process was demonstrated to be comparable. This process enabled screening a number of formulations of a protein at an early pharmaceutical development stage with a short sample preparation time.

  9. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

    SciTech Connect

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

  10. Stability of Magnetically-Suppressed Solutal Convection In Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    Leslie, F. W.; Ramachandran, N.

    2005-01-01

    The effect of convection during the crystallization of proteins is not very well understood. In a gravitational field, convection is caused by crystal sedimentation and by solutal buoyancy induced flow and these can lead to crystal imperfections. While crystallization in microgravity can approach diffusion limited growth conditions (no convection), terrestrially strong magnetic fields can be used to control fluid flow and sedimentation effects. In this work, a theory is presented on the stability of solutal convection of a magnetized fluid in the presence of a magnetic field. The requirements for stability are developed and compared to experiments performed within the bore of a superconducting magnet. The theoretical predictions are in good agreement with the experiments and show solutal convection can be stabilized if the surrounding fluid has larger magnetic susceptibility and the magnetic field has a specific structure. Discussion on the application of the technique to protein crystallization is also provided.

  11. Elucidating Protein Involvement in the Stabilization of the Biogenic Silver Nanoparticles

    NASA Astrophysics Data System (ADS)

    Ballottin, Daniela; Fulaz, Stephanie; Souza, Michele L.; Corio, Paola; Rodrigues, Alexandre G.; Souza, Ana O.; Gaspari, Priscyla M.; Gomes, Alexandre F.; Gozzo, Fábio; Tasic, Ljubica

    2016-06-01

    Silver nanoparticles (AgNPs) have been broadly used as antibacterial and antiviral agents. Further, interests for green AgNP synthesis have increased in recent years and several results for AgNP biological synthesis have been reported using bacteria, fungi and plant extracts. The understanding of the role and nature of fungal proteins, their interaction with AgNPs and the subsequent stabilization of nanosilver is yet to be deeply investigated. Therefore, in an attempt to better understand biogenic AgNP stabilization with the extracellular fungal proteins and to describe these supramolecular interactions between proteins and silver nanoparticles, AgNPs, produced extracellularly by Aspergillus tubingensis—isolated as an endophytic fungus from Rizophora mangle—were characterized in order to study their physical characteristics, identify the involved proteins, and shed light into the interactions among protein-NPs by several techniques. AgNPs of around 35 nm in diameter as measured by TEM and a positive zeta potential of +8.48 mV were obtained. These AgNPs exhibited a surface plasmon resonance (SPR) band at 440 nm, indicating the nanoparticles formation, and another band at 280 nm, attributed to the electronic excitations in tryptophan, tyrosine, and/or phenylalanine residues in fungal proteins. Fungal proteins were covalently bounded to the AgNPs, mainly through S-Ag bonds due to cysteine residues (HS-) and with few N-Ag bonds from H2N- groups, as verified by Raman spectroscopy. Observed supramolecular interactions also occur by electrostatic and other protein-protein interactions. Furthermore, proteins that remain free on AgNP surface may perform hydrogen bonds with other proteins or water increasing thus the capping layer around the AgNPs and consequently expanding the hydrodynamic diameter of the particles (~264 nm, measured by DLS). FTIR results enabled us to state that proteins adsorbed to the AgNPs did not suffer relevant secondary structure alteration upon

  12. Elucidating Protein Involvement in the Stabilization of the Biogenic Silver Nanoparticles.

    PubMed

    Ballottin, Daniela; Fulaz, Stephanie; Souza, Michele L; Corio, Paola; Rodrigues, Alexandre G; Souza, Ana O; Gaspari, Priscyla M; Gomes, Alexandre F; Gozzo, Fábio; Tasic, Ljubica

    2016-12-01

    Silver nanoparticles (AgNPs) have been broadly used as antibacterial and antiviral agents. Further, interests for green AgNP synthesis have increased in recent years and several results for AgNP biological synthesis have been reported using bacteria, fungi and plant extracts. The understanding of the role and nature of fungal proteins, their interaction with AgNPs and the subsequent stabilization of nanosilver is yet to be deeply investigated. Therefore, in an attempt to better understand biogenic AgNP stabilization with the extracellular fungal proteins and to describe these supramolecular interactions between proteins and silver nanoparticles, AgNPs, produced extracellularly by Aspergillus tubingensis-isolated as an endophytic fungus from Rizophora mangle-were characterized in order to study their physical characteristics, identify the involved proteins, and shed light into the interactions among protein-NPs by several techniques. AgNPs of around 35 nm in diameter as measured by TEM and a positive zeta potential of +8.48 mV were obtained. These AgNPs exhibited a surface plasmon resonance (SPR) band at 440 nm, indicating the nanoparticles formation, and another band at 280 nm, attributed to the electronic excitations in tryptophan, tyrosine, and/or phenylalanine residues in fungal proteins. Fungal proteins were covalently bounded to the AgNPs, mainly through S-Ag bonds due to cysteine residues (HS-) and with few N-Ag bonds from H2N- groups, as verified by Raman spectroscopy. Observed supramolecular interactions also occur by electrostatic and other protein-protein interactions. Furthermore, proteins that remain free on AgNP surface may perform hydrogen bonds with other proteins or water increasing thus the capping layer around the AgNPs and consequently expanding the hydrodynamic diameter of the particles (~264 nm, measured by DLS). FTIR results enabled us to state that proteins adsorbed to the AgNPs did not suffer relevant secondary structure alteration upon

  13. Elucidating Protein Involvement in the Stabilization of the Biogenic Silver Nanoparticles.

    PubMed

    Ballottin, Daniela; Fulaz, Stephanie; Souza, Michele L; Corio, Paola; Rodrigues, Alexandre G; Souza, Ana O; Gaspari, Priscyla M; Gomes, Alexandre F; Gozzo, Fábio; Tasic, Ljubica

    2016-12-01

    Silver nanoparticles (AgNPs) have been broadly used as antibacterial and antiviral agents. Further, interests for green AgNP synthesis have increased in recent years and several results for AgNP biological synthesis have been reported using bacteria, fungi and plant extracts. The understanding of the role and nature of fungal proteins, their interaction with AgNPs and the subsequent stabilization of nanosilver is yet to be deeply investigated. Therefore, in an attempt to better understand biogenic AgNP stabilization with the extracellular fungal proteins and to describe these supramolecular interactions between proteins and silver nanoparticles, AgNPs, produced extracellularly by Aspergillus tubingensis-isolated as an endophytic fungus from Rizophora mangle-were characterized in order to study their physical characteristics, identify the involved proteins, and shed light into the interactions among protein-NPs by several techniques. AgNPs of around 35 nm in diameter as measured by TEM and a positive zeta potential of +8.48 mV were obtained. These AgNPs exhibited a surface plasmon resonance (SPR) band at 440 nm, indicating the nanoparticles formation, and another band at 280 nm, attributed to the electronic excitations in tryptophan, tyrosine, and/or phenylalanine residues in fungal proteins. Fungal proteins were covalently bounded to the AgNPs, mainly through S-Ag bonds due to cysteine residues (HS-) and with few N-Ag bonds from H2N- groups, as verified by Raman spectroscopy. Observed supramolecular interactions also occur by electrostatic and other protein-protein interactions. Furthermore, proteins that remain free on AgNP surface may perform hydrogen bonds with other proteins or water increasing thus the capping layer around the AgNPs and consequently expanding the hydrodynamic diameter of the particles (~264 nm, measured by DLS). FTIR results enabled us to state that proteins adsorbed to the AgNPs did not suffer relevant secondary structure alteration upon

  14. Post-irradiation phosphorylation of structural maintenance chromosome 1 (SMC1) is independent of the Fanconi protein pathway

    SciTech Connect

    Nahas, Shareef A.; Lai, C.-H.; Gatti, Richard A. . E-mail: rgatti@mednet.ucla.edu

    2005-03-15

    Purpose: To confirm the sensitivity of cells from patients with Fanconi anemia (FA) to ionizing radiation, and to determine whether the phosphorylation of structural maintenance chromosome 1 (SMC1) was associated with radiosensitivity, as it is in other DNA repair disorders. Methods and materials: Using lymphoblastoid cell lines from FA patients before and after exposure to ionizing radiation, the colony survival assay, radioresistant DNA synthesis, and SMC1 phosphorylation were measured. FA lymphoblastoid cell lines that had been transfected with the wild-type FANC gene were used as controls. Results: Cells from FA patients of six complementation groups were radiosensitive. Despite this, SMC1 phosphorylation was normal in each case; radioresistant DNA synthesis, a measure of S phase checkpoint integrity, was defective in FANCD2 lymphoblastoid cell lines and was corrected in FANCD2 + D2 cells. Conclusions: The data indicate that the FANC pathway proteins play a major role in the cellular responses to ionizing radiation, but not in SMC1 phosphorylation or in the S phase checkpoint of FANCD2-deficient cells. Thus, SMC1 activation is not a common denominator of radiosensitivity, as has been suggested by radiation responses of cells from ataxia-telangiectasia, Nijmegen breakage syndrome, or Mre11 deficiency patients.

  15. Cryoprotectin: a plant lipid-transfer protein homologue that stabilizes membranes during freezing.

    PubMed Central

    Hincha, Dirk K

    2002-01-01

    Plants from temperate and cold climates are able to increase their freezing tolerance during exposure to low non-freezing temperatures. It has been shown that several genes are induced in a coordinated manner during this process of cold acclimation. The functional role of most of the corresponding cold-regulated proteins is not yet known. We summarize our knowledge of those cold-regulated proteins that are able to stabilize membranes during a freeze-thaw cycle. Special emphasis is placed on cryoprotectin, a lipid-transfer protein homologue that was isolated from cold-acclimated cabbage leaves and that protects isolated chloroplast thylakoid membranes from freeze-thaw damage. PMID:12171654

  16. Protein/Polysaccharide Electrostatic Complexes and Their Applications in Stabilizing Oil-in-Water Emulsions.

    PubMed

    Ai, Wenjia; Fang, Yapeng; Xiang, Shengping; Yao, Xiaolin; Nishinari, Katsuyoshi; Phillips, Glyn O

    2015-01-01

    Consumers are becoming increasingly fastidious in demanding food products with improved quality and functionality. This largely relies on rational design of food structures. As the two key food ingredients, protein and polysaccharides play important roles in food structuring. The combination of protein and polysaccharide provides rich opportunities for food structure and function designs through molecular interaction and assembly. This paper provides a brief review on the formation and characterization of protein/polysaccharide electrostatic complexes and their applications in stabilizing oil-in-water emulsions, particularly those containing polyunsaturated fatty acids. PMID:26598842

  17. Protein kinase C is likely to be involved in zoosporogenesis and maintenance of flagellar motility in the peronosporomycete zoospores.

    PubMed

    Islam, Md Tofazzal; von Tiedemann, Andreas; Laatsch, Hartmut

    2011-08-01

    The motility of zoospores is critical in the disease cycles of Peronosporomycetes that cause devastating diseases in plants, fishes, vertebrates, and microbes. In the course of screening for secondary metabolites, we found that ethyl acetate extracts of a marine Streptomyces sp. strain B5136 rapidly impaired the motility of zoospores of the grapevine downy mildew pathogen Plasmopara viticola at 0.1 μg/ml. The active principle in the extracts was identified as staurosporine, a known broad-spectrum inhibitor of protein kinases, including protein kinase C (PKC). In the presence of staurosporine (2 nM), zoospores moved very slowly in their axis or spun in tight circles, instead of displaying straight swimming in a helical fashion. Compounds such as K-252a, K-252b, and K-252c structurally related to staurosporine also impaired the motility of zoospores in a similar manner but at varying doses. Among the 22 known kinase inhibitors tested, the PKC inhibitor chelerythrine was the most potent to arrest the motility of zoospores at concentrations starting from 5 nM. Inhibitors that targeted kinase pathways other than PKC pathways did not practically show any activity in impairing zoospore motility. Interestingly, both staurosporine (5 nM) and chelerythrine (10 nM) also inhibited the release of zoospores from the P. viticola sporangia in a dose-dependent manner. In addition, staurosporine completely suppressed downy mildew disease in grapevine leaves at 2 μM, suggesting the potential of small-molecule PKC inhibitors for the control of peronosporomycete phytopathogens. Taken together, these results suggest that PKC is likely to be a key signaling mediator associated with zoosporogenesis and the maintenance of flagellar motility in peronosporomycete zoospores.

  18. Genetic Evidence for a Role for Protein Kinase A in the Maintenance of Sleep and Thalamocortical Oscillations

    PubMed Central

    Hellman, Kevin; Hernandez, Pepe; Park, Alice; Abel, Ted

    2010-01-01

    Study Objectives: Genetic manipulation of cAMP-dependent protein kinase A (PKA) in Drosophila has implicated an important role for PKA in sleep/wake state regulation. Here, we characterize the role of this signaling pathway in the regulation of sleep using electroencephalographic (EEG) and electromyographic (EMG) recordings in R(AB) transgenic mice that express a dominant negative form of the regulatory subunit of PKA in neurons within cortex and hippocampus. Previous studies have revealed that these mutant mice have reduced PKA activity that results in the impairment of hippocampus-dependent long-term memory and long-lasting forms of hippocampal synaptic plasticity. Design: PKA assays, in situ hybridization, immunoblots, and sleep studies were performed in R(AB) transgenic mice and wild-type control mice. Measurements and Results: We have found that R(AB) transgenic mice have reduced PKA activity within cortex and reduced Ser845 phosphorylation of the glutamate receptor subunit GluR1. R(AB) transgenic mice exhibit non-rapid eye movement (NREM) sleep fragmentation and increased amounts of rapid eye movement (REM) sleep relative to wild-type mice. Further, R(AB) transgenic mice have more delta power but less sigma power during NREM sleep relative to wild-type mice. After sleep deprivation, the amounts of NREM and REM sleep were comparable between wild-type and R(AB) transgenic mice. However, the homeostatic rebound of sigma power in R(AB) transgenic mice was reduced. Conclusions: Alterations in cortical synaptic receptors, impairments in sleep continuity, and alterations in sleep oscillations in R(AB) mice imply that PKA is involved not only in synaptic plasticity and memory storage but also in the regulation of sleep/wake states. Citation: Hellman K; Hernandez P; Park A; Abel T. Genetic evidence for a role for protein kinase a in the maintenance of sleep and thalamocortical oscillations. SLEEP 2010;33(1):19-28. PMID:20120617

  19. P22 coat protein structures reveal a novel mechanism for capsid maturation: stability without auxiliary proteins or chemical crosslinks.

    PubMed

    Parent, Kristin N; Khayat, Reza; Tu, Long H; Suhanovsky, Margaret M; Cortines, Juliana R; Teschke, Carolyn M; Johnson, John E; Baker, Timothy S

    2010-03-10

    Viral capsid assembly and stability in tailed, dsDNA phage and Herpesviridae are achieved by various means including chemical crosslinks (unique to HK97), or auxiliary proteins (lambda, T4, phi29, and herpesviruses). All these viruses have coat proteins (CP) with a conserved, HK97-like core structure. We used a combination of trypsin digestion, gold labeling, cryo-electron microscopy, 3D image reconstruction, and comparative modeling to derive two independent, pseudoatomic models of bacteriophage P22 CP: before and after maturation. P22 capsid stabilization results from intersubunit interactions among N-terminal helices and an extensive "P loop," which obviate the need for crosslinks or auxiliary proteins. P22 CP also has a telokin-like Ig domain that likely stabilizes the monomer fold so that assembly may proceed via individual subunit addition rather than via preformed capsomers as occurs in HK97. Hence, the P22 CP structure may be a paradigm for understanding how monomers assemble in viruses like phi29 and HSV-1.

  20. Influence of pea protein aggregates on the structure and stability of pea protein/soybean polysaccharide complex emulsions.

    PubMed

    Yin, Baoru; Zhang, Rujing; Yao, Ping

    2015-01-01

    The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI) with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS), and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

  1. Rational stabilization of the C-LytA affinity tag by protein engineering.

    PubMed

    Hernández-Rocamora, Víctor M; Maestro, Beatriz; Mollá-Morales, Almudena; Sanz, Jesús M

    2008-12-01

    The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 degrees C thermal stabilization compared with the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at approximately 65 degrees C, whereas C-LytAm7 may stand temperatures up to 90 degrees C. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C-LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization. PMID:18840883

  2. Quantifying the kinetic stability of hyperstable proteins via time-dependent SDS trapping.

    PubMed

    Xia, Ke; Zhang, Songjie; Bathrick, Brendan; Liu, Shuangqi; Garcia, Yeidaliz; Colón, Wilfredo

    2012-01-10

    Globular proteins are usually in equilibrium with unfolded conformations, whereas kinetically stable proteins (KSPs) are conformationally trapped by their high unfolding transition state energy. Kinetic stability (KS) could allow proteins to maintain their activity under harsh conditions, increase a protein's half-life, or protect against misfolding-aggregation. Here we show the development of a simple method for quantifying a protein's KS that involves incubating a protein in SDS at high temperature as a function of time, running the unheated samples on SDS-PAGE, and quantifying the bands to determine the time-dependent loss of a protein's SDS resistance. Six diverse proteins, including two monomer, two dimers, and two tetramers, were studied by this method, and the kinetics of the loss of SDS resistance correlated linearly with their unfolding rate determined by circular dichroism. These results imply that the mechanism by which SDS denatures proteins involves conformational trapping, with a trapping rate that is determined and limited by the rate of protein unfolding. We applied the SDS trapping of proteins (S-TraP) method to superoxide dismutase (SOD) and transthyretin (TTR), which are highly KSPs with native unfolding rates that are difficult to measure by conventional spectroscopic methods. A combination of S-TraP experiments between 75 and 90 °C combined with Eyring plot analysis yielded an unfolding half-life of 70 ± 37 and 18 ± 6 days at 37 °C for SOD and TTR, respectively. The S-TraP method shown here is extremely accessible, sample-efficient, cost-effective, compatible with impure or complex samples, and will be useful for exploring the biological and pathological roles of kinetic stability. PMID:22106876

  3. Quantifying the kinetic stability of hyperstable proteins via time-dependent SDS trapping.

    PubMed

    Xia, Ke; Zhang, Songjie; Bathrick, Brendan; Liu, Shuangqi; Garcia, Yeidaliz; Colón, Wilfredo

    2012-01-10

    Globular proteins are usually in equilibrium with unfolded conformations, whereas kinetically stable proteins (KSPs) are conformationally trapped by their high unfolding transition state energy. Kinetic stability (KS) could allow proteins to maintain their activity under harsh conditions, increase a protein's half-life, or protect against misfolding-aggregation. Here we show the development of a simple method for quantifying a protein's KS that involves incubating a protein in SDS at high temperature as a function of time, running the unheated samples on SDS-PAGE, and quantifying the bands to determine the time-dependent loss of a protein's SDS resistance. Six diverse proteins, including two monomer, two dimers, and two tetramers, were studied by this method, and the kinetics of the loss of SDS resistance correlated linearly with their unfolding rate determined by circular dichroism. These results imply that the mechanism by which SDS denatures proteins involves conformational trapping, with a trapping rate that is determined and limited by the rate of protein unfolding. We applied the SDS trapping of proteins (S-TraP) method to superoxide dismutase (SOD) and transthyretin (TTR), which are highly KSPs with native unfolding rates that are difficult to measure by conventional spectroscopic methods. A combination of S-TraP experiments between 75 and 90 °C combined with Eyring plot analysis yielded an unfolding half-life of 70 ± 37 and 18 ± 6 days at 37 °C for SOD and TTR, respectively. The S-TraP method shown here is extremely accessible, sample-efficient, cost-effective, compatible with impure or complex samples, and will be useful for exploring the biological and pathological roles of kinetic stability.

  4. Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?

    PubMed

    Sedlák, Erik; Stagg, Loren; Wittung-Stafshede, Pernilla

    2008-11-01

    We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge -19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT trs/d[ion] (T trs; thermal midpoint). We show that dT trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.

  5. Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?

    PubMed

    Sedlák, Erik; Stagg, Loren; Wittung-Stafshede, Pernilla

    2008-11-01

    We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge -19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT trs/d[ion] (T trs; thermal midpoint). We show that dT trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins. PMID:18782555

  6. Honey-Induced Protein Stabilization as Studied by Fluorescein Isothiocyanate Fluorescence

    PubMed Central

    Abdul Kadir, Habsah; Tayyab, Saad

    2013-01-01

    Protein stabilizing potential of honey was studied on a model protein, bovine serum albumin (BSA), using extrinsic fluorescence of fluorescein isothiocyanate (FITC) as the probe. BSA was labelled with FITC using chemical coupling, and urea and thermal denaturation studies were performed on FITC-labelled BSA (FITC-BSA) both in the absence and presence of 10% and 20% (w/v) honey using FITC fluorescence at 522 nm upon excitation at 495 nm. There was an increase in the FITC fluorescence intensity upon increasing urea concentration or temperature, suggesting protein denaturation. The results from urea and thermal denaturation studies showed increased stability of protein in the presence of honey as reflected from the shift in the transition curve along with the start point and the midpoint of the transition towards higher urea concentration/temperature. Furthermore, the increase in ΔGDH2O and ΔGD25°C in presence of honey also suggested protein stabilization. PMID:24222758

  7. On the Efficiency of NHS Ester Cross-Linkers for Stabilizing Integral Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Chen, Fan; Gerber, Sabina; Korkhov, Volodymyr M.; Mireku, Samantha; Bucher, Monika; Locher, Kaspar P.; Zenobi, Renato

    2015-03-01

    We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes. Here, we investigated the capability of N-hydroxysuccinimide (NHS) esters, which react much more specifically, to cross-link membrane protein complexes such as PglK and BtuC2D2. We present clear evidence that NHS esters are capable of stabilizing membrane protein complexes in situ, in the presence of detergents such as DDM, C12E8, and LDAO. The stabilization efficiency strongly depends on the membrane protein structure (i.e, the number of primary amine groups and the distances between primary amines). A minimum number of primary amine groups is required, and the distances between primary amines govern whether a cross-linker with a specific spacer arm length is able to bridge two amine groups.

  8. Start2Fold: a database of hydrogen/deuterium exchange data on protein folding and stability

    PubMed Central

    Pancsa, Rita; Varadi, Mihaly; Tompa, Peter; Vranken, Wim F.

    2016-01-01

    Proteins fulfil a wide range of tasks in cells; understanding how they fold into complex three-dimensional (3D) structures and how these structures remain stable while retaining sufficient dynamics for functionality is essential for the interpretation of overall protein behaviour. Since the 1950's, solvent exchange-based methods have been the most powerful experimental means to obtain information on the folding and stability of proteins. Considerable expertise and care were required to obtain the resulting datasets, which, despite their importance and intrinsic value, have never been collected, curated and classified. Start2Fold is an openly accessible database (http://start2fold.eu) of carefully curated hydrogen/deuterium exchange (HDX) data extracted from the literature that is open for new submissions from the community. The database entries contain (i) information on the proteins investigated and the underlying experimental procedures and (ii) the classification of the residues based on their exchange protection levels, also allowing for the instant visualization of the relevant residue groups on the 3D structures of the corresponding proteins. By providing a clear hierarchical framework for the easy sharing, comparison and (re-)interpretation of HDX data, Start2Fold intends to promote a better understanding of how the protein sequence encodes folding and structure as well as the development of new computational methods predicting protein folding and stability. PMID:26582925

  9. Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance

    PubMed Central

    Johnson, Neil; Johnson, Shawn F.; Yao, Wei; Li, Yu-Chen; Choi, Young-Eun; Bernhardy, Andrea J.; Wang, Yifan; Capelletti, Marzia; Sarosiek, Kristopher A.; Moreau, Lisa A.; Chowdhury, Dipanjan; Wickramanayake, Anneka; Harrell, Maria I.; Liu, Joyce F.; D’Andrea, Alan D.; Miron, Alexander; Swisher, Elizabeth M.; Shapiro, Geoffrey I.

    2013-01-01

    Breast Cancer Type 1 Susceptibility Protein (BRCA1)-deficient cells have compromised DNA repair and are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Despite initial responses, the development of resistance limits clinical efficacy. Mutations in the BRCA C-terminal (BRCT) domain of BRCA1 frequently create protein products unable to fold that are subject to protease-mediated degradation. Here, we show HSP90-mediated stabilization of a BRCT domain mutant BRCA1 protein under PARP inhibitor selection pressure. The stabilized mutant BRCA1 protein interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation, and conferred PARP inhibitor as well as cisplatin resistance. Treatment of resistant cells with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occur in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum. These results provide evidence for a two-event mechanism by which BRCA1-mutant tumors acquire anticancer therapy resistance. PMID:24085845

  10. The Collagen Receptor Discoidin Domain Receptor 2 Stabilizes Snail1 Protein to Facilitate Breast Cancer Metastasis

    PubMed Central

    Zhang, Kun; Corsa, Callie A.; Ponik, Suzanne M.; Prior, Julie L.; Piwnica-Worms, David; Eliceiri, Kevin W.; Keely, Patricia J.; Longmore, Gregory D.

    2013-01-01

    Increased stromal collagen deposition in human breast tumours correlates with metastases. We show that activation of the collagen I receptor DDR2 regulates Snail1 protein stability by stimulating ERK2 activity, in a Src-dependent manner. Activated ERK2 directly phosphorylates Snail1, leading to Snail1 nuclear accumulation, reduced ubiquitination, and increased protein half-life. DDR2-mediated stabilization of Snail1 promotes breast cancer cell invasion and migration in vitro, and metastasis in vivo. DDR2 expression was observed in the majority of human invasive ductal breast carcinomas studied, and was associated with nuclear Snail1 and absence of E-cadherin expression. We propose that DDR2 maintains Snail1 protein level and activity in tumor cells that have undergone EMT, thereby facilitating continued tumor cell invasion through collagen I-rich ECM by sustaining the EMT phenotype. As such, DDR2 could be an RTK target for the treatment of breast cancer metastasis. PMID:23644467

  11. Modification of the Sweetness and Stability of Sweet-Tasting Protein Monellin by Gene Mutation and Protein Engineering

    PubMed Central

    Liu, Qiulei; Li, Lei; Yang, Liu; Liu, Tianming; Cai, Chenggu; Liu, Bo

    2016-01-01

    Natural sweet protein monellin has a high sweetness and low calorie, suggesting its potential in food applications. However, due to its low heat and acid resistance, the application of monellin is limited. In this study, we show that the thermostability of monellin can be improved with no sweetness decrease by means of sequence, structure analysis, and site-directed mutagenesis. We analyzed residues located in the α-helix as well as an ionizable residue C41. Of the mutants investigated, the effects of E23A and C41A mutants were most remarkable. The former displayed significantly improved thermal stability, while its sweetness was not changed. The mutated protein was stable after 30 min incubation at 85°C. The latter showed increased sweetness and slight improvement of thermostability. Furthermore, we found that most mutants enhancing the thermostability of the protein were distributed at the two ends of α-helix. Molecular biophysics analysis revealed that the state of buried ionizable residues may account for the modulated properties of mutated proteins. Our results prove that the properties of sweet protein monellin can be modified by means of bioinformatics analysis, gene manipulation, and protein modification, highlighting the possibility of designing novel effective sweet proteins based on structure-function relationships. PMID:26881217

  12. Protein stability in mixed solvents: a balance of contact interaction and excluded volume.

    PubMed

    Schellman, John A

    2003-07-01

    Changes in excluded volume and contact interaction with the surface of a protein have been suggested as mechanisms for the changes in stability induced by cosolvents. The aim of the present paper is to present an analysis that combines both effects in a quantitative manner. The result is that both processes are present in both stabilizing and destabilizing interactions and neither can be ignored. Excluded volume was estimated using accessible surface area calculations of the kind introduced by Lee and Richards. The change in excluded volume on unfolding, deltaX, is quite large. For example, deltaX for ribonuclease is 6.7 L in urea and approximately 16 L in sucrose. The latter number is greater than the molar volume of the protein. Direct interaction with the protein is represented as the solvent exchange mechanism, which differs from ordinary association theory because of the weakness of the interaction and the high concentrations of cosolvents. The balance between the two effects and their contribution to overall stability are most simply presented as bar diagrams as in Fig. 3. Our finding for five proteins is that excluded volume contributes to the stabilization of the native structure and that contact interaction contributes to destabilization. This is true for five proteins and four cosolvents including both denaturants and osmolytes. Whether a substance stabilizes a protein or destabilizes it depends on the relative size of these two contributions. The constant for the cosolvent contact with the protein is remarkably uniform for four of the proteins, indicating a similarity of groups exposed during unfolding. One protein, staphylococcus nuclease, is anomalous in almost all respects. In general, the strength of the interaction with guanidinium is about twice that of urea, which is about twice that of trimethylamine-N-oxide and sucrose. Arguments are presented for the use of volume fractions in equilibrium equations and the ignoring of activity coefficients of

  13. An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii

    PubMed Central

    Liu, Jun; He, Yudou; Benmerzouga, Imaan; Sullivan, William J.; Morrissette, Naomi S.; Murray, John M.; Hu, Ke

    2016-01-01

    Although all microtubules within a single cell are polymerized from virtually identical subunits, different microtubule populations carry out specialized and diverse functions, including directional transport, force generation, and cellular morphogenesis. Functional differentiation requires specific targeting of associated proteins to subsets or even subregions of these polymers. The cytoskeleton of Toxoplasma gondii, an important human parasite, contains at least five distinct tubulin-based structures. In this work, we define the differential localization of proteins along the cortical microtubules of T. gondii, established during daughter biogenesis and regulated by protein expression and exchange. These proteins distinguish cortical from mitotic spindle microtubules, even though the assembly of these subsets is contemporaneous during cell division. Finally, proteins associated with cortical microtubules collectively protect the stability of the polymers with a remarkable degree of functional redundancy. PMID:26680740

  14. An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii.

    PubMed

    Liu, Jun; He, Yudou; Benmerzouga, Imaan; Sullivan, William J; Morrissette, Naomi S; Murray, John M; Hu, Ke

    2016-02-01

    Although all microtubules within a single cell are polymerized from virtually identical subunits, different microtubule populations carry out specialized and diverse functions, including directional transport, force generation, and cellular morphogenesis. Functional differentiation requires specific targeting of associated proteins to subsets or even subregions of these polymers. The cytoskeleton of Toxoplasma gondii, an important human parasite, contains at least five distinct tubulin-based structures. In this work, we define the differential localization of proteins along the cortical microtubules of T. gondii, established during daughter biogenesis and regulated by protein expression and exchange. These proteins distinguish cortical from mitotic spindle microtubules, even though the assembly of these subsets is contemporaneous during cell division. Finally, proteins associated with cortical microtubules collectively protect the stability of the polymers with a remarkable degree of functional redundancy. PMID:26680740

  15. Mitotic phosphorylation of Bloom helicase at Thr182 is required for its proteasomal degradation and maintenance of chromosomal stability.

    PubMed

    Kharat, S S; Tripathi, V; Damodaran, A P; Priyadarshini, R; Chandra, S; Tikoo, S; Nandhakumar, R; Srivastava, V; Priya, S; Hussain, M; Kaur, S; Fishman, J B; Sengupta, S

    2016-02-25

    Mutations in Bloom helicase (BLM) lead to Bloom Syndrome (BS). BS is characterized by multiple clinical manifestations including predisposition to a wide spectrum of cancers. Studies have revealed the mechanism of BLM recruitment after stalled replication and its role during the repair of DNA damage. We now provide evidence that BLM undergoes K48-linked ubiquitylation and subsequent degradation during mitosis due to the E3 ligase, Fbw7α. Fbw7α carries out its function after GSK3β- and CDK2/cyclin A2-dependent phosphorylation events on Thr171 and Ser175 of BLM which lies within a well-defined phosphodegron, a sequence which is conserved in all primates. Phosphorylation on BLM Thr171 and Ser175 depends on prior phosphorylation at Thr182 by Chk1/Chk2. Thr182 phosphorylation not only controls BLM ubiquitylation and degradation during mitosis but is also a determinant for its localization on the ultrafine bridges. Consequently lack of Thr182 phosphorylation leads to multiple manifestations of chromosomal instability including increased levels of DNA damage, lagging chromatin, micronuclei formation, breaks and quadriradials. Hence Thr182 phosphorylation on BLM has two functions-it regulates BLM turnover during mitosis and also helps to maintain the chromosomal stability. PMID:26028025

  16. Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein

    PubMed Central

    Picone, Delia

    2016-01-01

    MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques. PMID:27340829

  17. Estimating conformation content of a protein using citrate-stabilized Au nanoparticles

    NASA Astrophysics Data System (ADS)

    Deka, Jashmini; Paul, Anumita; Chattopadhyay, Arun

    2010-08-01

    Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration.Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for

  18. Cyclic-AMP-dependent protein kinase A regulates apoptosis by stabilizing the BH3-only protein Bim.

    PubMed

    Moujalled, Diane; Weston, Ross; Anderton, Holly; Ninnis, Robert; Goel, Pranay; Coley, Andrew; Huang, David C S; Wu, Li; Strasser, Andreas; Puthalakath, Hamsa

    2011-01-01

    The proapoptotic Bcl2 homology domain 3(BH3)-only protein Bim is controlled by stringent post-translational regulation, predominantly through alterations in phosphorylation status. To identify new kinases involved in its regulation, we carried out a yeast two-hybrid screen using a non-spliceable variant of the predominant isoform--Bim(EL)--as the bait and identified the regulatory subunit of cyclic-AMP-dependent protein kinase A--PRKAR1A--as an interacting partner. We also show that protein kinase A (PKA) is a Bim(EL) isoform-specific kinase that promotes its stabilization. Inhibition of PKA or mutation of the PKA phosphorylation site within Bim(EL) resulted in its accelerated proteasome-dependent degradation. These results might have implications for human diseases that are characterized by abnormally increased PKA activity, such as the Carney complex and dilated cardiomyopathy. PMID:21151042

  19. Conformational stability of HPr: the histidine-containing phosphocarrier protein from Bacillus subtilis.

    PubMed Central

    Scholtz, J. M.

    1995-01-01

    The conformational stability of the histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been determined using a combination of thermal unfolding and solvent denaturation experiments. The urea-induced denaturation of HPr was monitored spectroscopically at fixed temperatures and thermal unfolding was performed in the presence of fixed concentrations of urea. These data were analyzed in several different ways to afford a measure of the cardinal parameters (delta Hg, Tg, delta Sg, and delta Cp) that describe the thermodynamics of folding for HPr. The method of Pace and Laurents (Pace CN, Laurents DV, 1989, Biochemistry 28:2520-2525) was used to estimate delta Cp as was a global analysis of the thermal- and urea-induced unfolding data. Each method used to analyze the data gives a similar value for delta Cp (1,170 +/- 50 cal mol-1K-1). Despite the high melting temperature for HPr (Tg = 73.5 degrees C), the maximum stability of the protein, which occurs at 26 degrees C, is quite modest (delta Gs = 4.2 kcal mol-1). In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of delta Cp, can be achieved using the method of Chen and Schellman (Chen B, Schellman JA, 1989, Biochemistry 28:685-691). A comparison of the different methods for the analysis of solvent denaturation curves is provided and the effects of urea on the thermal stability of this small globular protein are discussed. The methods presented will be of general utility in the characterization of the stability curve for many small proteins. PMID:7773175

  20. The von Hippel-Lindau tumor suppressor stabilizes novel plant homeodomain protein Jade-1.

    PubMed

    Zhou, Mina I; Wang, Hongmei; Ross, Jonathan J; Kuzmin, Igor; Xu, Chengen; Cohen, Herbert T

    2002-10-18

    The von Hippel-Lindau disease gene (VHL) is the causative gene for most adult renal cancers. However, the mechanism by which VHL protein functions as a renal tumor suppressor remains largely unknown. To identify low occupancy VHL protein partners with potential relevance to renal cancer, we screened a human kidney library against human VHL p30 using a yeast two-hybrid approach. Jade-1 (gene for Apoptosis and Differentiation in Epithelia) encodes a previously uncharacterized 64-kDa protein that interacts strongly with VHL protein and is most highly expressed in kidney. Jade-1 protein is short-lived and contains a candidate destabilizing (PEST) motif and plant homeodomains that are not required for the VHL interaction. Jade-1 is abundant in proximal tubule cells, which are clear-cell renal cancer precursors, and expression increases with differentiation. Jade-1 is expressed in cytoplasm and the nucleus diffusely and in speckles, where it partly colocalizes with VHL. VHL reintroduction into renal cancer cells increases endogenous Jade-1 protein abundance up to 10-fold. Furthermore, VHL increases Jade-1 protein half-life up to 3-fold. Thus, direct protein stabilization is identified as a new VHL function. Moreover, Jade-1 protein represents a novel candidate regulatory factor in VHL-mediated renal tumor suppression.

  1. Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteins

    PubMed Central

    Lee, Sung Chang; Bennett, Brad C.; Hong, Wen-Xu; Fu, Yu; Baker, Kent A.; Marcoux, Julien; Robinson, Carol V.; Ward, Andrew B.; Halpert, James R.; Stevens, Raymond C.; Stout, Charles David; Yeager, Mark J.; Zhang, Qinghai

    2013-01-01

    Amphiphile selection is a critical step for structural studies of membrane proteins (MPs). We have developed a family of steroid-based facial amphiphiles (FAs) that are structurally distinct from conventional detergents and previously developed FAs. The unique FAs stabilize MPs and form relatively small protein–detergent complexes (PDCs), a property considered favorable for MP crystallization. We attempted to crystallize several MPs belonging to different protein families, including the human gap junction channel protein connexin 26, the ATP binding cassette transporter MsbA, the seven-transmembrane G protein-coupled receptor-like bacteriorhodopsin, and cytochrome P450s (peripheral MPs). Using FAs alone or mixed with other detergents or lipids, we obtained 3D crystals of the above proteins suitable for X-ray crystallographic analysis. The fact that FAs enhance MP crystallizability compared with traditional detergents can be attributed to several properties, including increased protein stability, formation of small PDCs, decreased PDC surface flexibility, and potential to mediate crystal lattice contacts. PMID:23479627

  2. iStable: off-the-shelf predictor integration for predicting protein stability changes

    PubMed Central

    2013-01-01

    Background Mutation of a single amino acid residue can cause changes in a protein, which could then lead to a loss of protein function. Predicting the protein stability changes can provide several possible candidates for the novel protein designing. Although many prediction tools are available, the conflicting prediction results from different tools could cause confusion to users. Results We proposed an integrated predictor, iStable, with grid computing architecture constructed by using sequence information and prediction results from different element predictors. In the learning model, several machine learning methods were evaluated and adopted the support vector machine as an integrator, while not just choosing the majority answer given by element predictors. Furthermore, the role of the sequence information played was analyzed in our model, and an 11-window size was determined. On the other hand, iStable is available with two different input types: structural and sequential. After training and cross-validation, iStable has better performance than all of the element predictors on several datasets. Under different classifications and conditions for validation, this study has also shown better overall performance in different types of secondary structures, relative solvent accessibility circumstances, protein memberships in different superfamilies, and experimental conditions. Conclusions The trained and validated version of iStable provides an accurate approach for prediction of protein stability changes. iStable is freely available online at: http://predictor.nchu.edu.tw/iStable. PMID:23369171

  3. Molecular Simulations of Cotranslational Protein Folding: Fragment Stabilities, Folding Cooperativity, and Trapping in the Ribosome

    PubMed Central

    Elcock, Adrian H

    2006-01-01

    Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these

  4. A role for naturally occurring variation of the murine coronavirus spike protein in stabilizing association with the cellular receptor.

    PubMed

    Gallagher, T M

    1997-04-01

    Murine hepatitis virus (MHV), a coronavirus, initiates infection by binding to its cellular receptor (MHVR) via spike (S) proteins projecting from the virion membrane. The structures of these S proteins vary considerably among MHV strains, and this variation is generally considered to be important in determining the strain-specific pathologies of MHV infection, perhaps by affecting the interaction between MHV and the MHVR. To address the relationships between S variation and receptor binding, assays capable of measuring interactions between MHV and MHVR were developed. The assays made use of a novel soluble form of the MHVR, sMHVR-Ig, which comprised the virus-binding immunoglobulin-like domain of MHVR fused to the Fc portion of human immunoglobulin G1. sMHVR-Ig was stably expressed as a disulfide-linked dimer in human 293 EBNA cells and was immobilized to Sepharose-protein G via the Fc domain. The resulting Sepharose beads were used to adsorb radiolabelled MHV particles. At 4 degrees C, the beads specifically adsorbed two prototype MHV strains, MHV JHM (strain 4) and a tissue culture-adapted mutant of MHV JHM, the JHMX strain. A shift to 37 degrees C resulted in elution of JHM but not JHMX. This in vitro observation of JHM (but not JHMX) elution from its receptor at 37 degrees C was paralleled by a corresponding 37 degrees C elution of receptor-associated JHM (but not JHMX) from tissue culture cells. The basis for this difference in maintenance of receptor association was correlated with a large deletion mutation present within the JHMX S protein, as sMHVR-Ig exhibited relatively thermostable binding to vaccinia virus-expressed S proteins containing the deletion. These results indicate that naturally occurring mutations in the coronavirus S protein affect the stability of the initial interaction with the host cell and thus contribute to the likelihood of successful infection by incoming virions. These changes in virus entry features may result in coronaviruses

  5. MEK1 signaling promotes self-renewal and tumorigenicity of liver cancer stem cells via maintaining SIRT1 protein stabilization

    PubMed Central

    Cheng, Jiamin; Liu, Chungang; Liu, Limei; Chen, Xuejiao; Shan, Juanjuan; Shen, Junjie; Zhu, Wei; Qian, Cheng

    2016-01-01

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. This high mortality has been commonly attributed to the presence of residual cancer stem cells (CSCs). Meanwhile, MEK1 signaling is regarded as a key molecular in HCC maintenance and development. However, nobody has figured out the particular mechanisms that how MEK1 signaling regulates liver CSCs self-renewal. In this study, we show that inhibition or depletion of MEK1 can significantly decrease liver CSCs self-renewal and tumor growth both in vitro and vivo conditions. Furthermore, we demonstrate that MEK1 signaling promotes liver CSCs self-renewal and tumorigenicity by maintaining SIRT1 level. Mechanistically, MEK1 signaling keeps SIRT1 protein stabilization through activating SIRT1 ubiquitination, which inhibits proteasomal degradation. Clinical analysis shows that patients co-expression of MEK1 and SIRT1 are associated with poor survival. Our finding indicates that MEK1-SIRT1 can act as a novel diagnostic biomarker and inhibition of MEK1 may be a viable therapeutic option for targeting liver CSCs treatment. PMID:26967560

  6. Recombinant Protein-Stabilized Monodisperse Microbubbles with Tunable Size Using a Valve-Based Microfluidic Device

    PubMed Central

    2015-01-01

    Microbubbles are used as contrast enhancing agents in ultrasound sonography and more recently have shown great potential as theranostic agents that enable both diagnostics and therapy. Conventional production methods lead to highly polydisperse microbubbles, which compromise the effectiveness of ultrasound imaging and therapy. Stabilizing microbubbles with surfactant molecules that can impart functionality and properties that are desirable for specific applications would enhance the utility of microbubbles. Here we generate monodisperse microbubbles with a large potential for functionalization by combining a microfluidic method and recombinant protein technology. Our microfluidic device uses an air-actuated membrane valve that enables production of monodisperse microbubbles with narrow size distribution. The size of microbubbles can be precisely tuned by dynamically changing the dimension of the channel using the valve. The microbubbles are stabilized by an amphiphilic protein, oleosin, which provides versatility in controlling the functionalization of microbubbles through recombinant biotechnology. We show that it is critical to control the composition of the stabilizing agents to enable formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. Our protein-shelled microbubbles based on the combination of microfluidic generation and recombinant protein technology provide a promising platform for ultrasound-related applications. PMID:25265041

  7. Protein arginylation regulates cellular stress response by stabilizing HSP70 and HSP40 transcripts

    PubMed Central

    Deka, Kamalakshi; Singh, Archana; Chakraborty, Surajit; Mukhopadhyay, Rupak; Saha, Sougata

    2016-01-01

    ATE1-mediated post-translational addition of arginine to a protein has been shown to regulate activity, interaction, and stability of the protein substrates. Arginylation has been linked to many different stress conditions, namely ER stress, cytosolic misfolded protein stress, and nitrosative stress. However, clear understanding about the effect of arginylation in cellular stress responses is yet to emerge. In this study, we investigated the role of arginylation in heat-stress response. Our findings suggest that Ate1 knock out (KO) cells are more susceptible to heat stress compared with its wild-type counterparts due to the induction of apoptosis in KO cells. Gene expression analysis of inducible heat-shock proteins (HSP70.1, HSP70.3, and HSP40) showed induction of these genes in KO cells early in the heat shock, but were drastically diminished at the later period of heat shock. Further analysis revealed that loss of ATE1 drastically reduced the stability of all three HSP mRNAs. These phenotypes were greatly restored by overexpression of Ate1 in KO cells. Our findings show that arginylation plays a protective role during heat stress by regulating HSP gene expression and mRNA stability. PMID:27752365

  8. Effect of the compatible solute ectoine on the stability of the membrane proteins.

    PubMed

    Roychoudhury, Arpita; Haussinger, Dieter; Oesterhelt, Filipp

    2012-08-01

    Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.

  9. Tracking evolution of myoglobin stability in cetaceans using experimentally calibrated computational methods that account for generic protein relaxation.

    PubMed

    Holm, Jeppe; Dasmeh, Pouria; Kepp, Kasper P

    2016-07-01

    The evolution of cetaceans (whales, dolphins, and porpoises) from land to water is one of the most spectacular events in mammal evolution. It has been suggested that selection for higher myoglobin stability (∆G of folding) allowed whales to conquer the deep-diving niche. The stability of multi-site protein variants, including ancient proteins, is however hard to describe theoretically. From a compilation of experimental ∆∆G vs. ∆G we first find that protein substitutions are subject to large generic protein relaxation effects. Using this discovery, we develop a simple two-parameter model that predicts multi-site ∆∆G as accurately as standard methods do for single-site mutations and reproduces trends in contemporary myoglobin stabilities. We then apply this new method to the study of the evolution of Mb stability in cetaceans: With both methods the main change in stability (about 1kcal/mol) occurred very early, and stability was later relaxed in dolphins and porpoises, but was further increased in the sperm whales. This suggests that single proteins can affect whole organism evolution and indicates a role of Mb stability in the evolution of cetaceans. Transition to the deep-diving niche probably occurred already in the ancestor of contemporary baleen and toothed whales. In summary, we have discovered generic stability relaxation effects in proteins that, when incorporated into a simple model, improves the description of multi-site protein variants. PMID:27068539

  10. Tracking evolution of myoglobin stability in cetaceans using experimentally calibrated computational methods that account for generic protein relaxation.

    PubMed

    Holm, Jeppe; Dasmeh, Pouria; Kepp, Kasper P

    2016-07-01

    The evolution of cetaceans (whales, dolphins, and porpoises) from land to water is one of the most spectacular events in mammal evolution. It has been suggested that selection for higher myoglobin stability (∆G of folding) allowed whales to conquer the deep-diving niche. The stability of multi-site protein variants, including ancient proteins, is however hard to describe theoretically. From a compilation of experimental ∆∆G vs. ∆G we first find that protein substitutions are subject to large generic protein relaxation effects. Using this discovery, we develop a simple two-parameter model that predicts multi-site ∆∆G as accurately as standard methods do for single-site mutations and reproduces trends in contemporary myoglobin stabilities. We then apply this new method to the study of the evolution of Mb stability in cetaceans: With both methods the main change in stability (about 1kcal/mol) occurred very early, and stability was later relaxed in dolphins and porpoises, but was further increased in the sperm whales. This suggests that single proteins can affect whole organism evolution and indicates a role of Mb stability in the evolution of cetaceans. Transition to the deep-diving niche probably occurred already in the ancestor of contemporary baleen and toothed whales. In summary, we have discovered generic stability relaxation effects in proteins that, when incorporated into a simple model, improves the description of multi-site protein variants.

  11. Effect of lead on cytoskeletal protein stability in crucian carp Carassius auratus

    NASA Astrophysics Data System (ADS)

    Cheng, Jia; Zhang, Dongyi; Chu, Wuying; Liu, Fang; Liu, Zhen; Zhou, Ruixue; Meng, Tao; Zhang, Jianshe

    2008-11-01

    Inorganic lead (Pb) is one of the most common environmental pollutants. Much evidence indicates that Pb exposure could directly affect fish growth and development. In this study, we investigated the cytotoxic effects of Pb on cytoskeletal protein stability at both protein and mRNA level in crucian carp Carassius auratus. Pb(NO3)2 treatment in concentration of 100 μmol/L resulted in decreased expression of both α- and β-tubulin but γ-tubulin as assayed with SDS-PAGE, Western Blot, and ELISA. In vivo and in vitro analyses on protein expression of tubulins are consistent. The effect of Pb on mRNA expression varied among different tissues. Our results suggest that cytotoxicity of Pb at protein translation level is stronger than at mRNA expression level.

  12. Stability of some Cactaceae proteins based on fluorescence, circular dichroism, and differential scanning calorimetry measurements.

    PubMed

    Gorinstein, S; Zemser, M; Vargas-Albores, F; Ochoa, J L; Paredes-Lopez, O; Scheler, C; Aksu, S; Salnikow, J

    1999-02-01

    Characterization of three cactus proteins (native and denatured) from Machaerocereus gummosus (Pitahaya agria), Lophocereu schottii (Garambullo), and Cholla opuntia (Cholla), was based on electrophoretic, fluorescence, CD (circular dichroism), DSC (differential scanning calorimetry), and FT-IR (Fourier transform infrared) measurements. The obtained results of intrinsic fluorescence, DSC, and CD were dissimilar for the three species of cactus, providing evidence of differences in secondary and tertiary structures. Cactus proteins may be situated in the following order corresponding to their relative stability: Machaerocereus gummosus (Pitahaya agria) > Cholla opuntia (Cholla) > Lophocereu schottii (Garambullo). Thermodynamic properties of proteins and their changes upon denaturation (temperature of denaturation, enthalphy, and the number of ruptured hydrogen bonds) were correlated with the secondary structure of proteins and disappearance of alpha-helix.

  13. Stability of some Cactaceae proteins based on fluorescence, circular dichroism, and differential scanning calorimetry measurements.

    PubMed

    Gorinstein, S; Zemser, M; Vargas-Albores, F; Ochoa, J L; Paredes-Lopez, O; Scheler, C; Aksu, S; Salnikow, J

    1999-02-01

    Characterization of three cactus proteins (native and denatured) from Machaerocereus gummosus (Pitahaya agria), Lophocereu schottii (Garambullo), and Cholla opuntia (Cholla), was based on electrophoretic, fluorescence, CD (circular dichroism), DSC (differential scanning calorimetry), and FT-IR (Fourier transform infrared) measurements. The obtained results of intrinsic fluorescence, DSC, and CD were dissimilar for the three species of cactus, providing evidence of differences in secondary and tertiary structures. Cactus proteins may be situated in the following order corresponding to their relative stability: Machaerocereus gummosus (Pitahaya agria) > Cholla opuntia (Cholla) > Lophocereu schottii (Garambullo). Thermodynamic properties of proteins and their changes upon denaturation (temperature of denaturation, enthalphy, and the number of ruptured hydrogen bonds) were correlated with the secondary structure of proteins and disappearance of alpha-helix. PMID:10333299

  14. Understanding the role of hydrogen bonds in water dynamics and protein stability.

    PubMed

    Bianco, Valentino; Iskrov, Svilen; Franzese, Giancarlo

    2012-01-01

    The mechanisms of cold and pressure denaturation of proteins are a matter of debate, but it is commonly accepted that water plays a fundamental role in the process. It has been proposed that the denaturation process is related to an increase of hydrogen bonds among hydration water molecules. Other theories suggest that the causes of denaturation are the density fluctuations of surface water, or the destabilization of hydrophobic contacts as a consequence of water molecule inclusions inside the protein, especially at high pressures. We review some theories that have been proposed to give insight into this problem, and we describe a coarse-grained model of water that compares well with experiments for proteins' hydration water. We introduce its extension for a homopolymer in contact with the water monolayer and study it by Monte Carlo simulations in an attempt to understand how the interplay of water cooperativity and interfacial hydrogen bonds affects protein stability. PMID:23277668

  15. Protein Kinase M[Zeta] Is Essential for the Induction and Maintenance of Dopamine-Induced Long-Term Potentiation in Apical CA1 Dendrites

    ERIC Educational Resources Information Center

    Navakkode, Sheeja; Sajikumar, Sreedharan; Sacktor, Todd Charlton; Frey, Julietta U.

    2010-01-01

    Dopaminergic D1/D5-receptor-mediated processes are important for certain forms of memory as well as for a cellular model of memory, hippocampal long-term potentiation (LTP) in the CA1 region of the hippocampus. D1/D5-receptor function is required for the induction of the protein synthesis-dependent maintenance of CA1-LTP (L-LTP) through activation…

  16. Novel characterization of the HSPA2-stabilizing protein BAG6 in human spermatozoa.

    PubMed

    Bromfield, Elizabeth; Aitken, R John; Nixon, Brett

    2015-10-01

    While a large cohort of sperm surface receptors underpin sperm-oocyte adhesion processes, our recent work has revealed that the molecular chaperone Heat Shock Protein A2 (HSPA2) is a key regulator of zona pellucida-receptor complex assembly in our own species. Indeed, in the infertile population, spermatozoa that fail to interact with the zona pellucida of the oocyte consistently lack HSPA2 protein expression. While the mechanisms behind this protein deficiency are under consideration, BCL2-associated athanogene 6 (BAG6) has been identified as a key regulator of HSPA2 stability in mouse germ cells. However, in the human, the presence of BAG family proteins remains completely uncharacterized. Consequently, this study aimed to determine the presence of BAG6 in human sperm cells and to characterize its putative interaction with HSPA2 throughout sperm cell development. BAG6 was shown to co-localize with HSPA2 in human testicular germ cells and epididymal spermatozoa. Similarly, BAG6 was identified in the equatorial region of non-capacitated spermatozoa but underwent a marked relocation to the anterior region of the head upon the induction of capacitation in these cells. Protein-protein interaction assays revealed the stable interaction of BAG6 and HSPA2 proteins in mature spermatozoa. Furthermore, examination of the spermatozoa of infertile men with zona pellucida binding defects, related to a lack of HSPA2, revealed a concomitant deficiency in BAG6 protein expression. In view of the findings described in this study, we propose that BAG6 is likely a key regulator of HSPA2 stability/function in human germ cells. Moreover, its under-representation in spermatozoa with zona pellucida binding deficiency suggests that BAG6 may be an important candidate to study for a further understanding of male idiopathic infertility.

  17. Integrated Stability and Activity Control of the Drosophila Rbf1 Retinoblastoma Protein*

    PubMed Central

    Zhang, Liang; Wei, Yiliang; Pushel, Irina; Heinze, Karolin; Elenbaas, Jared; Henry, R. William; Arnosti, David N.

    2014-01-01

    The retinoblastoma (RB) family transcriptional corepressors regulate diverse cellular events including cell cycle, senescence, and differentiation. The activity and stability of these proteins are mediated by post-translational modifications; however, we lack a general understanding of how distinct modifications coordinately impact both of these properties. Previously, we showed that protein turnover and activity are tightly linked through an evolutionarily conserved C-terminal instability element (IE) in the Drosophila RB-related protein Rbf1; surprisingly, mutant proteins with enhanced stability were less, not more active. To better understand how activity and turnover are controlled in this model RB protein, we assessed the impact of Cyclin-Cdk kinase regulation on Rbf1. An evolutionarily conserved N-terminal threonine residue is required for Cyclin-Cdk response and showed a dominant impact on turnover and activity; however, specific residues in the C-terminal IE differentially impacted Rbf1 activity and turnover, indicating an additional level of regulation. Strikingly, specific IE mutations that impaired turnover but not activity induced dramatic developmental phenotypes in the Drosophila eye. Mutation of the highly conserved Lys-774 residue induced hypermorphic phenotypes that mimicked the loss of phosphorylation control; mutation of the corresponding codon of the human RBL2 gene has been reported in lung tumors. Our data support a model in which closely intermingled residues within the conserved IE govern protein turnover, presumably through interactions with E3 ligases, and protein activity via contacts with E2F transcription partners. Such functional relationships are likely to similarly impact mammalian RB family proteins, with important implications for development and disease. PMID:25049232

  18. Conformational Stability and Pathogenic Misfolding of the Integral Membrane Protein PMP22

    PubMed Central

    2016-01-01

    Despite broad biochemical relevance, our understanding of the physiochemical reactions that limit the assembly and cellular trafficking of integral membrane proteins remains superficial. In this work, we report the first experimental assessment of the relationship between the conformational stability of a eukaryotic membrane protein and the degree to which it is retained by cellular quality control in the secretory pathway. We quantitatively assessed both the conformational equilibrium and cellular trafficking of 12 variants of the α-helical membrane protein peripheral myelin protein 22 (PMP22), the intracellular misfolding of which is known to cause peripheral neuropathies associated with Charcot–Marie–Tooth disease (CMT). We show that the extent to which these mutations influence the energetics of Zn(II)-mediated PMP22 folding is proportional to the observed reduction in cellular trafficking efficiency. Strikingly, quantitative analyses also reveal that the reduction of motor nerve conduction velocities in affected patients is proportional to the extent of the mutagenic destabilization. This finding provides compelling evidence that the effects of these mutations on the energetics of PMP22 folding lie at the heart of the molecular basis of CMT. These findings highlight conformational stability as a key factor governing membrane protein biogenesis and suggest novel therapeutic strategies for CMT. PMID:26102530

  19. Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion.

    PubMed

    Le, T T; Van de Wiele, T; Do, T N H; Debyser, G; Struijs, K; Devreese, B; Dewettinck, K; Van Camp, J

    2012-05-01

    The milk fat globule membrane (MFGM) fraction refers to the thin film of polar lipids and membrane proteins that surrounds fat globules in milk. It is its unique biochemical composition that renders MFGM with some beneficial biological activities, such as anti-adhesive effects toward pathogens. However, a prerequisite for the putative bioactivity of MFGM is its stability during gastrointestinal digestion. We, therefore, subjected MFGM material, isolated from raw milk, to an in vitro enzymatic gastrointestinal digestion. Sodium dodecyl sulfate PAGE, in combination with 2 staining methods, Coomassie Blue and periodic acid Schiff staining, was used to evaluate polypeptide patterns of the digest, whereas mass spectrometry was used to confirm the presence of specific MFGM proteins. Generally, it was observed that glycoproteins showed higher resistance to endogenous proteases compared with non-glycosylated proteins. Mucin 1 displayed the highest resistance to digestion and a considerable part of this protein was still detected at its original molecular weight after gastric and small intestine digestion. Cluster of differentiation 36 was also quite resistant to pepsin. A significant part of periodic acid Schiff 6/7 survived the gastric digestion, provided that the lipid moiety was not removed from the MFGM material. Overall, MFGM glycoproteins are generally more resistant to gastrointestinal digestion than serum milk proteins and the presence of lipids, besides glycosylation, may protect MFGM glycoproteins from gastrointestinal digestion. This gastrointestinal stability makes MFGM glycoproteins amenable to further studies in which their putative health-promoting effects can be explored.

  20. Ccdc13 is a novel human centriolar satellite protein required for ciliogenesis and genome stability.

    PubMed

    Staples, Christopher J; Myers, Katie N; Beveridge, Ryan D D; Patil, Abhijit A; Howard, Anna E; Barone, Giancarlo; Lee, Alvin J X; Swanton, Charles; Howell, Michael; Maslen, Sarah; Skehel, J Mark; Boulton, Simon J; Collis, Spencer J

    2014-07-01

    Here, we identify coiled-coil domain-containing protein 13 (Ccdc13) in a genome-wide RNA interference screen for regulators of genome stability. We establish that Ccdc13 is a newly identified centriolar satellite protein that interacts with PCM1, Cep290 and pericentrin and prevents the accumulation of DNA damage during mitotic transit. Depletion of Ccdc13 results in the loss of microtubule organisation in a manner similar to PCM1 and Cep290 depletion, although Ccdc13 is not required for satellite integrity. We show that microtubule regrowth is enhanced in Ccdc13-depleted cells, but slowed in cells that overexpress Ccdc13. Furthermore, in serum-starved cells, Ccdc13 localises to the basal body, is required for primary cilia formation and promotes the localisation of the ciliopathy protein BBS4 to both centriolar satellites and cilia. These data highlight the emerging link between DNA damage response factors, centriolar and peri-centriolar satellites and cilia-associated proteins and implicate Ccdc13 as a centriolar satellite protein that functions to promote both genome stability and cilia formation. PMID:24816561

  1. Elongator Protein 3 (Elp3) stabilizes Snail1 and regulates neural crest migration in Xenopus

    PubMed Central

    Yang, Xiangcai; Li, Jiejing; Zeng, Wanli; Li, Chaocui; Mao, Bingyu

    2016-01-01

    Elongator protein 3 (Elp3) is the enzymatic unit of the elongator protein complex, a histone acetyltransferase complex involved in transcriptional elongation. It has long been shown to play an important role in cell migration; however, the underlying mechanism is unknown. Here, we showed that Elp3 is expressed in pre-migratory and migrating neural crest cells in Xenopus embryos, and knockdown of Elp3 inhibited neural crest cell migration. Interestingly, Elp3 binds Snail1 through its zinc-finger domain and inhibits its ubiquitination by β-Trcp without interfering with the Snail1/Trcp interaction. We showed evidence that Elp3-mediated stabilization of Snail1 was likely involved in the activation of N-cadherin in neural crest cells to regulate their migratory ability. Our findings provide a new mechanism for the function of Elp3 in cell migration through stabilizing Snail1, a master regulator of cell motility. PMID:27189455

  2. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes

    PubMed Central

    Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

    2016-01-01

    The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains—In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model. PMID:27763556

  3. Folding 19 proteins to their native state and stability of large proteins from a coarse-grained model.

    PubMed

    Kapoor, Abhijeet; Travesset, Alex

    2014-03-01

    We develop an intermediate resolution model, where the backbone is modeled with atomic resolution but the side chain with a single bead, by extending our previous model (Proteins (2013) DOI: 10.1002/prot.24269) to properly include proline, preproline residues and backbone rigidity. Starting from random configurations, the model properly folds 19 proteins (including a mutant 2A3D sequence) into native states containing β sheet, α helix, and mixed α/β. As a further test, the stability of H-RAS (a 169 residue protein, critical in many signaling pathways) is investigated: The protein is stable, with excellent agreement with experimental B-factors. Despite that proteins containing only α helices fold to their native state at lower backbone rigidity, and other limitations, which we discuss thoroughly, the model provides a reliable description of the dynamics as compared with all atom simulations, but does not constrain secondary structures as it is typically the case in more coarse-grained models. Further implications are described.

  4. Brh2 and Rad51 promote telomere maintenance in Ustilago maydis, a new model system of DNA repair proteins at telomeres.

    PubMed

    Yu, Eun Young; Kojic, Milorad; Holloman, William K; Lue, Neal F

    2013-07-01

    Recent studies implicate a number of DNA repair proteins in mammalian telomere maintenance. However, because several key repair proteins in mammals are missing from the well-studied budding and fission yeast, their roles at telomeres cannot be modeled in standard fungi. In this report, we explored the dimorphic fungus Ustilago maydis as an alternative model for telomere research. This fungus, which belongs to the phylum Basidiomycota, has a telomere repeat unit that is identical to the mammalian repeat, as well as a constellation of DNA repair proteins that more closely mimic the mammalian collection. We showed that the two core components of homology-directed repair (HDR) in U. maydis, namely Brh2 and Rad51, both promote telomere maintenance in telomerase positive cells, just like in mammals. In addition, we found that Brh2 is localized to telomeres in vivo, suggesting that it acts directly at chromosome ends. We surveyed a series of mutants with DNA repair defects, and found many of them to have short telomeres. Our results indicate that factors involved in DNA repair are probably also needed for optimal telomere maintenance in U. maydis, and that this fungus is a useful alternative model system for telomere research.

  5. Alternative splicing of parathyroid hormone-related protein mRNA: expression and stability

    PubMed Central

    Sellers, R S; Luchin, A I; Richard, V; Brena, R M; Lima, D; Rosol, T J

    2011-01-01

    Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that is often dysregulated in cancer. The human PTHrP gene is alternatively spliced into three isoforms, each with a unique 3′-untranslated region (3′-UTR), encoding 139, 173 and 141 amino acid proteins. The regulation of PTHrP mRNA isoform expression has not been completely elucidated, but it may be affected by transforming growth factor-β1 (TGF-β1). In this study, we examined differences in the PTHrP mRNA isoform expression in two squamous carcinoma cell lines (SCC2/88 and HARA), an immortalized keratinocyte cell line (HaCaT), and spontaneous human lung cancer with adjacent normal tissue. In addition, the effect of TGF-β1 on PTHrP mRNA isoform expression and stability was examined. Cell-type specific expression of PTHrP mRNA isoforms occurred between the various cell lines, normal human lung, and immortalized human keratinocytes (HaCaT). PTHrP isoform expression pattern was significantly altered between normal lung tissue and the adjacent lung cancer. In vitro studies revealed that TGF-β1 differentially altered the mRNA steady-state levels and mRNA stability of the PTHrP isoforms. Protein–RNA binding studies identified different proteins binding to the 3′-UTR of the PTHrP isoforms (139) and (141), which may be important in the differential mRNA stability and response to cytokines between the PTHrP isoforms. The data demonstrate that there is cell-type specific expression of PTHrP mRNA isoforms, and disruption of the normal regulation during cancer progression may in part be associated with TGF-β1-induced changes in PTHrP mRNA isoform expression and stability. PMID:15291755

  6. Co-encapsulation of lyoprotectants improves the stability of protein-loaded PLGA nanoparticles upon lyophilization.

    PubMed

    Fonte, Pedro; Araújo, Francisca; Seabra, Vítor; Reis, Salette; van de Weert, Marco; Sarmento, Bruno

    2015-12-30

    The purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants with insulin into PLGA nanoparticles, on the stability of the protein and nanoparticles upon lyophilization. Different lyoprotectants were used, namely trehalose, glucose, sucrose, fructose and sorbitol at 10% (w/v). Insulin-loaded PLGA nanoparticles with co-encapsulated lyoprotectants achieved a mean particle size of 386-466nm, and a zeta potential ranging between -34 and -38mV, dependent on the lyoprotectant used. Formulations had association efficiencies and loading capacities of 85-91% and 10-12%, respectively. The lyophilization process increased the colloidal stability of nanoparticles, and maintained their spherical shape and smooth surface, particularly in presence of lyoprotectants. XRPD revealed that the lyophilizates of nanoparticles with co-encapsulated lyoprotectants were amorphous, whereas formulations with externally added lyoprotectants, except trehalose, showed crystallinity. FTIR assessment showed that co-encapsulating lyoprotectants better preserved insulin structure upon lyophilization with a spectral area overlap of 82-87%, compared to only 72% in lyoprotectant absence. These results were confirmed by circular dichroism spectroscopy. Surprisingly, the simultaneous co-encapsulation and addition of lyoprotectants was detrimental to protein stabilization. The insulin in vitro release studies demonstrated that formulations with co-encapsulated trehalose, glucose, sucrose, fructose and sorbitol achieved 83%, 69%, 70%, 77% and 74%, respectively after 48h. In contrast, formulations added with those lyoprotectants prior lyophilization showed a lower release rate not higher than 60% after 48h. This work gives rise to a different promising strategy of co-encapsulating lyoprotectants and therapeutic proteins, to better stabilize protein structure upon lyophilization.

  7. Sequence-only evolutionary and predicted structural features for the prediction of stability changes in protein mutants

    PubMed Central

    2013-01-01

    Background Even a single amino acid substitution in a protein sequence may result in significant changes in protein stability, structure, and therefore in protein function as well. In the post-genomic era, computational methods for predicting stability changes from only the sequence of a protein are of importance. While evolutionary relationships of protein mutations can be extracted from large protein databases holding millions of protein sequences, relevant evolutionary features for the prediction of stability changes have not been proposed. Also, the use of predicted structural features in situations when a protein structure is not available has not been explored. Results We proposed a number of evolutionary and predicted structural features for the prediction of stability changes and analysed which of them capture the determinants of protein stability the best. We trained and evaluated our machine learning method on a non-redundant data set of experimentally measured stability changes. When only the direction of the stability change was predicted, we found that the best performance improvement can be achieved by the combination of the evolutionary features mutation likelihood and SIFTscore in conjunction with the predicted structural feature secondary structure. The same two evolutionary features in the combination with the predicted structural feature accessible surface area achieved the lowest error when the prediction of actual values of stability changes was assessed. Compared to similar studies, our method achieved improvements in prediction performance. Conclusion Although the strongest feature for the prediction of stability changes appears to be the vector of amino acid identities in the sequential neighbourhood of the mutation, the most relevant combination of evolutionary and predicted structural features further improves prediction performance. Even the predicted structural features, which did not perform well on their own, turn out to be beneficial

  8. How hydrophobicity and the glycosylation site of glycans affect protein folding and stability: a molecular dynamics simulation.

    PubMed

    Lu, Diannan; Yang, Cheng; Liu, Zheng

    2012-01-12

    Glycosylation is one of the most common post-translational modifications in the biosynthesis of protein, but its effect on the protein conformational transitions underpinning folding and stabilization is poorly understood. In this study, we present a coarse-grained off-lattice 46-β barrel model protein glycosylated by glycans with different hydrophobicity and glycosylation sites to examine the effect of glycans on protein folding and stabilization using a Langevin dynamics simulation, in which an H term was proposed as the index of the hydrophobicity of glycan. Compared with its native counterpart, introducing glycans of suitable hydrophobicity (0.1 < H < 0.4) at flexible peptide residues of this model protein not only facilitated folding of the protein but also increased its conformation stability significantly. On the contrary, when glycans were introduced at the restricted peptide residues of the protein, only those hydrophilic (H = 0) or very weak hydrophobic (H < 0.2) ones contributed slightly to protein stability but hindered protein folding due to increased free energy barriers. The glycosylated protein retained the two-step folding mechanism in terms of hydrophobic collapse and structural rearrangement. Glycan chains located in a suitable site with an appropriate hydrophobicity facilitated both collapse and rearrangement, whereas others, though accelerating collapse, hindered rearrangement. In addition to entropy effects, that is, narrowing the space of the conformations of the unfolded state, the presence of glycans with suitable hydrophobicity at suitable glycosylation site strengthened the folded state via hydrophobic interaction, that is, the enthalpy effect. The simulations have shown both the stabilization and the destabilization effects of glycosylation, as experimentally reported in the literature, and provided molecular insight into glycosylated proteins. The understanding of the effects of glycans with different hydrophobicities on the folding

  9. Insights into Hemoglobin Assembly through in Vivo Mutagenesis of α-Hemoglobin Stabilizing Protein*

    PubMed Central

    Khandros, Eugene; Mollan, Todd L.; Yu, Xiang; Wang, Xiaomei; Yao, Yu; D'Souza, Janine; Gell, David A.; Olson, John S.; Weiss, Mitchell J.

    2012-01-01

    α-Hemoglobin stabilizing protein (AHSP) is believed to facilitate adult Hemoglobin A assembly and protect against toxic free α-globin subunits. Recombinant AHSP binds multiple forms of free α-globin to stabilize their structures and inhibit precipitation. However, AHSP also stimulates autooxidation of αO2 subunit and its rapid conversion to a partially unfolded bishistidyl hemichrome structure. To investigate these biochemical properties, we altered the evolutionarily conserved AHSP proline 30 in recombinantly expressed proteins and introduced identical mutations into the endogenous murine Ahsp gene. In vitro, the P30W AHSP variant bound oxygenated α chains with 30-fold increased affinity. Both P30W and P30A mutant proteins also caused decreased rates of αO2 autooxidation as compared with wild-type AHSP. Despite these abnormalities, mice harboring P30A or P30W Ahsp mutations exhibited no detectable defects in erythropoiesis at steady state or during induced stresses. Further biochemical studies revealed that the AHSP P30A and P30W substitutions had minimal effects on AHSP interactions with ferric α subunits. Together, our findings indicate that the ability of AHSP to stabilize nascent α chain folding intermediates prior to hemin reduction and incorporation into adult Hemoglobin A is physiologically more important than AHSP interactions with ferrous αO2 subunits. PMID:22287545

  10. Insights into hemoglobin assembly through in vivo mutagenesis of α-hemoglobin stabilizing protein.

    PubMed

    Khandros, Eugene; Mollan, Todd L; Yu, Xiang; Wang, Xiaomei; Yao, Yu; D'Souza, Janine; Gell, David A; Olson, John S; Weiss, Mitchell J

    2012-03-30

    α-Hemoglobin stabilizing protein (AHSP) is believed to facilitate adult Hemoglobin A assembly and protect against toxic free α-globin subunits. Recombinant AHSP binds multiple forms of free α-globin to stabilize their structures and inhibit precipitation. However, AHSP also stimulates autooxidation of αO(2) subunit and its rapid conversion to a partially unfolded bishistidyl hemichrome structure. To investigate these biochemical properties, we altered the evolutionarily conserved AHSP proline 30 in recombinantly expressed proteins and introduced identical mutations into the endogenous murine Ahsp gene. In vitro, the P30W AHSP variant bound oxygenated α chains with 30-fold increased affinity. Both P30W and P30A mutant proteins also caused decreased rates of αO(2) autooxidation as compared with wild-type AHSP. Despite these abnormalities, mice harboring P30A or P30W Ahsp mutations exhibited no detectable defects in erythropoiesis at steady state or during induced stresses. Further biochemical studies revealed that the AHSP P30A and P30W substitutions had minimal effects on AHSP interactions with ferric α subunits. Together, our findings indicate that the ability of AHSP to stabilize nascent α chain folding intermediates prior to hemin reduction and incorporation into adult Hemoglobin A is physiologically more important than AHSP interactions with ferrous αO(2) subunits.

  11. Impact of extractables/leachables from filters on stability of protein formulations.

    PubMed

    Huang, Min; Horwitz, Teresa S; Zweiben, Cindy; Singh, Satish K

    2011-11-01

    Aqueous extractables/leachables from three sterilizing-grade filter membranes [polyvinylidene fluoride (PVDF), polyethersulfone (PES), and mixed cellulose ester (MCE)] were found to significantly reduce the surface tension of aqueous solutions. To evaluate the effect of these extractables/leachables from filter membranes on stability of protein formulations, model IgG2 formulations (with or without added surfactant) were spiked with different levels of filter extractables from stock solutions as a stress study. The stock solutions of extractables were created by processing the filter membranes through autoclaving and soaking steps. The IgG2 formulations were subsequently subject to agitation and temperature stress. Extractables/leachables from the filters were found to have a significant protective (PVDF, PES) and destabilizing (MCE) impact on both visible and subvisible particulates formation under agitation stress for formulations that did not contain any additional surfactant such as polysorbate 80. The impact of filter extractables/leachables on chemical stability of the antibody formulation displayed a more complicated pattern, but was generally destabilizing, causing increases in aggregation, oxidation, and acidic species. In conclusion, extractables/leachables from filter membranes may have impact on protein formulation stability and caution should be exercised during protein filtration, especially when filtering small volumes and in preformulation or high-throughput screening studies.

  12. Stabilizing effects of G protein on the active conformation of adenosine A1 receptor differ depending on G protein type.

    PubMed

    Tateyama, Michihiro; Kubo, Yoshihiro

    2016-10-01

    G protein coupled receptors (GPCRs) trigger various cellular and physiological responses upon the ligand binding. The ligand binding induces conformational change in GPCRs which allows G protein to interact with the receptor. The interaction of G protein also affects the active conformation of GPCRs. In this study, we have investigated the effects of Gαi1, Gαo and chimeric Gαqi5 on the active conformation of the adenosine A1 receptor, as each Gα showed difference in the interaction with adenosine A1 receptor. The conformational changes in the adenosine A1 receptor were detected as the agonist-induced decreases in efficiency of Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) fused at the two intracellular domains of the adenosine A1 receptor. Amplitudes of the agonist-induced FRET decreases were subtle when the FP-tagged adenosine A1 receptor was expressed alone, whereas they were significantly enhanced when co-expressed with Gαi1Gβ1Gγ22 (Gi1) or Gαqi5Gβ1Gγ22 (Gqi5) but not with GαοGβ1Gγ22 (Go). The enhancement of the agonist-induced FRET decrease in the presence of Gqi5 was significantly larger than that of Gi1. Furthermore, the FRET recovery upon the agonist removal in the presence of Gqi5 was significantly slower than that of Gi1. From these results it was revealed that the agonist-bound active conformation of adenosine A1 receptor is unstable without the binding of G protein and that the stabilizing effects of G protein differ depending on the types of G protein.

  13. Stability and immunogenicity of hypoallergenic peanut protein-polyphenol complexes during in vitro pepsin digestion.

    PubMed

    Plundrich, Nathalie J; White, Brittany L; Dean, Lisa L; Davis, Jack P; Foegeding, E Allen; Lila, Mary Ann

    2015-07-01

    Allergenic peanut proteins are relatively resistant to digestion, and if digested, metabolized peptides tend to remain large and immunoreactive, triggering allergic reactions in sensitive individuals. In this study, the stability of hypoallergenic peanut protein-polyphenol complexes was evaluated during simulated in vitro gastric digestion. When digested with pepsin, the basic subunit of the peanut allergen Ara h 3 was more rapidly hydrolyzed in peanut protein-cranberry or green tea polyphenol complexes compared to uncomplexed peanut flour. Ara h 2 was also hydrolyzed more quickly in the peanut protein-cranberry polyphenol complex than in uncomplexed peanut flour. Peptides from peanut protein-cranberry polyphenol complexes and peanut protein-green tea polyphenol complexes were substantially less immunoreactive (based on their capacity to bind to peanut-specific IgE from patient plasma) compared to peptides from uncomplexed peanut flour. These results suggest that peanut protein-polyphenol complexes may be less immunoreactive passing through the digestive tract in vivo, contributing to their attenuated allergenicity.

  14. Effect of Temperature and Pressure on the Stability of Protein Microbubbles.

    PubMed

    Rovers, Tijs A M; Sala, Guido; van der Linden, Erik; Meinders, Marcel B J

    2016-01-13

    Protein microbubbles are air bubbles with a network of interacting proteins at the air-water interface. Protein microbubbles are commonly used in medical diagnostic and therapeutic research. They have also recently gained interest in the research area of food as they can be used as structural elements to control texture, allowing for the manufacture of healthier foods with increased consumer perception. For the application of microbubbles in the food industry, it is important to gain insights into their stability under food processing conditions. In this study, we tested the stability of protein microbubbles against heating and pressurization. Microbubbles could be heated to 50 °C for 2 min or pressurized to 100 kPa overpressure for 15 s without significantly affecting their stability. At higher pressures and temperatures, the microbubbles became unstable and buckled. Buckling was observed above a critical pressure and was influenced by the shell modulus. The addition of cross-linkers like glutaraldehyde and tannic acid resulted in microbubbles that were stable against all tested temperatures and overpressures, more specifically, up to 120 °C and 470 kPa, respectively. We found a relation between the storage temperatures of microbubble dispersions (4, 10, 15, and 21 °C) and a decrease in the number of microbubbles with the highest decrease at the highest storage temperature. The average rupture time of microbubbles stored at different storage temperatures followed an Arrhenius relation with an activation energy for rupture of the shell of approximately 27 kT. This strength ensures applicability of microbubbles in food processes only at moderate temperatures and storage for a moderate period of time. After the proteins in the shell are cross-linked, the microbubbles can withstand pressures and temperatures that are representative of food processes.

  15. Effect of Temperature and Pressure on the Stability of Protein Microbubbles.

    PubMed

    Rovers, Tijs A M; Sala, Guido; van der Linden, Erik; Meinders, Marcel B J

    2016-01-13

    Protein microbubbles are air bubbles with a network of interacting proteins at the air-water interface. Protein microbubbles are commonly used in medical diagnostic and therapeutic research. They have also recently gained interest in the research area of food as they can be used as structural elements to control texture, allowing for the manufacture of healthier foods with increased consumer perception. For the application of microbubbles in the food industry, it is important to gain insights into their stability under food processing conditions. In this study, we tested the stability of protein microbubbles against heating and pressurization. Microbubbles could be heated to 50 °C for 2 min or pressurized to 100 kPa overpressure for 15 s without significantly affecting their stability. At higher pressures and temperatures, the microbubbles became unstable and buckled. Buckling was observed above a critical pressure and was influenced by the shell modulus. The addition of cross-linkers like glutaraldehyde and tannic acid resulted in microbubbles that were stable against all tested temperatures and overpressures, more specifically, up to 120 °C and 470 kPa, respectively. We found a relation between the storage temperatures of microbubble dispersions (4, 10, 15, and 21 °C) and a decrease in the number of microbubbles with the highest decrease at the highest storage temperature. The average rupture time of microbubbles stored at different storage temperatures followed an Arrhenius relation with an activation energy for rupture of the shell of approximately 27 kT. This strength ensures applicability of microbubbles in food processes only at moderate temperatures and storage for a moderate period of time. After the proteins in the shell are cross-linked, the microbubbles can withstand pressures and temperatures that are representative of food processes. PMID:26619225

  16. Stability of buffer-free freeze-dried formulations: A feasibility study of a monoclonal antibody at high protein concentrations.

    PubMed

    Garidel, Patrick; Pevestorf, Benjamin; Bahrenburg, Sven

    2015-11-01

    We studied the stability of freeze-dried therapeutic protein formulations over a range of initial concentrations (from 40 to 160 mg/mL) and employed a variety of formulation strategies (including buffer-free freeze dried formulations, or BF-FDF). Highly concentrated, buffer-free liquid formulations of therapeutic monoclonal antibodies (mAbs) have been shown to be a viable alternative to conventionally buffered preparations. We considered whether it is feasible to use the buffer-free strategy in freeze-dried formulations, as an answer to some of the known drawbacks of conventional buffers. We therefore conducted an accelerated stability study (24 weeks at 40 °C) to assess the feasibility of stabilizing freeze-dried formulations without "classical" buffer components. Factors monitored included pH stability, protein integrity, and protein aggregation. Because the protein solutions are inherently self-buffering, and the system's buffer capacity scales with protein concentration, we included highly concentrated buffer-free freeze-dried formulations in the study. The tested formulations ranged from "fully formulated" (containing both conventional buffer and disaccharide stabilizers) to "buffer-free" (including formulations with only disaccharide lyoprotectant stabilizers) to "excipient-free" (with neither added buffers nor stabilizers). We evaluated the impacts of varying concentrations, buffering schemes, pHs, and lyoprotectant additives. At the end of 24 weeks, no change in pH was observed in any of the buffer-free formulations. Unbuffered formulations were found to have shorter reconstitution times and lower opalescence than buffered formulations. Protein stability was assessed by visual inspection, sub-visible particle analysis, protein monomer content, charge variants analysis, and hydrophobic interaction chromatography. All of these measures found the stability of buffer-free formulations that included a disaccharide stabilizer comparable to buffer

  17. Comparative effects of cryosolvents on tubulin association, thermal stability, and binding of microtubule-associated proteins.

    PubMed

    Pajot-Augy, E

    1993-06-01

    Organic cryosolvents essential for cryopreservation of living cells have a colligative effect on water properties, but also affect cellular structures such as the membrane, actin, or tubulin cytoskeleton. The effects of cryosolvents on actin and its binding proteins are starting to be well investigated. In parallel, tubulin assembly characteristics were investigated comparatively, with 0-30% 1,2-propanediol, dimethyl sulfoxide, or glycerol, and with or without microtubule-associated proteins, at 37 or 4 degrees C. Tubulin association was monitored by spectrometry and sedimentation, providing the concentration in free protein, cold-depolymerizable microtubules, and cold-resistant associations. At 37 degrees C, 1,2-propanediol and dimethyl sulfoxide induce a similar association level and cold stability of the assemblies. Glycerol yields a lower level of tubulin association. Cold stability of the assemblies requires the presence of solvent, the amount of which is modulated by microtubule-associated proteins (MAPs): 15% 1,2-propanediol or dimethyl sulfoxide, decreasing down to 10% with MAPs, or 10% glycerol with MAPs only. At 4 degrees C, some cold-stable association is promoted by 1,2-propanediol or dimethyl sulfoxide above 10-15%, in the presence or absence of MAPs, but not with glycerol. In addition, protein content of the various fractions obtained with MAPs and 30% solvent was examined by densitometry of electrophoresis gels. Cold-labile associations obtained at 37 degrees C with 1,2-propanediol or dimethyl sulfoxide are lacking in tubulin and enriched in tau proteins relative to control or glycerol. Associations formed at 37 degrees C and stable to subsequent cold treatment, or at 4 degrees C, regardless of the solvent, present a large tubulin content, as well as few tau proteins and high-molecular-weight MAPs.

  18. Wheat proteins enhance stability and function of adhesion molecules in cryopreserved hepatocytes.

    PubMed

    Grondin, Mélanie; Hamel, Francine; Averill-Bates, Diana A; Sarhan, Fathey

    2009-01-01

    Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell-cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (beta1-integrin, E-cadherin, and beta-catenin). Immunoblot analyses revealed that the levels of beta1-integrin, E-cadherin, and beta-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for beta1-integrin (62-74%) > beta-catenin (51-58%) > E-cadherin (21-37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of beta1-integrin, E-cadherin, and beta-catenin. Protein expression was only 10-20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good

  19. A novel mechanism for antiglycative action of limonene through stabilization of protein conformation.

    PubMed

    Joglekar, Madhav M; Panaskar, Shrimant N; Chougale, Ashok D; Kulkarni, Mahesh J; Arvindekar, Akalpita U

    2013-10-01

    Inhibition of protein glycation is known to ameliorate secondary complications in diabetes. In the present study antiglycative properties of limonene, a natural product, were evaluated using BSA as a model protein. AMG (aminoguanidine) was used as a positive control. Measurement of total AGEs (Advanced Glycation End-products) and specific AGEs revealed that limonene could inhibit protein glycation to the extent of 56.3% and 75.1% respectively at 50 μM concentration as against 54.4% and 82.2% by AMG at 1 mM. Congo red binding and CD (Circular Dichroism) analysis revealed inhibition of α-helix to β-sheet transition wherein 18.5% β-sheet structures were observed in glycated BSA (bovine serum albumin) as against 4.9% with limonene. Glycation of protein in the presence of urea was enhanced by 18%, while in the presence of limonene it was reduced by 23% revealing the stabilizing effect of limonene. Electrophoretic mobility was similar to the normal control and a zeta potential value of -12.1 mV as against -15.1 mV in diabetic control was observed. Inhibition of glycation in limonene treated samples was confirmed through LC-MS analysis wherein AGEs such as pentosidine, CML (N(ε)-(carboxymethyl)lysine), CEL (N(ε)-(carboxyethyl)lysine), MOLD (methylglyoxal-lysine dimer) and imidazolone observed in glycated samples were absent in limonene treated samples. PatchDock studies revealed that limonene could bind to the major glycation sites IB, IIA and IIB sub domains and AMG to the IIIA sub domain. Thus limonene is a potent protein glycation inhibitor that prevents protein glycation through a novel mechanism of stabilization of protein structure through hydrophobic interactions.

  20. Mollusc-Algal Chloroplast Endosymbiosis. Photosynthesis, Thylakoid Protein Maintenance, and Chloroplast Gene Expression Continue for Many Months in the Absence of the Algal Nucleus1

    PubMed Central

    Green, Brian J.; Li, Wei-Ye; Manhart, James R.; Fox, Theodore C.; Summer, Elizabeth J.; Kennedy, Robert A.; Pierce, Sidney K.; Rumpho, Mary E.

    2000-01-01

    Early in its life cycle, the marine mollusc Elysia chlorotica Gould forms an intracellular endosymbiotic association with chloroplasts of the chromophytic alga Vaucheria litorea C. Agardh. As a result, the dark green sea slug can be sustained in culture solely by photoautotrophic CO2 fixation for at least 9 months if provided with only light and a source of CO2. Here we demonstrate that the sea slug symbiont chloroplasts maintain photosynthetic oxygen evolution and electron transport activity through photosystems I and II for several months in the absence of any external algal food supply. This activity is correlated to the maintenance of functional levels of chloroplast-encoded photosystem proteins, due in part at least to de novo protein synthesis of chloroplast proteins in the sea slug. Levels of at least one putative algal nuclear encoded protein, a light-harvesting complex protein homolog, were also maintained throughout the 9-month culture period. The chloroplast genome of V. litorea was found to be 119.1 kb, similar to that of other chromophytic algae. Southern analysis and polymerase chain reaction did not detect an algal nuclear genome in the slug, in agreement with earlier microscopic observations. Therefore, the maintenance of photosynthetic activity in the captured chloroplasts is regulated solely by the algal chloroplast and animal nuclear genomes. PMID:10982447

  1. Memory reconsolidation and its maintenance depend on L-voltage-dependent calcium channels and CaMKII functions regulating protein turnover in the hippocampus.

    PubMed

    Da Silva, Weber Cláudio; Cardoso, Gabriela; Bonini, Juliana Sartori; Benetti, Fernando; Izquierdo, Ivan

    2013-04-16

    Immediate postretrieval bilateral blockade of long-acting voltage-dependent calcium channels (L-VDCCs), but not of glutamatergic NMDA receptors, in the dorsal CA1 region of the hippocampus hinders retention of long-term spatial memory in the Morris water maze. Immediate postretrieval bilateral inhibition of calcium/calmodulin-dependent protein kinase (CaMK) II in dorsal CA1 does not affect retention of this task 24 h later but does hinder it 5 d later. These two distinct amnesic effects are abolished if protein degradation by proteasomes is inhibited concomitantly. These results indicate that spatial memory reconsolidation depends on the functionality of L-VDCC in dorsal CA1, that maintenance of subsequent reconsolidated memory trace depends on CaMKII, and these results also suggest that the role played by both L-VDCC and CaMKII is to promote the retrieval-dependent, synaptically localized enhancement of protein synthesis necessary to counteract a retrieval-dependent, synaptic-localized enhancement of protein degradation, which has been described as underlying the characteristic labilization of the memory trace triggered by retrieval. Thus, conceivably, L-VDCC and CaMKII would enhance activity-dependent localized protein renewal, which may account for the improvement of the long-term efficiency of the synapses responsible for the maintenance of reactivated long-term spatial memory.

  2. Identification of Stabilizing Mutations in an H5 Hemagglutinin Influenza Virus Protein

    PubMed Central

    Hanson, Anthony; Imai, Masaki; Hatta, Masato; McBride, Ryan; Imai, Hirotaka; Taft, Andrew; Zhong, Gongxun; Watanabe, Tokiko; Suzuki, Yasuo; Neumann, Gabriele; Paulson, James C.

    2015-01-01

    ABSTRACT Highly pathogenic avian influenza viruses of the H5N1 subtype continue to circulate in poultry in Asia, Africa, and the Middle East. Recently, outbreaks of novel reassortant H5 viruses have also occurred in North America. Although the number of human infections with highly pathogenic H5N1 influenza viruses continues to rise, these viruses remain unable to efficiently transmit between humans. However, we and others have identified H5 viruses capable of respiratory droplet transmission in ferrets. Two experimentally introduced mutations in the viral hemagglutinin (HA) receptor-binding domain conferred binding to human-type receptors but reduced HA stability. Compensatory mutations in HA (acquired during virus replication in ferrets) were essential to restore HA stability. These stabilizing mutations in HA also affected the pH at which HA undergoes an irreversible switch to its fusogenic form in host endosomes, a crucial step for virus infectivity. To identify additional stabilizing mutations in an H5 HA, we subjected a virus library possessing random mutations in the ectodomain of an H5 HA (altered to bind human-type receptors) to three rounds of treatment at 50°C. We isolated several mutants that maintained their human-type receptor-binding preference but acquired an appreciable increase in heat stability and underwent membrane fusion at a lower pH; collectively, these properties may aid H5 virus respiratory droplet transmission in mammals. IMPORTANCE We have identified mutations in HA that increase its heat stability and affect the pH that triggers an irreversible conformational change (a prerequisite for virus infectivity). These mutations were identified in the genetic background of an H5 HA protein that was mutated to bind to human cells. The ability to bind to human-type receptors, together with physical stability and an altered pH threshold for HA conformational change, may facilitate avian influenza virus transmission via respiratory droplets in

  3. Computational study of elements of stability of a four-helix bundle protein biosurfactant.

    PubMed

    Schaller, Andrea; Connors, Natalie K; Dwyer, Mirjana Dimitrijev; Oelmeier, Stefan A; Hubbuch, Jürgen; Middelberg, Anton P J

    2015-01-01

    Biosurfactants are surface-active molecules produced principally by microorganisms. They are a sustainable alternative to chemically-synthesized surfactants, having the advantages of being non-toxic, highly functional, eco-friendly and biodegradable. However they are currently only used in a few industrial products due to costs associated with production and purification, which exceed those for commodity chemical surfactants. DAMP4, a member of a four-helix bundle biosurfactant protein family, can be produced in soluble form and at high yield in Escherichia coli, and can be recovered using a facile thermal phase-separation approach. As such, it encompasses an interesting synergy of biomolecular and chemical engineering with prospects for low-cost production even for industrial sectors. DAMP4 is highly functional, and due to its extraordinary thermal stability it can be purified in a simple two-step process, in which the combination of high temperature and salt leads to denaturation of all contaminants, whereas DAMP4 stays stable in solution and can be recovered by filtration. This study aimed to characterize and understand the fundamental drivers of DAMP4 stability to guide further process and surfactant design studies. The complementary use of experiments and molecular dynamics simulation revealed a broad pH and temperature tolerance for DAMP4, with a melting point of 122.4 °C, suggesting the hydrophobic core as the major contributor to thermal stability. Simulation of systematically created in silico variants of DAMP4 showed an influence of number and location of hydrophilic mutations in the hydrophobic core on stability, demonstrating a tolerance of up to three mutations before a strong loss in stability occurred. The results suggest a consideration of a balance of stability, functionality and kinetics for new designs according to their application, aiming for maximal functionality but at adequate stability to allow for cost-efficient production using thermal

  4. Rapid and Adaptable Measurement of Protein Thermal Stability by Differential Scanning Fluorimetry: Updating a Common Biochemical Laboratory Experiment

    ERIC Educational Resources Information Center

    Johnson, R. Jeremy; Savas, Christopher J.; Kartje, Zachary; Hoops, Geoffrey C.

    2014-01-01

    Measurement of protein denaturation and protein folding is a common laboratory technique used in undergraduate biochemistry laboratories. Differential scanning fluorimetry (DSF) provides a rapid, sensitive, and general method for measuring protein thermal stability in an undergraduate biochemistry laboratory. In this method, the thermal…

  5. Using protein-fatty acid complexes to improve vitamin D stability.

    PubMed

    Pedersen, Jannik Nedergaard; Frislev, Henriette Søster; Pedersen, Jan Skov; Otzen, Daniel E

    2016-10-01

    Liprotides are complexes between lipids and partially denatured proteins in which the protein forms a stabilizing shell around a fatty acid micelle core. We have previously shown that liprotides stabilize small aliphatic molecules such as retinal and tocopherol by sequestering these molecules in the fatty acid core. This opens up the use of liprotides to formulate food additives. Here, we expand our investigations to the large and bulky molecule vitamin D3 (vitD), motivated by the population-wide occurrence of vitD deficiency. We prepared liprotides using different proteins and fatty acids and evaluated their ability to protect vitD upon exposure to heating or intense UV light. Additionally, we determined the stability of liprotides toward pH, Ca(2+), and BSA. The best results were obtained with liprotides made from α-lactalbumin and oleate. These liprotides were able to completely solubilize vitD, increase the stability toward UV light 9-fold, and increase the long-term stability at 37°C up to 1,000-fold. Native α-lactalbumin binds Ca(2+), making Ca(2+) potentially disruptive toward liprotides. However, liprotides prepared by incubation at 80°C were stable toward Ca(2+), in contrast to those made at 20°C. Nevertheless, the fatty acid binding protein BSA reduced the ability of both liprotides to protect vitD; the amount of vitD remianing after 20d at 20°C decreased from 79±3% in the absence of BSA to 49±4 and 23±3% in the presence of BSA for liprotides made at 80 and 20°C, respectively. Both classes of liprotides were able to release their vitD content, as demonstrated by the transfer of vitD encapsulated in liprotides to phospholipid vesicles. Importantly, liprotides were not stable at pH 6 and below, limiting the useful pH range of the liprotides to >pH 6. Our results indicate that vitD may be encapsulated and stabilized for enrichment of clear beverages at neutral pH to improve the intake and bioavailability of vitD. PMID:27474981

  6. Monitoring the stability of crosslinked protein crystals biotemplates: a feasibility study.

    PubMed

    Cohen-Hadar, Noa; Wine, Yariv; Nachliel, Esther; Huppert, Danny; Gutman, Menahem; Frolow, Felix; Freeman, Amihay

    2006-08-01

    Protein crystals, routinely prepared for the elucidation of protein 3D structures by X-ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein molecules in the crystal is a complementary 3D array of voids made of interconnected cavities and exhibiting highly ordered porosity. The permeability of the porosity of chemically crosslinked enzyme protein crystals to low molecular weight solutes, was used for enzyme mediated organic synthesis and size exclusion chromatography. This permeability might be extended to explore new potential applications for protein crystals, for example, their use as bio-templates for the fabrication of novel, nano-structured composite materials. The quality of composites obtained from "filling" of the ordered voids in protein crystals and their potential applications will be strongly dependent upon an accurate preservation of the order in the original protein crystal 3D array during the "filling" process. Here we propose and demonstrate the feasibility of monitoring the changes in 3D order of the protein array by a step-by-step molecular level monitoring of a model system for hydrogel bio-templating by glutaraldehyde crosslinked lysozyme crystals. This monitoring is based on step-by-step comparative analysis of data obtained from (i) X-ray crystallography: resolution, unit cell dimensions and B-factor values and (ii) fluorescence decay kinetics of ultra-fast laser activated dye, impregnated within these crystals. Our results demonstrated feasibility of the proposed monitoring approach and confirmed that the stabilized protein crystal template retained its 3D structure throughout the process.

  7. The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability.

    PubMed

    Rowland, Elden; Kim, Jitae; Bhuiyan, Nazmul H; van Wijk, Klaas J

    2015-11-01

    Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed. PMID:26371235

  8. Polar interactions trump hydrophobicity in stabilizing the self-inserting membrane protein Mistic.

    PubMed

    Broecker, Jana; Fiedler, Sebastian; Gimpl, Katharina; Keller, Sandro

    2014-10-01

    Canonical integral membrane proteins are attached to lipid bilayers through hydrophobic transmembrane helices, whose topogenesis requires sophisticated insertion machineries. By contrast, membrane proteins that, for evolutionary or functional reasons, cannot rely on these machineries need to resort to driving forces other than hydrophobicity. A striking example is the self-inserting Bacillus subtilis protein Mistic, which is involved in biofilm formation and has found application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins. Although this unusual protein contains numerous polar and charged residues and lacks characteristic membrane-interaction motifs, it is tightly bound to membranes in vivo and membrane-mimetic systems in vitro. Therefore, we set out to quantify the contributions from polar and nonpolar interactions to the coupled folding and insertion of Mistic. To this end, we defined conditions under which the protein can be unfolded completely and reversibly from various detergent micelles by urea in a two-state equilibrium and where the unfolded state is independent of the detergent used for solubilizing the folded state. This enabled equilibrium unfolding experiments previously used for soluble and β-barrel membrane proteins, revealing that polar interactions with ionic and zwitterionic headgroups and, presumably, the interfacial dipole potential stabilize the protein much more efficiently than nonpolar interactions with the micelle core. These findings unveil the forces that allow a protein to tightly interact with a membrane-mimetic environment without major hydrophobic contributions and rationalize the differential suitability of detergents for the extraction and solubilization of Mistic-tagged membrane proteins.

  9. Salt Potentiates Methylamine Counteraction System to Offset the Deleterious Effects of Urea on Protein Stability and Function

    PubMed Central

    Singh, Laishram R.; Warepam, Marina; Ahmad, Faizan; Dar, Tanveer Ali

    2015-01-01

    Cellular methylamines are osmolytes (low molecular weight organic compounds) believed to offset the urea’s harmful effects on the stability and function of proteins in mammalian kidney and marine invertebrates. Although urea and methylamines are found at 2:1 molar ratio in tissues, their opposing effects on protein structure and function have been questioned on several grounds including failure to counteraction or partial counteraction. Here we investigated the possible involvement of cellular salt, NaCl, in urea-methylamine counteraction on protein stability and function. We found that NaCl mediates methylamine counteracting system from no or partial counteraction to complete counteraction of urea’s effect on protein stability and function. These conclusions were drawn from the systematic thermodynamic stability and functional activity measurements of lysozyme and RNase-A. Our results revealed that salts might be involved in protein interaction with charged osmolytes and hence in the urea-methylamine counteraction. PMID:25793733

  10. In Vitro Properties of the Conserved Mammalian Protein hnRNP D Suggest a Role in Telomere Maintenance

    PubMed Central

    Eversole, Ashley; Maizels, Nancy

    2000-01-01

    Mammalian chromosomes terminate with a 3′ tail which consists of reiterations of the G-rich repeat, d(TTAGGG). The telomeric tail is the primer for replication by telomerase, and it may also invade telomeric duplex DNA to form terminal lariat structures, or T loops. Here we show that the ubiquitous and highly conserved mammalian protein hnRNP D interacts specifically with the G-rich strand of the telomeric repeat. A single gene encodes multiple isoforms of hnRNP D. All isoforms bind comparably to the G-rich strand, and certain isoforms can also bind tightly and specifically to the C-rich telomeric strand. G-rich telomeric sequences readily form structures stabilized by G-G pairing, which can interfere with telomere replication by telomerase. We show that hnRNP D binding to the G-rich strand destabilizes intrastrand G-G pairing and that hnRNP D interacts specifically with telomerase in human cell extracts. This biochemical analysis suggest that hnRNP D could function in vivo to destabilize structures formed by telomeric G-rich tails and facilitate their extension by telomerase. PMID:10891483

  11. Influence of cysteine and methionine availability on protein peroxide scavenging activity and phenolic stability in emulsions.

    PubMed

    Zhou, Lisa; Elias, Ryan J

    2014-03-01

    Plant phenolics are secondary metabolites that have been shown to confer beneficial health effects in humans. However, many of these compounds undergo metal-catalysed oxidation reactions, leading to the generation of hydrogen peroxide (H2O2) and other reactive oxygen species that may negatively impact product stability. In proteins, methionine (Met) and cysteine (Cys) are capable of reacting directly with peroxides. Thus, the dairy proteins, casein (CAS) and β-lactoglobulin (BLG), were examined for their ability to scavenge H2O2 (400μM) and influence (-)-epigallocatechin-3-gallate (EGCG) oxidation (400μM) in Tween- or sodium dodecyl sulphate (SDS)-stabilised hexadecane emulsions. To examine the effect that the accessibility of these amino acids have on their peroxide scavenging activities, proteins were pre-treated with tert-butyl hydroperoxide (TBHP), a bulky peroxide, to oxidise only solvent accessible Met residues or H2O2, the smallest peroxide, to oxidise buried Met residues. In CAS treatments, higher Met content yielded greater peroxide scavenging activity and EGCG stability. CAS treatments also showed significantly higher peroxide scavenging activity compared to the corresponding BLG treatment. However, BLG peroxide scavenging activity was greatly enhanced in SDS-stabilised emulsions due to protein denaturation and subsequent exposure of previously buried Cys residues.

  12. A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state.

    PubMed

    Tolleter, Dimitri; Hincha, Dirk K; Macherel, David

    2010-10-01

    Late embryogenesis abundant (LEA) proteins are a highly diverse group of polypeptides expected to play important roles in desiccation tolerance of plant seeds. They are also found in other plant tissues and in some anhydrobotic invertebrates, fungi, protists and prokaryotes. The LEA protein LEAM accumulates in the matrix space of pea (Pisum sativum) mitochondria during late seed maturation. LEAM is an intrinsically disordered protein folding into amphipathic alpha-helix upon desiccation. This suggests that it could interact with the inner mitochondrial membrane, providing structural protection in dry seeds. Here, we have used Fourier-transform infrared and fluorescence spectroscopy to gain insight into the molecular details of interactions of LEAM with phospholipid bilayers in the dry state and their effects on liposome stability. LEAM interacted specifically with negatively charged phosphate groups in dry phospholipids, increasing fatty acyl chain mobility. This led to an enhanced stability of liposomes during drying and rehydration, but also upon freezing. Protection depended on phospholipid composition and was strongly enhanced in membranes containing the mitochondrial phospholipid cardiolipin. Collectively, the results provide strong evidence for a function of LEAM as a mitochondrial membrane protectant during desiccation and highlight the role of lipid composition in the interactions between LEA proteins and membranes.

  13. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins

    PubMed Central

    Nijtmans, Leo G.J.; de Jong, Liesbeth; Artal Sanz, Marta; Coates, Philip J.; Berden, Jan A.; Willem Back, Jaap; Muijsers, Anton O.; van der Spek, Hans; Grivell, Les A.

    2000-01-01

    Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co-overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse–chase experiments with mitochondria isolated from phb1/phb2-null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins. PMID:10835343

  14. Factors contributing to decreased protein stability when aspartic acid residues are in {beta}-sheet regions.

    SciTech Connect

    Pokkuluri, P. R.; Cai, X.; Raffen, R.; Gu, M.; Stevens, F. J.; Schiffer, M.

    2002-07-01

    Asp residues are significantly under represented in {beta}-sheet regions of proteins, especially in the middle of {beta}-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a {beta}-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V{sub L}) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a {beta}-strand, and that of Q89D, located in the middle of a {beta}-strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V{sub L}-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V{sub L}-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V{sub L}-Len domain. The structures of mutants V{sub L}-Len Q38D and V{sub L}-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these QD mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a {beta}-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a {beta}-strand residue to Asp could prevent the expression of the

  15. Role of alpha-hemoglobin-stabilizing protein in normal erythropoiesis and beta-thalassemia.

    PubMed

    Weiss, Mitchell J; Zhou, Suiping; Feng, Liang; Gell, David A; Mackay, Joel P; Shi, Yigong; Gow, Andrew J

    2005-01-01

    Hemoglobin (Hb) synthesis is coordinated by homeostatic mechanisms to limit the accumulation of free alpha or beta subunits, which are cytotoxic. Alpha hemoglobin-stabilizing protein (AHSP) is an abundant erythroid protein that specifically binds free alphaHb, stabilizes its structure, and limits its ability to participate in chemical reactions that generate reactive oxygen species. Gene ablation studies in mice demonstrate that AHSP is required for normal erythropoiesis. AHSP-null erythrocytes are short-lived, contain Hb precipitates, and exhibit signs of oxidative damage. Loss of AHSP exacerbates beta-thalassemia in mice, indicating that altered AHSP expression or function could modify thalassemia phenotypes in humans, a topic that is beginning to be explored in clinical studies. We used biochemical, spectroscopic, and crystallographic methods to examine how AHSP stabilizes alphaHb. AHSP binds the G and H helices of alphaHb on a surface that largely overlaps with the alpha1-beta1 interface of HbA. This result explains previous findings that betaHb can competitively displace AHSP from alphaHb to form HbA tetramer. Remarkably, binding of AHSP to oxygenated alphaHb induces dramatic conformational changes and converts the heme-bound iron to an oxidized hemichrome state in which all six coordinate positions are occupied. This structure limits the reactivity of heme iron, providing a mechanism by which AHSP stabilizes alphaHb. These findings suggest a biochemical pathway through which AHSP might participate in normal Hb synthesis and modulate the severity of thalassemias. Moreover, understanding how AHSP stabilizes alphaHb provides a theoretical basis for new strategies to inhibit the damaging effects of free alphaHb that accumulates in beta-thalassemia.

  16. Hydrolysis of proteins by immobilized-stabilized alcalase-glyoxyl agarose.

    PubMed

    Tardioli, Paulo W; Pedroche, Justo; Giordano, Raquel L C; Fernández-Lafuente, Roberto; Guisán, José M

    2003-01-01

    This paper presents stable Alcalase-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced by immobilizing-stabilizing Alcalase on cross-linked 10% agarose beads, using low and high activation grades of the support and different immobilization times. The Alcalase glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde and CNBr as activation reactants. The performance of derivatives in the hydrolysis of casein was also tested. At pH 8.0 and 50 degrees C, Alcalase derivatives produced with 1 h of immobilization time on agarose activated with glutaraldehyde, CNBr, and low and high glyoxyl groups concentration presented half-lives of ca. 10, 29, 60, and 164 h, respectively. More extensive immobilization monotonically led to higher stabilization. The most stabilized Alcalase-glyoxyl derivative was produced using 96 h of immobilization time and high activation grade of the support. It presented half-life of ca. 23 h, at pH 8.0 and 63 degrees C and was ca. 500-fold more stable than the soluble enzyme. Thermal inactivation of all derivatives followed a single-step non-first-order kinetics. The most stable derivative presented ca. 54% of the activity of the soluble enzyme for the hydrolysis of casein and of the small substrate Boc-Ala-ONp. This behavior suggests that the decrease in activity was due to enzyme distortion but not to wrong orientation. The hydrolysis degree of casein at 80 degrees C with the most stabilized enzyme was 2-fold higher than that achieved using soluble enzyme, as a result of the thermal inactivation of the latter. Therefore, the high stability of the new Alcalase-glyoxyl derivative allows the design of continuous processes to hydrolyze proteins at temperatures that avoid microbial growth.

  17. The Nicastrin-like protein Nicalin regulates assembly and stability of the Nicalin-nodal modulator (NOMO) membrane protein complex.

    PubMed

    Haffner, Christof; Dettmer, Ulf; Weiler, Timotheus; Haass, Christian

    2007-04-01

    The assembly of the gamma-secretase complex, an Alzheimer disease-related protease required for beta-amyloid generation, is tightly regulated and predominantly limited by the stoichiometrical availability of its components. We have identified a novel endoplasmic reticulum-located protein complex that is regulated in a similar fashion. It contains the recently identified Nodal signaling antagonists Nicalin (a distant homolog of the gamma-secretase component Nicastrin) and NOMO (Nodal modulator). Using an RNA interference approach, we found that Nicalin and NOMO became unstable in the absence of the respective binding partner, suggesting that complex formation has a stabilizing effect. Overexpression of Nicalin resulted in an increase in NOMO, whereas endogenous Nicalin was reduced below the detection limit. Both effects were shown to occur at a post-transcriptional level. Thus, NOMO is most likely produced in excess amounts and either stabilized by Nicalin or rapidly degraded. In contrast, Nicalin levels are limited independently of NOMO. We, therefore, propose that Nicalin controls the assembly and stability of the Nicalin-NOMO complex. PMID:17261586

  18. Assessing Predictors of Changes in Protein Stability upon Mutation Using Self-Consistency

    PubMed Central

    Thiltgen, Grant; Goldstein, Richard A.

    2012-01-01

    The ability to predict the effect of mutations on protein stability is important for a wide range of tasks, from protein engineering to assessing the impact of SNPs to understanding basic protein biophysics. A number of methods have been developed that make these predictions, but assessing the accuracy of these tools is difficult given the limitations and inconsistencies of the experimental data. We evaluate four different methods based on the ability of these methods to generate consistent results for forward and back mutations, and examine how this ability varies with the nature and location of the mutation. We find that, while one method seems to outperform the others, the ability of these methods to make accurate predictions is limited. PMID:23144695

  19. The BRCA1-interacting protein, Abraxas, is required for genomic stability and tumor suppression

    PubMed Central

    Castillo, Andy; Paul, Atanu; Sun, Baohua; Huang, Ting Hsiang; Wang, Yucai; Yazinski, Stephanie A.; Tyler, Jessica; Li, Lei; You, M James; Zou, Lee; Yao, Jun; Wang, Bin

    2014-01-01

    Summary Germline mutations of BRCA1 confer hereditary susceptibility to breast and ovarian cancer. However, somatic mutation of BRCA1 is infrequent in sporadic breast cancers. The BRCA1 protein C-terminus BRCT domains interact with multiple proteins and are required for BRCA1's tumor suppressor function. In this study, we demonstrated that Abraxas, a BRCA1 BRCT domain-interacting protein, plays a role in tumor suppression. Abraxas exerts its function through binding to BRCA1 to regulate DNA repair and maintain genome stability. Both homozygous and heterozygous Abraxas knockout mice exhibited decreased survival and increased tumor incidence. The gene encoding Abraxas suffers from gene copy loss and somatic mutations in multiple human cancers including breast, ovarian, and endometrial cancers, suggesting that mutation and loss of function of Abraxas may contribute to tumor development in human patients. PMID:25066119

  20. Exploiting Transient Protein States for the Design of Small-Molecule Stabilizers of Mutant p53

    PubMed Central

    Joerger, Andreas C.; Bauer, Matthias R.; Wilcken, Rainer; Baud, Matthias G.J.; Harbrecht, Hannes; Exner, Thomas E.; Boeckler, Frank M.; Spencer, John; Fersht, Alan R.

    2015-01-01

    Summary The destabilizing p53 cancer mutation Y220C creates an extended crevice on the surface of the protein that can be targeted by small-molecule stabilizers. Here, we identify different classes of small molecules that bind to this crevice and determine their binding modes by X-ray crystallography. These structures reveal two major conformational states of the pocket and a cryptic, transiently open hydrophobic subpocket that is modulated by Cys220. In one instance, specifically targeting this transient protein state by a pyrrole moiety resulted in a 40-fold increase in binding affinity. Molecular dynamics simulations showed that both open and closed states of this subsite were populated at comparable frequencies along the trajectories. Our data extend the framework for the design of high-affinity Y220C mutant binders for use in personalized anticancer therapy and, more generally, highlight the importance of implementing protein dynamics and hydration patterns in the drug-discovery process. PMID:26636255

  1. Controlled formation of emulsion gels stabilized by salted myofibrillar protein under malondialdehyde (MDA)-induced oxidative stress.

    PubMed

    Zhou, Feibai; Sun, Weizheng; Zhao, Mouming

    2015-04-15

    This study presented the cold-set gelation of emulsions stabilized by salted myofibrillar protein (MP) under oxidative stress originated from malondialdehyde (MDA). Gel properties were compared over a range of MDA/NaCl concentrations including gel viscoelastic properties, strength, water-holding capacity (WHC), amount of protein entrapped, and microstructure. The oxidative stability of emulsion gels as indicated by lipid hydroperoxide was further determined and compared. Results indicated that emulsion stabilized by MP at swollen state under certain ionic strengths (0.2-0.6 M) was the premise of gel formation under MDA. In the presence of intermediate MDA concentrations (2.5-10 mM), the emulsion gels showed an improved elasticity, strength, WHC, and oxidative stability. This improvement should be mainly attributed to the enhanced protein-protein cross-linkings via MDA, which were homogeneously formed among absorbed and/or unabsorbed proteins, entrapping a greater amount and fractions of protein within network. Therefore, the oil droplets were better adherent to the gel matrix. Nevertheless, addition of high MDA concentrations (25-50 mM) led to the formation of excessive covalent bonds, which might break protein-protein bonds and trigger the desorption of protein from the interface. This ultimately caused "oil leak" phenomena as well as the collapse of gel structure and, thus, overall decreased gel properties and oxidative stability. PMID:25749308

  2. Different Stability and Proteasome-Mediated Degradation Rate of SMN Protein Isoforms

    PubMed Central

    Locatelli, Denise; Terao, Mineko; Kurosaki, Mami; Zanellati, Maria Clara; Pletto, Daniela Rita; Finardi, Adele; Colciaghi, Francesca; Garattini, Enrico; Battaglia, Giorgio Stefano

    2015-01-01

    The key pathogenic steps leading to spinal muscular atrophy (SMA), a genetic disease characterized by selective motor neuron degeneration, are not fully clarified. The full-length SMN protein (FL-SMN), the main protein product of the disease gene SMN1, plays an established role in the cytoplasm in snRNP biogenesis ultimately leading to mRNA splicing within the nucleus. It is also involved in the mRNA axonal transport. However, to what extent the impairment of these two SMN functions contributes to SMA pathogenesis remains unknown. A shorter SMN isoform, axonal-SMN or a-SMN, with more specific axonal localization, has been discovered, but whether it might act in concert with FL-SMN in SMA pathogenesis is not known. As a first step in defining common or divergent intracellular roles of FL-SMN vs a-SMN proteins, we here characterized the turn-over of both proteins and investigated which pathway contributed to a-SMN degradation. We performed real time western blot and confocal immunofluorescence analysis in easily controllable in vitro settings. We analyzed co-transfected NSC34 and HeLa cells and cell clones stably expressing both a-SMN and FL-SMN proteins after specific blocking of transcript or protein synthesis and inhibition of known intracellular degradation pathways. Our data indicated that whereas the stability of both FL-SMN and a-SMN transcripts was comparable, the a-SMN protein was characterized by a much shorter half-life than FL-SMN. In addition, as already demonstrated for FL-SMN, the Ub/proteasome pathway played a major role in the a-SMN protein degradation. We hypothesize that the faster degradation rate of a-SMN vs FL-SMN is related to the protection provided by the protein complex in which FL-SMN is assembled. The diverse a-SMN vs FL-SMN C-terminus may dictate different protein interactions and complex formation explaining the different localization and role in the neuronal compartment, and the lower expression and stability of a-SMN. PMID:26214005

  3. Critical lysine residues of Klf4 required for protein stabilization and degradation

    SciTech Connect

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  4. Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein

    PubMed Central

    Perugino, Giuseppe; Miggiano, Riccardo; Serpe, Mario; Vettone, Antonella; Valenti, Anna; Lahiri, Samarpita; Rossi, Franca; Rossi, Mosè; Rizzi, Menico; Ciaramella, Maria

    2015-01-01

    Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins. PMID:26227971

  5. Discrimination of Proteins Using an Array of Surfactant-Stabilized Gold Nanoparticles.

    PubMed

    Rogowski, Jacob L; Verma, Mohit S; Gu, Frank X

    2016-08-01

    Protein analysis is a fundamental aspect of biochemical research. Gold nanoparticles are an emerging platform for various biological applications given their high surface area, biocompatibility, and unique optical properties. The colorimetric properties of gold nanoparticles make them ideal for point-of-care diagnostics. Different aspects of gold nanoparticle-protein interactions have been investigated to predict the effect of protein adsorption on colloidal stability, but the role of surfactants is often overlooked, despite their potential to alter both protein and nanoparticle properties. Herein we present a method by which gold nanoparticles can be prepared in various surfactants and used for array-based quantification and identification of proteins. The exchange of surfactant not only changed the zeta potential of those gold nanoparticles but also drastically altered their aggregation response to five different proteins (bovine serum albumin, human serum albumin, immunoglobulin G, lysozyme, and hemoglobin) in a concentration-dependent manner. Finally, we demonstrate that varying surfactant concentration can be used to control assay sensitivity. PMID:27399345

  6. Why has porcine VEG protein unusually high stability and suppressed binding ability?

    PubMed

    Burova, T V; Rabesona, H; Choiset, Y; Jankowski, C K; Sawyer, L; Haertlé, T

    2000-05-23

    Von Ebner gland protein (VEGP) and odorant-binding protein (OBP) were purified from porcine lingual epithelium and nasal mucosa, respectively. Both VEGP and OBP preparations were homogeneous as indicated by SDS-PAGE, isoelectric focusing, gel-filtration and electrospray mass spectrometry. However, high-sensitivity differential scanning calorimetry (HS-DSC) yielded multiphasic denaturation thermograms for both proteins indicating their conformational heterogeneity. The unfolding transition of VEGP is observed at extremely high temperatures (about 110 degrees C), which is unexpected for a protein with significant structural homology to OBP and other lipocalins. Isothermal titration calorimetry (ITC) did not detect the binding of either aspartame or denatonium saccharide to VEGP nor did it detect binding of 2-isobutyl-3-methoxypyrazine (IBMP) to OBP. Extraction of OBP with mixed organic solvents eliminated the conformational heterogeneity and the protein showed a reversible two-state transition in HS-DSC thereafter. ITC also showed that the extracted OBP was able to bind IBMP. These results imply that tightly bound endogenous ligands increase the thermal stability of OBP and block the binding of other ligands. In contrast to OBP, the extraction of VEGP with organic solvents failed to promote binding or to establish thermal homogeneity, most likely because of the irreversible denaturation of VEGP. Thus, the elucidation of the functional behaviour of VEGP is closely related to the exhaustive purging of its endogenous ligands which otherwise very efficiently mask ligand binding sites of this protein.

  7. The effect of microgravity on the stability and assembly of viral proteins

    SciTech Connect

    Gillock, E.; Rottinghaus, S.; Paulsen, A.; Smiley, S.; Chang, D.; Consigli, R.; Chang, D.

    1997-01-01

    The coat protein VP1 of polyomavirus was utilized as a model protein to determine the effects of microgravity on the stability of the protein, as well as its ability to self-assemble into capsid-like particles that resemble the intact virus. Our laboratory has previously reported that microgravity, under physiological conditions, caused the capsomere subunits to swell and lose their ability to assemble into capsid-like particles in the presence of calcium. When subjected to prolonged exposure to microgravity, the capsomeres swelled even further, becoming amorphous, losing their characteristic capsomere-like structure, which is highly indicative of protein unfolding. Other experiments, which utilized high ionic conditions (2.0 M NaCl) in the assembly reaction mixture, exhibited the presence of capsomeres which appeared more stable and also retained the capability of self assembly into capsid-like particles in microgravity. The high salt conditions apparently prevented the unfolding of the recombinant VP1 protein in microgravity. In subsequent studies, involving native polyomavirus virions or empty capsids, it was revealed that when these native particles were subjected to microgravity, they retained their characteristic structures but were found to have swollen in diameter by approximately 10{percent}. This observation also seems to be indicative of the occurrence of a protein unfolding phenomenon. {copyright} {ital 1997 American Institute of Physics.}

  8. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A

    PubMed Central

    Wu, Yuanzhong; Zhou, Liwen; Wang, Xin; Lu, Jinping; Zhang, Ruhua; Liang, Xiaoting; Wang, Li; Deng, Wuguo; Zeng, Yi-Xin; Huang, Haojie; Kang, Tiebang

    2016-01-01

    The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be developed. Recently, a genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss-of-function screening. Here, we developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1DCAF8 was identified as a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was acetylated by p300/CBP and deacetylated by HDAC3, prevented the ubiquitin-mediated degradation of Cdc25A by the proteasome. This is the first study to report that acetylation, as a novel posttranslational modification, modulates Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening method at the genome scale may be widely used to globally identify regulators of protein stability. PMID:27462461

  9. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A.

    PubMed

    Wu, Yuanzhong; Zhou, Liwen; Wang, Xin; Lu, Jinping; Zhang, Ruhua; Liang, Xiaoting; Wang, Li; Deng, Wuguo; Zeng, Yi-Xin; Huang, Haojie; Kang, Tiebang

    2016-01-01

    The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be developed. Recently, a genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss-of-function screening. Here, we developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1(DCAF8) was identified as a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was acetylated by p300/CBP and deacetylated by HDAC3, prevented the ubiquitin-mediated degradation of Cdc25A by the proteasome. This is the first study to report that acetylation, as a novel posttranslational modification, modulates Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening method at the genome scale may be widely used to globally identify regulators of protein stability. PMID:27462461

  10. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A.

    PubMed

    Wu, Yuanzhong; Zhou, Liwen; Wang, Xin; Lu, Jinping; Zhang, Ruhua; Liang, Xiaoting; Wang, Li; Deng, Wuguo; Zeng, Yi-Xin; Huang, Haojie; Kang, Tiebang

    2016-01-01

    The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be developed. Recently, a genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss-of-function screening. Here, we developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1(DCAF8) was identified as a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was acetylated by p300/CBP and deacetylated by HDAC3, prevented the ubiquitin-mediated degradation of Cdc25A by the proteasome. This is the first study to report that acetylation, as a novel posttranslational modification, modulates Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening method at the genome scale may be widely used to globally identify regulators of protein stability.

  11. A Three-protein Charge Zipper Stabilizes a Complex Modulating Bacterial Gene Silencing*

    PubMed Central

    Cordeiro, Tiago N.; García, Jesús; Bernadó, Pau; Millet, Oscar; Pons, Miquel

    2015-01-01

    The Hha/YmoA nucleoid-associated proteins help selectively silence horizontally acquired genetic material, including pathogenicity and antibiotic resistance genes and their maintenance in the absence of selective pressure. Members of the Hha family contribute to gene silencing by binding to the N-terminal dimerization domain of H-NS and modifying its selectivity. Hha-like proteins and the H-NS N-terminal domain are unusually rich in charged residues, and their interaction is mostly electrostatic-driven but, nonetheless, highly selective. The NMR-based structural model of the complex between Hha/YmoA and the H-NS N-terminal dimerization domain reveals that the origin of the selectivity is the formation of a three-protein charge zipper with interdigitated complementary charged residues from Hha and the two units of the H-NS dimer. The free form of YmoA shows collective microsecond-millisecond dynamics that can by measured by NMR relaxation dispersion experiments and shows a linear dependence with the salt concentration. The number of residues sensing the collective dynamics and the population of the minor form increased in the presence of H-NS. Additionally, a single residue mutation in YmoA (D43N) abolished H-NS binding and the dynamics of the apo-form, suggesting the dynamics and binding are functionally related. PMID:26085102

  12. ATP-stabilized amorphous calcium carbonate nanospheres and their application in protein adsorption.

    PubMed

    Qi, Chao; Zhu, Ying-Jie; Lu, Bing-Qiang; Zhao, Xin-Yu; Zhao, Jing; Chen, Feng; Wu, Jin

    2014-05-28

    Calcium carbonate is a common substance found in rocks worldwide, and is the main biomineral formed in shells of marine organisms and snails, pearls and eggshells. Amorphous calcium carbonate (ACC) is the least stable polymorph of calcium carbonate, which is so unstable under normal conditions that it is difficult to be prepared in vitro because it rapidly crystallizes to form one of the more stable polymorphs in aqueous solution. Herein, we report the successful synthesis of highly stable ACC nanospheres in vitro using adenosine 5'-triphosphate disodium salt (ATP) as a stabilizer. The effect of ATP on the stability of ACC nanospheres is investigated. Our experiments show that ATP plays an unique role in the stabilization of ACC nanospheres in aqueous solution. Moreover, the as-prepared ACC nanospheres are highly stable in phosphate buffered saline for a relatively long period of time (12 days) even under relatively high concentrations of calcium and p