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Sample records for 3a4 protein stability

  1. Altered CYP2C9 Activity Following Modulation of CYP3A4 Levels in Human Hepatocytes: an Example of Protein-Protein Interactions

    PubMed Central

    Tweedie, Donald J.; Chan, Tom S.; Tracy, Timothy S.

    2014-01-01

    Cytochrome P450 (P450) protein-protein interactions resulting in modulation of enzyme activities have been well documented using recombinant isoforms. This interaction has been less clearly demonstrated in a more physiologic in vitro system such as human hepatocytes. As an expansion of earlier work (Subramanian et al., 2010), in which recombinant CYP2C9 activity decreased with increasing levels of CYP3A4, the current study modulated CYP3A4 content in human hepatocytes to determine the impact on CYP2C9. Modulation of CYP3A4 levels in situ was enabled by the use of a long-term human hepatocyte culture model (HepatoPac) shown to retain phenotypic hepatocyte function over a number of weeks. The extended period of culture allowed time for knockdown of CYP3A4 protein by small interfering RNA (siRNA) with subsequent recovery, as well as upregulation through induction with a recovery period. CYP3A4 gene silencing resulted in a 60% decrease in CYP3A4 activity and protein levels with a concomitant 74% increase in CYP2C9 activity, with no change in CYP2C9 mRNA levels. Upon removal of siRNA, both CYP2C9 and CYP3A4 activities returned to pre-knockdown levels. Importantly, modulation of CYP3A4 protein levels had no impact on cytochrome P450 reductase activities or levels. However, the possibility for competition for limiting reductase cannot be ruled out. Interestingly, lowering CYP3A4 levels also increased UDP-glucuronosyltransferase 2B7 activity. These studies clearly demonstrate that alterations in CYP3A4 levels can modulate CYP2C9 activity in situ and suggest that further studies are warranted to evaluate the possible clinical consequences of these findings. PMID:25157098

  2. [Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

    PubMed

    Gnedenko, O V; Ivanov, A S; Iablokov, E O; Usanov, S A; Mukha, D V; Sergeev, G V; Kuzikov, A V; Moskaleva, N E; Bulko, T V; Shumiantseva, V V; Archakov, A I

    2014-01-01

    Molecular interactions between proteins redox partners (cytochromes P450 3A4, 3A5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 - -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 to testosterone.

  3. [Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

    PubMed

    Gnedenko, O V; Ivanov, A S; Yablokov, E O; Usanov, S A; Mukha, D V; Sergeev, G V; Kuzikov, A V; Bulko, T V; Moskaleva, N E; Shumyantseva, V V; Archakov, A I

    2015-01-01

    Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3А4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435  -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 in the presence of its substrate testosterone.

  4. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    PubMed Central

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien; Schuetz, Erin G.; Chen, Taosheng

    2013-01-01

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotics detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet-drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that cautions should be taken for PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. PMID:23707768

  5. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    SciTech Connect

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien; Schuetz, Erin G.; Chen, Taosheng

    2013-10-01

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. - Highlights: • Piperine induces PXR-mediated CYP3A4 and MDR1 expression. • Piperine activates PXR by binding to PXR and recruiting coactivator SRC-1. • Piperine induces PXR activation in vivo. • Caution should be taken in piperine consumption during drug treatment.

  6. 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.

  7. 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

  8. Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1

    PubMed Central

    Banerjee, Monimoy; Chen, Taosheng

    2014-01-01

    Human pregnane X receptor (hPXR) regulates the expression of drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and drug transporters such as multidrug-resistance protein 1 (MDR1). PXR can be modulated by small molecules, including Federal Drug Administration (FDA)–approved drugs, thus altering drug metabolism and causing drug-drug interactions. To determine the role of FDA-approved drugs in PXR-mediated regulation of drug metabolism and clearance, we screened 1481 FDA-approved small-molecule drugs by using a luciferase reporter assay in HEK293T cells and identified the diuretic drug metolazone as an activator of PXR. Our data showed that metolazone activated hPXR-mediated expression of CYP3A4 and MDR1 in human hepatocytes and intestine cells and increased CYP3A4 promoter activity in various cell lines. Mammalian two-hybrid assays showed that hPXR recruits its co-activator SRC-1 upon metolazone binding in HepG2 cells, explaining the mechanism of hPXR activation. To understand the role of other commonly-used diuretics in PXR activation and the structure-activity relationship of metolazone, thiazide and non-thiazide diuretics drugs were also tested but only metolazone activates PXR. To understand the molecular mechanism, docking studies and mutational analysis were carried out and showed that metolazone binds in the ligand-binding pocket and interacts with mostly hydrophobic amino acid residues. This is the first report showing that metolazone activates PXR. Because activation of hPXR might cause drug-drug interactions, metolazone should be used with caution for drug treatment in patients undergoing combination therapy. PMID:25181459

  9. Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1.

    PubMed

    Banerjee, Monimoy; Chen, Taosheng

    2014-11-15

    Human pregnane X receptor (hPXR) regulates the expression of drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and drug transporters such as multidrug-resistance protein 1 (MDR1). PXR can be modulated by small molecules, including Federal Drug Administration (FDA)-approved drugs, thus altering drug metabolism and causing drug-drug interactions. To determine the role of FDA-approved drugs in PXR-mediated regulation of drug metabolism and clearance, we screened 1481 FDA-approved small-molecule drugs by using a luciferase reporter assay in HEK293T cells and identified the diuretic drug metolazone as an activator of hPXR. Our data showed that metolazone activated hPXR-mediated expression of CYP3A4 and MDR1 in human hepatocytes and intestine cells and increased CYP3A4 promoter activity in various cell lines. Mammalian two-hybrid assays showed that hPXR recruits its co-activator SRC-1 upon metolazone binding in HepG2 cells, explaining the mechanism of hPXR activation. To understand the role of other commonly-used diuretics in hPXR activation and the structure-activity relationship of metolazone, thiazide and non-thiazide diuretics drugs were also tested but only metolazone activates hPXR. To understand the molecular mechanism, docking studies and mutational analysis were carried out and showed that metolazone binds in the ligand-binding pocket and interacts with mostly hydrophobic amino acid residues. This is the first report showing that metolazone activates hPXR. Because activation of hPXR might cause drug-drug interactions, metolazone should be used with caution for drug treatment in patients undergoing combination therapy.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. Hyperconjugation with lone pair of morpholine nitrogen stabilizes transition state for phenyl hydroxylation in CYP3A4 metabolism of ( S)- N-[1-(3-morpholin-4-yl phenyl) ethyl]-3-phenylacrylamide

    NASA Astrophysics Data System (ADS)

    Shaikh, Abdul Rajjak; Broclawik, Ewa; Ismael, Mohamed; Tsuboi, Hideyuki; Koyama, Michihisa; Kubo, Momoji; Del Carpio, Carlos A.; Miyamoto, Akira

    2006-02-01

    Using quantum chemical modelling we describe a novel effect in the mechanism of CYP3A4 metabolism for the arene substrate with o-substituent yielding a lone pair donation to conjugate π system; this will compensate for the loss of aromaticity on formation of the tetrahedral complex and lower the rate-determining energy barrier.

  18. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

    Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G.

    2010-11-16

    Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. UNDERSTANDING THE MECHANISM OF CYTOCHROME P450 3A4: RECENT ADVANCES AND REMAINING PROBLEMS

    PubMed Central

    Sevrioukova, Irina F.; Poulos, Thomas L.

    2013-01-01

    Cytochromes P450 (CYPs) represent a diverse group of heme-thiolate proteins found in almost all organisms. CYPs share a common protein fold but differ in substrate selectivity and catalyze a wide variety of monooxygenation reactions via activation of molecular oxygen. Among 57 human P450s, the 3A4 isoform (CYP3A4) is the most abundant and the most important because it metabolizes the majority of the administered drugs. A remarkable feature of CYP3A4 is its extreme promiscuity in substrate specificity and cooperative substrate binding, which often leads to undesirable drug-drug interactions and toxic side effects. Owing to its importance in drug development and therapy, CYP3A4 has been the most extensively studied mammalian P450. In this review we provide an overview on recent progress and remaining problems in the CYP3A4 research. PMID:23018626

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. High frequency and founder effect of the CYP3A4*20 loss-of-function allele in the Spanish population classifies CYP3A4 as a polymorphic enzyme.

    PubMed

    Apellániz-Ruiz, M; Inglada-Pérez, L; Naranjo, M E G; Sánchez, L; Mancikova, V; Currás-Freixes, M; de Cubas, A A; Comino-Méndez, I; Triki, S; Rebai, A; Rasool, M; Moya, G; Grazina, M; Opocher, G; Cascón, A; Taboada-Echalar, P; Ingelman-Sundberg, M; Carracedo, A; Robledo, M; Llerena, A; Rodríguez-Antona, C

    2015-06-01

    Cytochrome P450 3A4 (CYP3A4) is a key drug-metabolizing enzyme. Loss-of-function variants have been reported as rare events, and the first demonstration of a CYP3A4 protein lacking functional activity is caused by CYP3A4*20 allele. Here we characterized the world distribution and origin of CYP3A4*20 mutation. CYP3A4*20 was determined in more than 4000 individuals representing different populations, and haplotype analysis was performed using CYP3A polymorphisms and microsatellite markers. CYP3A4*20 allele was present in 1.2% of the Spanish population (up to 3.8% in specific regions), and all CYP3A4*20 carriers had a common haplotype. This is compatible with a Spanish founder effect and classifies CYP3A4 as a polymorphic enzyme. This constitutes the first description of a CYP3A4 loss-of-function variant with high frequency in a population. CYP3A4*20 results together with the key role of CYP3A4 in drug metabolism support screening for rare CYP3A4 functional alleles among subjects with adverse drug events in certain populations. PMID:25348618

  19. Interactions between CYP3A4 and Dietary Polyphenols

    PubMed Central

    Basheer, Loai; Kerem, Zohar

    2015-01-01

    The human cytochrome P450 enzymes (P450s) catalyze oxidative reactions of a broad spectrum of substrates and play a critical role in the metabolism of xenobiotics, such as drugs and dietary compounds. CYP3A4 is known to be the main enzyme involved in the metabolism of drugs and most other xenobiotics. Dietary compounds, of which polyphenolics are the most studied, have been shown to interact with CYP3A4 and alter its expression and activity. Traditionally, the liver was considered the prime site of CYP3A-mediated first-pass metabolic extraction, but in vitro and in vivo studies now suggest that the small intestine can be of equal or even greater importance for the metabolism of polyphenolics and drugs. Recent studies have pointed to the role of gut microbiota in the metabolic fate of polyphenolics in human, suggesting their involvement in the complex interactions between dietary polyphenols and CYP3A4. Last but not least, all the above suggests that coadministration of drugs and foods that are rich in polyphenols is expected to stimulate undesirable clinical consequences. This review focuses on interactions between dietary polyphenols and CYP3A4 as they relate to structural considerations, food-drug interactions, and potential negative consequences of interactions between CYP3A4 and polyphenols. PMID:26180597

  20. Interactions between CYP3A4 and Dietary Polyphenols.

    PubMed

    Basheer, Loai; Kerem, Zohar

    2015-01-01

    The human cytochrome P450 enzymes (P450s) catalyze oxidative reactions of a broad spectrum of substrates and play a critical role in the metabolism of xenobiotics, such as drugs and dietary compounds. CYP3A4 is known to be the main enzyme involved in the metabolism of drugs and most other xenobiotics. Dietary compounds, of which polyphenolics are the most studied, have been shown to interact with CYP3A4 and alter its expression and activity. Traditionally, the liver was considered the prime site of CYP3A-mediated first-pass metabolic extraction, but in vitro and in vivo studies now suggest that the small intestine can be of equal or even greater importance for the metabolism of polyphenolics and drugs. Recent studies have pointed to the role of gut microbiota in the metabolic fate of polyphenolics in human, suggesting their involvement in the complex interactions between dietary polyphenols and CYP3A4. Last but not least, all the above suggests that coadministration of drugs and foods that are rich in polyphenols is expected to stimulate undesirable clinical consequences. This review focuses on interactions between dietary polyphenols and CYP3A4 as they relate to structural considerations, food-drug interactions, and potential negative consequences of interactions between CYP3A4 and polyphenols. PMID:26180597

  1. 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.

  2. 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

  3. Characterization of the genetic variation present in CYP3A4 in three South African populations.

    PubMed

    Drögemöller, Britt; Plummer, Marieth; Korkie, Lundi; Agenbag, Gloudi; Dunaiski, Anke; Niehaus, Dana; Koen, Liezl; Gebhardt, Stefan; Schneider, Nicol; Olckers, Antonel; Wright, Galen; Warnich, Louise

    2013-01-01

    The CYP3A4 enzyme is the most abundant human cytochrome P450 (CYP) and is regarded as the most important enzyme involved in drug metabolism. Inter-individual and inter-population variability in gene expression and enzyme activity are thought to be influenced, in part, by genetic variation. Although Southern African individuals have been shown to exhibit the highest levels of genetic diversity, they have been under-represented in pharmacogenetic research to date. Therefore, the aim of this study was to identify genetic variation within CYP3A4 in three South African population groups comprising of 29 Khoisan, 65 Xhosa and 65 Mixed Ancestry (MA) individuals. To identify known and novel CYP3A4 variants, 15 individuals were randomly selected from each of the population groups for bi-directional Sanger sequencing of ~600 bp of the 5'-upstream region and all thirteen exons including flanking intronic regions. Genetic variants detected were genotyped in the rest of the cohort. In total, 24 SNPs were detected, including CYP3A4(*)12, CYP3A4(*)15, and the reportedly functional CYP3A4(*)1B promoter polymorphism, as well as two novel non-synonymous variants. These putatively functional variants, p.R162W and p.Q200H, were present in two of the three populations and all three populations, respectively, and in silico analysis predicted that the former would damage the protein product. Furthermore, the three populations were shown to exhibit distinct genetic profiles. These results confirm that South African populations show unique patterns of variation in the genes encoding xenobiotic metabolizing enzymes. This research suggests that population-specific genetic profiles for CYP3A4 and other drug metabolizing genes would be essential to make full use of pharmacogenetics in Southern Africa. Further investigation is needed to determine if the identified genetic variants influence CYP3A4 metabolism phenotype in these populations. PMID:23423246

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  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. 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

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. Diindolylmethane, a naturally occurring compound, induces CYP3A4 and MDR1 gene expression by activating human PXR

    PubMed Central

    Pondugula, Satyanarayana R.; Flannery, Patrick C.; Abbott, Kodye L.; Coleman, Elaine S.; Mani, Sridhar; Samuel, Temesgen; Xie, Wen

    2015-01-01

    Activation of human pregnane X receptor (hPXR)-regulated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1) plays an important role in mediating adverse drug interactions. Given the common use of natural products as part of adjunct human health behavior, there is a growing concern about natural products for their potential to induce undesired drug interactions through the activation of hPXR-regulated CYP3A4 and MDR1. Here, we studied whether 3,3′-diindolylmethane (DIM), a natural health supplement, could induce hPXR-mediated regulation of CYP3A4 and MDR1 in human hepatocytes and intestinal cells. DIM, at its physiologically relevant concentrations, not only induced hPXR transactivation of CYP3A4 promoter activity but also induced gene expression of CYP3A4 and MDR1. DIM decreased intracellular accumulation of MDR1 substrate rhodamine 123, suggesting that DIM induces the functional expression of MDR1. Pharmacologic inhibition or genetic knockdown of hPXR resulted in attenuation of DIM induced CYP3A4 and MDR1 gene expression, suggesting that DIM induces CYP3A4 and MDR1 in an hPXR-dependent manner. Together, these results support our conclusion that DIM induces hPXR-regulated CYP3A4 and MDR1 gene expression. The inductive effects of DIM on CYP3A4 and MDR1 expression caution the use of DIM in conjunction with other medications metabolized and transported via CYP3A4 and MDR1, respectively. PMID:25542144

  9. Comparison of Paeoniflorin and Albiflorin on Human CYP3A4 and CYP2D6

    PubMed Central

    Gao, Li-Na; Zhang, Ye; Cui, Yuan-Lu; Akinyi, Olunga Mary

    2015-01-01

    Peony (Paeonia lactiflora Pall-) is a plant medicine and a functional food ingredient with wide application for more than 2000 years. It can be coadministrated with many other drugs, composed of traditional Chinese medicine compound such as shaoyao-gancao decoction. In order to explore the efficacy and safety of peony, effects of paeoniflorin and albiflorin (the principal components of peony) on cytochrome P450 (CYP) 3A4 and CYP2D6 were analyzed in human hepatoma HepG2 cells and evaluated from the level of recombinant CYP enzymes in vitro. The findings indicated that albiflorin possessed stronger regulation on the mRNA expression of CYP3A4 and CYP2D6 than paeoniflorin. For the protein level of CYP3A4, albiflorin showed significant induction or inhibition with the concentration increasing from 10−7 M to 10−5 M, but no remarkable variation was observed in paeoniflorin-treated group. Enzyme activity assay implied that both paeoniflorin and albiflorin could regulate CYP3A4 and CYP2D6 with varying degrees. The results showed that albiflorin should be given more attention because it may play a vital role on the overall efficacy of peony. The whole behavior of both paeoniflorin and albiflorin should be focused on ensuring the rationality and effectiveness of clinical application. PMID:26089940

  10. Comparison of Paeoniflorin and Albiflorin on Human CYP3A4 and CYP2D6.

    PubMed

    Gao, Li-Na; Zhang, Ye; Cui, Yuan-Lu; Akinyi, Olunga Mary

    2015-01-01

    Peony (Paeonia lactiflora Pall-) is a plant medicine and a functional food ingredient with wide application for more than 2000 years. It can be coadministrated with many other drugs, composed of traditional Chinese medicine compound such as shaoyao-gancao decoction. In order to explore the efficacy and safety of peony, effects of paeoniflorin and albiflorin (the principal components of peony) on cytochrome P450 (CYP) 3A4 and CYP2D6 were analyzed in human hepatoma HepG2 cells and evaluated from the level of recombinant CYP enzymes in vitro. The findings indicated that albiflorin possessed stronger regulation on the mRNA expression of CYP3A4 and CYP2D6 than paeoniflorin. For the protein level of CYP3A4, albiflorin showed significant induction or inhibition with the concentration increasing from 10(-7) M to 10(-5) M, but no remarkable variation was observed in paeoniflorin-treated group. Enzyme activity assay implied that both paeoniflorin and albiflorin could regulate CYP3A4 and CYP2D6 with varying degrees. The results showed that albiflorin should be given more attention because it may play a vital role on the overall efficacy of peony. The whole behavior of both paeoniflorin and albiflorin should be focused on ensuring the rationality and effectiveness of clinical application. PMID:26089940

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  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. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. Arsenite and its metabolites, MMA{sup III} and DMA{sup III}, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

    SciTech Connect

    Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.; Ramirez, P.; Vega, L.; Elizondo, G.

    2009-09-01

    Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA{sup III} induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA{sup III} increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA{sup III} induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. Mechanism-Based Inactivation of Human Cytochrome P450 3A4 by Two Piperazine-Containing Compounds

    PubMed Central

    Bolles, Amanda K.; Fujiwara, Rina; Briggs, Erran D.; Nomeir, Amin A.

    2014-01-01

    Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of more than half of pharmaceutic drugs, and inactivation of CYP3A4 can lead to adverse drug-drug interactions. The substituted imidazole compounds 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) and 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) have been previously identified as mechanism-based inactivators (MBI) of CYP2D6. The present study shows that both SCH 66712 and EMTPP are also MBIs of CYP3A4. Inhibition of CYP3A4 by SCH 66712 and EMTPP was determined to be concentration, time, and NADPH dependent. In addition, inactivation of CYP3A4 by SCH 66712 was shown to be unaffected by the presence of electrophile scavengers. SCH 66712 displays type I binding to CYP3A4 with a spectral binding constant (Ks) of 42.9 ± 2.9 µM. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. Whole protein mass spectrum analysis revealed 1:1 binding stoichiometry of SCH 66712 and EMTPP to CYP3A4 and a mass increase consistent with adduction by the inactivators without addition of oxygen. Heme adduction was not apparent. Multiple mono-oxygenation products with each inactivator were observed; no other products were apparent. These are the first MBIs to be shown to be potent inactivators of both CYP2D6 and CYP3A4. PMID:25273356

  13. Mechanism-based inactivation of human cytochrome P450 3A4 by two piperazine-containing compounds.

    PubMed

    Bolles, Amanda K; Fujiwara, Rina; Briggs, Erran D; Nomeir, Amin A; Furge, Laura Lowe

    2014-12-01

    Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of more than half of pharmaceutic drugs, and inactivation of CYP3A4 can lead to adverse drug-drug interactions. The substituted imidazole compounds 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) and 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) have been previously identified as mechanism-based inactivators (MBI) of CYP2D6. The present study shows that both SCH 66712 and EMTPP are also MBIs of CYP3A4. Inhibition of CYP3A4 by SCH 66712 and EMTPP was determined to be concentration, time, and NADPH dependent. In addition, inactivation of CYP3A4 by SCH 66712 was shown to be unaffected by the presence of electrophile scavengers. SCH 66712 displays type I binding to CYP3A4 with a spectral binding constant (Ks) of 42.9 ± 2.9 µM. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. Whole protein mass spectrum analysis revealed 1:1 binding stoichiometry of SCH 66712 and EMTPP to CYP3A4 and a mass increase consistent with adduction by the inactivators without addition of oxygen. Heme adduction was not apparent. Multiple mono-oxygenation products with each inactivator were observed; no other products were apparent. These are the first MBIs to be shown to be potent inactivators of both CYP2D6 and CYP3A4. PMID:25273356

  14. The effect of interferon-{alpha} on the expression of cytochrome P450 3A4 in human hepatoma cells

    SciTech Connect

    Flaman, Anathea S.; Gravel, Caroline; Hashem, Anwar M.; Tocchi, Monika; Li Xuguang

    2011-06-01

    Interferon {alpha} (IFN{alpha}) is used to treat malignancies and chronic viral infections. It has been found to decrease the rate of drug metabolism by acting on cytochrome P450 enzymes, but no studies have investigated the consequences of IFN{alpha} treatment on the CYP3A4 isoform, responsible for the metabolism of a majority of drugs. In this study, we have examined the effect of IFN{alpha} on CYP3A4 catalytic activity and expression in human hepatoma cells. We found that IFN{alpha} inhibits CYP3A4 activity and rapidly down-regulates the expression of CYP3A4, independent of de novo protein synthesis. Pharmacologic inhibitors and a dominant-negative mutant expression plasmid were used to dissect the molecular pathway required for CYP3A4 suppression, revealing roles for Jak1 and Stat1 and eliminating the involvement of the p38 mitogen-activated and extracellular regulated kinases. Treatment of hepatoma cells with IFN{alpha} did not affect the nuclear localization or relative abundance of Sp1 and Sp3 transcription factors, suggesting that the suppression of CYP3A4 by IFN{alpha} does not result from inhibitory Sp3 out-competing Sp1. To our knowledge, this is the first report that IFN{alpha} down-regulates CYP3A4 expression largely through the JAK-STAT pathway. Since IFN{alpha} suppresses CYP3A4 expression, caution is warranted when IFN{alpha} is administered in combination with CYP3A4 substrates to avoid the occurrence of adverse drug interactions.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. A Large-Scale Allosteric Transition in Cytochrome P450 3A4 Revealed by Luminescence Resonance Energy Transfer (LRET)

    PubMed Central

    Sineva, Elena V.; Rumfeldt, Jessica A. O.; Halpert, James R.; Davydov, Dmitri R.

    2013-01-01

    Effector-induced allosteric transitions in cytochrome P450 3A4 (CYP3A4) were investigated by luminescence resonance energy transfer (LRET) between two SH-reactive probes attached to various pairs of distantly located cysteine residues, namely the double-cysteine mutants CYP3A4(C64/C468), CYP3A4(C377/C468) and CYP3A4(C64/C121). Successive equimolar labeling of these proteins with the phosphorescent probe erythrosine iodoacetamide (donor) and the near-infrared fluorophore DY-731 maleimide (acceptor) allowed us to establish donor/acceptor pairs sensitive to conformational motions. The interactions of all three double-labeled mutants with the allosteric activators α-naphthoflavone and testosterone resulted in an increase in the distance between the probes. A similar effect was elicited by cholesterol. These changes in distance vary from 1.3 to 8.5 Å, depending on the position of the donor/acceptor pair and the nature of the effector. In contrast, the changes in the interprobe distance caused by such substrates as bromocriptine or 1-pyrenebutanol were only marginal. Our results provide a decisive support to the paradigm of allosteric modulation of CYP3A4 and indicate that the conformational transition caused by allosteric effectors increases the spatial separation between the beta-domain of the enzyme (bearing residues Cys64 and Cys377) and the alpha-domain, where Cys121 and Cys468 are located. PMID:24376769

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Pivotal Role of P450-P450 Interactions in CYP3A4 Allostery: the Case of α-Naphthoflavone

    PubMed Central

    Davydov, Dmitri R.; Davydova, Nadezhda Y.; Sineva, Elena V.; Kufareva, Irina; Halpert, James R.

    2014-01-01

    SYNOPSIS We investigated the relationship between oligomerization of cytochrome P450 3A4 (CYP3A4) and its response to α-naphthoflavone (ANF), a prototypical heterotropic activator. Addition of ANF resulted in over a two-fold increase in the rate of CYP3A4-dependent debenzylation of 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC) in human liver microsomes (HLM) but failed to produce activation in BD Supersomes™ or Baculosomes® containing recombinant CYP3A4 and NADPH-cytochrome P450 reductase (CPR). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behavior observed with HLM. The activation in this system was dependent on the surface density of the enzyme. While no activation was detectable at a lipid:P450 (L/P) ratio ≥ 750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization we probed P450-P450 interactions with a new technique based on luminescence resonance energy transfer (LRET). The amplitude of LRET in mixed oligomers of the heme protein labeled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes. Addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore, the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions. PMID:23651100

  10. 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.

  11. 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.

  12. In Silico Docking of Ligands to Drug Oxidation Enzymes Cytochrome P450 3A4 and Cytochrome P450 1A2.

    NASA Astrophysics Data System (ADS)

    Smith, David; Guglielmon, Jonathan; Glenn, Marsch; Peter, Guengerich F.

    2009-03-01

    Cytochrome P450 3A4 (CYP3A4) and Cytochrome P450 1A2 (CYP1A2) oxidize most drugs in humans. Protein modeling toolkits from OpenEye Scientific Software were used to examine the interaction of drug substrates with CYP3A4 and CYP1A2. Conformers and partial atomic charges were generated for each drug molecule. User-defined volumes were defined around CYP3A4 and CYP1A2 active sites. Ligands were docked assuming protein and substrates as rigid bodies. To assess rigid docking accuracy, x-ray diffraction coordinates of CYP3A4-erythromycin and CYP3A4-metyrapone complexes were obtained. Rigid re-docking of erythromycin and metyrapone into CYP3A4 yielded poses similar to the crystal structures. Rigid docking revealed two other energetically-favorable CYP3A4-metyrapone poses. The best poses were obtained by using all the Open Eye scoring functions. Optimization of protein-ligand interactions within 5-10 Angstroms of the docked ligand was then performed using the Merck Molecular Force Field in which the protein was assumed to be flexible and the ligand to be rigid. Nearby protein residues pulled slightly closer to the substrate, reducing the volume of the active site.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. Drug membrane transporters and CYP3A4 are affected by hypericin, hyperforin or aristoforin in colon adenocarcinoma cells.

    PubMed

    Šemeláková, M; Jendželovský, R; Fedoročko, P

    2016-07-01

    Our previous results have shown that the combination of hypericin-mediated photodynamic therapy (HY-PDT) at sub-optimal dose with hyperforin (HP) (compounds of Hypericum sp.), or its stable derivative aristoforin (AR) stimulates generation of reactive oxygen species (ROS) leading to antitumour activity. This enhanced oxidative stress evoked the need for an explanation for HY accumulation in colon cancer cells pretreated with HP or AR. Generally, the therapeutic efficacy of chemotherapeutics is limited by drug resistance related to the overexpression of drug efflux transporters in tumour cells. Therefore, the impact of non-activated hypericin (HY), HY-PDT, HP and AR on cell membrane transporter systems (Multidrug resistance-associated protein 1-MRP1/ABCC1, Multidrug resistance-associated protein 2-MRP2/ABCC2, Breast cancer resistance protein - BCRP/ABCG2, P-glycoprotein-P-gp/ABCC1) and cytochrome P450 3A4 (CYP3A4) was evaluated. The different effects of the three compounds on their expression, protein level and activity was determined under specific PDT light (T0+, T6+) or dark conditions (T0- T6-). We found that HP or AR treatment affected the protein levels of MRP2 and P-gp, whereas HP decreased MRP2 and P-gp expression mostly in the T0+ and T6+ conditions, while AR decreased MRP2 in T0- and T6+. Moreover, HY-PDT treatment induced the expression of MRP1. Our data demonstrate that HP or AR treatment in light or dark PDT conditions had an inhibitory effect on the activity of individual membrane transport proteins and significantly decreased CYP3A4 activity in HT-29 cells. We found that HP or AR significantly affected intracellular accumulation of HY in HT-29 colon adenocarcinoma cells. These results suggest that HY, HP and AR might affect the efficiency of anti-cancer drugs, through interaction with membrane transporters and CYP3A4. PMID:27261575

  6. Effects of Commonly Used Excipients on the Expression of CYP3A4 in Colon and Liver Cells

    PubMed Central

    Tompkins, Leslie; Lynch, Caitlin; Haidar, Sam; Polli, James; Wang, Hongbing

    2013-01-01

    Purpose The objective of this investigation was to assess whether common pharmaceutical excipients regulate the expression of drug-metabolizing enzymes in human colon and liver cells. Methods Nineteen commonly used excipients were evaluated using a panel of experiments including cell-based human PXR activation assays, real-time RT-PCR assays for CYP3A4 mRNA expression, and immunoblot analysis of CYP3A4 protein expression in immortalized human liver cells (HepG2 and Fa2N4), human primary hepatocytes, and the intestinal LS174T cell models. Results No excipient activated human PXR or practically induced CYP3A4. However, three excipients (polysorbate 80, pregelatinized starch, and hydroxypropyl methylcellulose) tended to decrease mRNA and protein expression across experimental models. Conclusion This study represents the first investigation of the potential role of excipients in the expression of drug-metabolizing enzymes. Findings imply that some excipients may hold potential for excipient-drug interactions by repression of CYP3A4 expression. PMID:20503067

  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. 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

  12. 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…

  13. 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.

  14. Inhibitory Effects of Vegetable Juices on CYP3A4 Activity in Recombinant CYP3A4 and LS180 Cells.

    PubMed

    Tsujimoto, Masayuki; Uchida, Tomoe; Kozakai, Hiroyuki; Yamamoto, Saori; Minegaki, Tetsuya; Nishiguchi, Kohshi

    2016-01-01

    It is thought that eating habits induces individual variation in intestinal absorption and metabolism of drugs. The objective of this research was to clarify the influence of vegetables juices on CYP3A4 activity, which is an important enzyme in intestine. Five vegetables juices (VJ-o, Kagome Original(®); VJ-g, Kagome 30 kinds of vegetables and fruits(®); VJ-p, Kagome Purple vegetables(®); VJ-r, Kagome Sweet Tomato(®); and VJ-y, Kagome Fruity Salada(®); KAGOME Co., Ltd., Aichi, Japan) were centrifuged (1630×g, 10 min) and filtered using filter paper and 0.45-µm membrane filters. In this study, recombinant CYP3A4 and LS180 cells were used for the evaluation of CYP3A4 activity. The metabolisms to 6β-hydroxytestosterone by recombinant CYP3A4 were significantly inhibited by VJ-o, VJ-g, and VJ-y in a preincubation time-dependent manner, and CYP3A4 activity in LS180 cells were significantly inhibited by VJ-o and VJ-y. These results show that the difference in ingestion volume of vegetable juices and vegetables might partially induce individual difference in intestinal drug metabolism.

  15. Inhibitory Effects of Vegetable Juices on CYP3A4 Activity in Recombinant CYP3A4 and LS180 Cells.

    PubMed

    Tsujimoto, Masayuki; Uchida, Tomoe; Kozakai, Hiroyuki; Yamamoto, Saori; Minegaki, Tetsuya; Nishiguchi, Kohshi

    2016-01-01

    It is thought that eating habits induces individual variation in intestinal absorption and metabolism of drugs. The objective of this research was to clarify the influence of vegetables juices on CYP3A4 activity, which is an important enzyme in intestine. Five vegetables juices (VJ-o, Kagome Original(®); VJ-g, Kagome 30 kinds of vegetables and fruits(®); VJ-p, Kagome Purple vegetables(®); VJ-r, Kagome Sweet Tomato(®); and VJ-y, Kagome Fruity Salada(®); KAGOME Co., Ltd., Aichi, Japan) were centrifuged (1630×g, 10 min) and filtered using filter paper and 0.45-µm membrane filters. In this study, recombinant CYP3A4 and LS180 cells were used for the evaluation of CYP3A4 activity. The metabolisms to 6β-hydroxytestosterone by recombinant CYP3A4 were significantly inhibited by VJ-o, VJ-g, and VJ-y in a preincubation time-dependent manner, and CYP3A4 activity in LS180 cells were significantly inhibited by VJ-o and VJ-y. These results show that the difference in ingestion volume of vegetable juices and vegetables might partially induce individual difference in intestinal drug metabolism. PMID:27582329

  16. In vitro inhibition of cytochrome P450 3A4 by Aronia melanocarpa constituents.

    PubMed

    Bräunlich, Marie; Christensen, Hege; Johannesen, Siri; Slimestad, Rune; Wangensteen, Helle; Malterud, Karl E; Barsett, Hilde

    2013-01-01

    Extracts, subfractions, isolated anthocyanins and procyanidins, and two phenolic acids from aronia [Aronia melanocarpa] were investigated for their CYP3A4 inhibitory effects, using midazolam as the probe substrate and recombinant insect cell microsomes expressing CYP3A4 as the enzyme source. Procyanidin B5 was a considerably stronger CYP3A4 inhibitor in vitro than the isomeric procyanidin B2 and comparable to bergamottin, a known CYP3A4 inhibitor from grapefruit juice. The inhibitory activity of proanthocyanidin-containing fractions was correlated to the degree of polymerization. Among the anthocyanins, cyanidin 3-arabinoside showed stronger CYP3A4 inhibition than cyanidin 3-galactoside and cyanidin 3-glucoside. Thus, the ability to inhibit CYP3A4 in vitro seems to be influenced by the sugar unit linked to the anthocyanidin.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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...

  2. 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.

  3. 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.

  4. 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.

  5. Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis.

    PubMed

    Wang, Hai-Yan; Zhang, Ji; Zhang, Yue-Jing; Zhang, Bo; Liu, Chong-Xi; He, Hai-Rong; Wang, Xiang-Jing; Xiang, Wen-Sheng

    2014-12-01

    Milbemycin oxime has been commercialized as effective anthelmintics in the fields of animal health, agriculture, and human infections. Currently, milbemycin oxime is synthesized by a two-step chemical reaction, which involves the ketonization of milbemycins A3/A4 to yield the intermediates 5-oxomilbemycins A3/A4 using CrO3 as catalyst. Due to the low efficiency and environmental unfriendliness of the ketonization of milbemycins A3/A4, it is imperative to develop alternative strategies to produce 5-oxomilbemycins A3/A4. In this study, the atmospheric and room temperature plasma (ARTP) mutation system was first employed to treat milbemycin-producing strain Streptomyces bingchenggensis, and a mutant strain BC-120-4 producing milbemycins A3, A4, B2, and B3 as main components was obtained, which favors the construction of genetically engineered strains producing 5-oxomilbemycins. Importantly, the milbemycins A3/A4 yield of BC-120-4 reached 3,890 ± 52 g/l, which was approximately two times higher than that of the initial strain BC-109-6 (1,326 ± 37 g/l). The subsequent interruption of the gene milF encoding a C5-ketoreductase responsible for the ketonization of milbemycins led to strain BCJ60 (∆milF) with the production of 5-oxomilbemycins A3/A4 and the elimination of milbemycins A3, A4, B2, and B3. The high 5-oxomilbemycins A3/A4 yield (3,470 ± 147 g/l) and genetic stability of BCJ60 implied the potential use in industry to prepare 5-oxomilbemycins A3/A4 for the semisynthesis of milbemycins oxime. PMID:25081559

  6. Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations.

    PubMed

    Müller, Christian S; Knehans, Tim; Davydov, Dmitri R; Bounds, Patricia L; von Mandach, Ursula; Halpert, James R; Caflisch, Amedeo; Koppenol, Willem H

    2015-01-27

    Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V.

  7. 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

  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.

  9. Norcocaine and N-hydroxynorcocaine formation in human liver microsomes: role of cytochrome P-450 3A4.

    PubMed

    LeDuc, B W; Sinclair, P R; Shuster, L; Sinclair, J F; Evans, J E; Greenblatt, D J

    1993-05-01

    Cocaine was metabolized to norcocaine by microsomes prepared from lymphoblastoid cells expressing transfected human P-450 3A4. The specific activities of norcocaine formation by microsomes prepared from three human liver samples correlated with the amount of P-450 3A immunoreactive protein detected by immunoblot. Triacetyloleandomycin, a specific inhibitor of P-450 3A isoforms, inhibited formation of norcocaine from cocaine, but not formation of N-hydroxynorcocaine from norcocaine. The chemical identity of the norcocaine and N-hydroxynorcocaine produced by human liver microsomes was established by combination of gas chromatography and mass spectrometry. Thus, human P-450 3A4 is a cocaine demethylase, and P-450 isoforms of the 3A family are responsible for the majority of norcocaine production by human hepatic microsomes.

  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. Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions

    PubMed Central

    Poulos, Thomas L.

    2015-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major and most important drug-metabolizing enzyme in humans that oxidizes and clears over a half of all administered pharmaceuticals. This is possible because CYP3A4 is promiscuous with respect to substrate binding and has the ability to catalyze diverse oxidative chemistries in addition to traditional hydroxylation reactions. Furthermore, CYP3A4 binds and oxidizes a number of substrates in a cooperative manner and can be both induced and inactivated by drugs. In vivo, CYP3A4 inhibition could lead to undesired drug-drug interactions and drug toxicity, a major reason for late-stage clinical failures and withdrawal of marketed pharmaceuticals. Owing to its central role in drug metabolism, many aspects of CYP3A4 catalysis have been extensively studied by various techniques. Here, we give an overview of experimental and theoretical methods currently used for investigation and prediction of CYP3A4-ligand interactions, a defining factor in drug metabolism, with an emphasis on the problems addressed and conclusions derived from the studies. PMID:26002732

  12. A comparative study of CYP3A4 polymorphisms in Mexican Amerindian and Mestizo populations.

    PubMed

    Reyes-Hernández, Octavio D; Lares-Asseff, Ismael; Sosa-Macias, Martha; Vega, Libia; Albores, Arnulfo; Elizondo, Guillermo

    2008-01-01

    Cytochrome P-450 3A4 (CYP3A4) contributes to the metabolism of approximately half the drugs in clinical use today. The aim of the present study was to determine the frequency of the CYP3A4*1B, *2, *4, *5, and *18 alleles amongst both Tepehuan Amerindians, a native group that has inhabited northern Mexico for thousands of years, and Mestizo Mexicans, and to compare the data with those of other populations. Genotyping experiments revealed that 8.8 and 8.0% of the Mestizo and Tepehuano subjects, respectively, carried the CYP3A4*1B allele. Only one Mestizo subject was heterozygous for the CYP3A4*2 variant, while CYP3A4*4, *5 and *18 allelic variants were not detected in either group. On the other hand, the frequencies of the CYP3A4*1B variant in Mestizos and Tepehuanos were similar to those reported for Caucasians, but different from those observed for African and Asian populations.

  13. Quantitative Prediction of Regioselectivity Toward Cytochrome P450/3A4 Using Machine Learning Approaches.

    PubMed

    Hasegawa, Kiyoshi; Koyama, Michio; Funatsu, Kimito

    2010-03-15

    In the drug discovery process, it is important to know the properties of both drug candidates and their metabolites. Fast and precise prediction of metabolites is essential. However, it has been difficult to predict metabolites because of the complexity of the mechanism of cytochrome P450/3A4 (CYP 3A4), which is the main metabolite enzyme of drugs. In this study, we focus on the regioselectivity of CYP 3A4, i.e., the selectivity of metabolic sites. We have developed a model to predict the regioselectivity of drug candidates by using machine learning (ML) approaches.

  14. 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

  15. 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

  16. 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

  17. A neural network based virtual screening of cytochrome P450 3A4 inhibitors.

    PubMed

    Molnar, László; Keseru, György M

    2002-02-11

    A virtual screening test to identify potential CP450 3A4 inhibitors has been developed. Molecular structures of inhibitors and non-inhibitors available in the Genetest database were represented using 2D Unity fingerprints and a feedforward neural network was trained to classify molecules regarding their inhibitory activity. Validation tests revealed that our neural net recognizes at least 89% of 3A4 inhibitors and suggest using this methodology in our virtual screening protocol.

  18. CYP3A4 drug interactions: correlation of 10 in vitro probe substrates

    PubMed Central

    Kenworthy, K E; Bloomer, J C; Clarke, S E; Houston, J B

    1999-01-01

    Aims Many substrates of cytochrome P450 (CYP) 3A4 are used for in vitro investigations of drug metabolism and potential drug–drug interactions. The aim of the present study was to determine the relationship between 10 commonly used CYP3A4 probes using modifiers with a range of inhibitory potency. Methods The effects of 34 compounds on CYP3A4-mediated metabolism were investigated in a recombinant CYP3A4 expression system. Inhibition of erythromycin, dextromethorphan and diazepam N-demethylation, testosterone 6β-hydroxylation, midazolam 1-hydroxylation, triazolam 4-hydroxylation, nifedipine oxidation, cyclosporin oxidation, terfenadine C-hydroxylation and N-dealkylation and benzyloxyresorufin O-dealkylation was evaluated at the apparent Km or S50 (for substrates showing sigmoidicity) value for each substrate and at an inhibitor concentration of 30 μm. Results While all CYP3A4 probe substrates demonstrate some degree of similarity, examination of the coefficients of determination, together with difference and cluster analysis highlighted that seven substrates can be categorized into two distinct substrate groups. Erythromycin, cyclosporin and testosterone form the most closely related group and dextromethorphan, diazepam, midazolam and triazolam form a second group. Terfenadine can be equally well placed in either group, while nifedipine shows a distinctly different relationship. Benzyloxyresorufin shows the weakest correlation with all the other CYP3A4 probes. Modifiers that caused negligible inhibition or potent inhibition are generally comparable in all assays, however, the greatest variability is apparent with compounds causing, on average, intermediate inhibition. Modifiers of this type may cause substantial inhibition, no effect or even activation depending on the substrate employed. Conclusions It is recommended that multiple CYP3A4 probes, representing each substrate group, are used for the in vitro assessment of CYP3A4-mediated drug interactions. PMID

  19. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

    SciTech Connect

    Casabar, Richard C.T.; Das, Parikshit C.; DeKrey, Gregory K.; Gardiner, Catherine S.; Cao Yan; Rose, Randy L.; Wallace, Andrew D.

    2010-06-15

    Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 {mu}M. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 {mu}M. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 {mu}M, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 {mu}M and 10 {mu}M, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. Prediction of small-molecule binding to cytochrome P450 3A4: flexible docking combined with multidimensional QSAR.

    PubMed

    Lill, Markus A; Dobler, Max; Vedani, Angelo

    2006-01-01

    The inhibition of cytochrome P450 3A4 (CYP3A4) by small molecules is a major mechanism associated with undesired drug-drug interactions, which are responsible for a substantial number of late-stage failures in the pharmaceutical drug-development process. For a quantitative prediction of associated pharmacokinetic parameters, a computational model was developed that allows prediction of the inhibitory potential of 48 structurally diverse molecules. Based on the experimental structure of CYP3A4, possible binding modes were first sampled by using automated docking (Yeti software) taking protein flexibility into account. The results are consistent with both X-ray crystallographic data and data from metabolic studies. Next, an ensemble of energetically favorable orientations was composed into a 4D dataset for use as input for a multidimensional QSAR technique (Raptor software). A dual-shell binding-site model that allows an explicit induced fit was then generated by using hydrophobicity scoring and hydrogen-bond propensity. The simulation reached a cross-validated r2 value of 0.825 and a predictive r2 value of 0.659. On average, the predicted binding affinity of the training ligands deviates by a factor of 2.7 from the experiment; those of the test set deviate by a factor of 3.8 in Ki.

  5. 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

  6. Dual effects of ketoconazole cis-enantiomers on CYP3A4 in human hepatocytes and HepG2 Cells.

    PubMed

    Novotná, Aneta; Krasulová, Kristýna; Bartoňková, Iveta; Korhoňová, Martina; Bachleda, Petr; Anzenbacher, Pavel; Dvořák, Zdeněk

    2014-01-01

    Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (-)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (-)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC₅₀ 1.69 µM, Ki 0.92 µM for testosterone, IC₅₀ 1.46 µM, Ki 2.52 µM for midazolam; (-)-ketoconazole IC₅₀ 0.90 µM, Ki 0.17 µM for testosterone, IC₅₀ 1.04 µM, Ki 1.51 µM for midazolam).

  7. Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib.

    PubMed

    Towles, Joanna K; Clark, Rebecca N; Wahlin, Michelle D; Uttamsingh, Vinita; Rettie, Allan E; Jackson, Klarissa D

    2016-10-01

    Metabolic activation of the dual-tyrosine kinase inhibitor lapatinib by cytochromes CYP3A4 and CYP3A5 has been implicated in lapatinib-induced idiosyncratic hepatotoxicity; however, the relative enzyme contributions have not been established. The objective of this study was to examine the roles of CYP3A4 and CYP3A5 in lapatinib bioactivation leading to a reactive, potentially toxic quinoneimine. Reaction phenotyping experiments were performed using individual human recombinant P450 enzymes and P450-selective chemical inhibitors. Lapatinib metabolites and quinoneimine-glutathione (GSH) adducts were analyzed using liquid chromatography-tandem mass spectrometry. A screen of cDNA-expressed P450s confirmed that CYP3A4 and CYP3A5 are the primary enzymes responsible for quinoneimine-GSH adduct formation using lapatinib or O-dealkylated lapatinib as the substrate. The mean kinetic parameters (Km and kcat) of lapatinib O-dealkylation revealed that CYP3A4 was 5.2-fold more efficient than CYP3A5 at lapatinib O-dealkylation (CYP3A4 kcat/Km = 6.8 μM(-1) min(-1) versus CYP3A5 kcat/Km = 1.3 μM(-1) min(-1)). Kinetic analysis of GSH adduct formation indicated that CYP3A4 was also 4-fold more efficient at quinoneimine-GSH adduct formation as measured by kcat (maximum relative GSH adduct levels)/Km (CYP3A4 = 0.0082 vs. CYP3A5 = 0.0021). In human liver microsomal (HLM) incubations, CYP3A4-selective inhibitors SR-9186 and CYP3cide reduced formation of GSH adducts by 78% and 72%, respectively, compared with >90% inhibition by the pan-CYP3A inhibitor ketoconazole. The 16%-22% difference between CYP3A- and CYP3A4-selective inhibition indicates the involvement of remaining CYP3A5 activity in generating reactive metabolites from lapatinib in pooled HLMs. Collectively, these findings support the conclusion that both CYP3A4 and CYP3A5 are quantitatively important contributors to lapatinib bioactivation. PMID:27450182

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  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. PMID:26896315

  16. 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.

  17. 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

  18. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    NASA Astrophysics Data System (ADS)

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-02-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

  19. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    PubMed Central

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-01-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification. PMID:26899743

  20. 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.

  1. 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

  2. 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.

  3. Hemopericardium with tamponade following rivaroxaban administration and its attenuation by CYP3A4 inhibitors

    PubMed Central

    Menendez, Denisse

    2016-01-01

    Novel oral anticoagulants including the factor Xa inhibitor rivaroxaban are important alternatives to warfarin for the prevention of thromboembolic stroke in patients with nonvalvular atrial fibrillation. The pharmacology and metabolism of these agents differ from those of the vitamin K antagonists used over the decades preceding their introduction. We present a case of spontaneous hemopericardium and cardiac tamponade following administration of rivaroxaban. A review of the patient's medications revealed a total of seven agents known to be metabolized through cytochrome P450 3A4 (CYP3A4), the major pathway for rivaroxaban metabolism. While most physicians are familiar with recommendations to monitor renal function in patients prescribed rivaroxaban, we suspect that many fail to evaluate possible interactions with other agents having CYP3A4 inhibitory or inducer activity. PMID:27695181

  4. Hemopericardium with tamponade following rivaroxaban administration and its attenuation by CYP3A4 inhibitors

    PubMed Central

    Menendez, Denisse

    2016-01-01

    Novel oral anticoagulants including the factor Xa inhibitor rivaroxaban are important alternatives to warfarin for the prevention of thromboembolic stroke in patients with nonvalvular atrial fibrillation. The pharmacology and metabolism of these agents differ from those of the vitamin K antagonists used over the decades preceding their introduction. We present a case of spontaneous hemopericardium and cardiac tamponade following administration of rivaroxaban. A review of the patient's medications revealed a total of seven agents known to be metabolized through cytochrome P450 3A4 (CYP3A4), the major pathway for rivaroxaban metabolism. While most physicians are familiar with recommendations to monitor renal function in patients prescribed rivaroxaban, we suspect that many fail to evaluate possible interactions with other agents having CYP3A4 inhibitory or inducer activity.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. Functional analysis of Pid3-A4, an ortholog of rice blast resistance gene Pid3 revealed by allele mining in common wild rice.

    PubMed

    Lv, Qiming; Xu, Xiao; Shang, Junjun; Jiang, Guanghuai; Pang, Zhiqian; Zhou, Zhuangzhi; Wang, Jing; Liu, Ya; Li, Ting; Li, Xiaobing; Xu, Jichen; Cheng, Zhukuan; Zhao, Xianfeng; Li, Shigui; Zhu, Lihuang

    2013-06-01

    The rice blast resistance gene Pid3 encodes a nucleotide-binding-site leucine-rich repeat (NBS-LRR) protein. This gene was cloned from the rice 'Digu' (indica) by performing a genome-wide comparison of the NBS-LRR gene family between two genome-sequenced varieties, '9311' (indica) and 'Nipponbare' (japonica). In this study, we performed functional analysis of Pid3-A4, an ortholog of Pid3 revealed by allele mining in the common wild rice A4 (Oryza rufipogon). The predicted protein encoded by Pid3-A4 shares 99.03% sequence identity with Pid3, with only nine amino-acid substitutions. In wild rice plants, Pid3-A4 is constitutively expressed, and its expression is not induced by Magnaporthe oryzae isolate Zhong-10-8-14 infection. Importantly, in transgenic plants, Pid3-A4, as compared with Pid3, displays a distinct resistance spectrum to a set of M. oryzae isolates, including those that prevail in the rice fields of Sichuan Province. Therefore, Pid3-A4 should be quite useful for the breeding of rice blast resistance, especially in southwestern China.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. Polychlorinated biphenyl (PCB) induction of CYP3A4 enzyme activity in healthy Faroese adults

    SciTech Connect

    Petersen, Maria Skaalum Halling, Jonrit; Damkier, Per; Nielsen, Flemming; Grandjean, Philippe; Weihe, Pal; Brosen, Kim

    2007-10-15

    The CYP3A4 enzyme is, along with other cytochrome P450 enzymes, involved in the metabolism of environmental pollutants and is highly inducible by these substances. A commercial polychlorinated biphenyl (PCB) mixture, 1,1,1,-trichloro-2-(o-chlorophenyl), 2-(p'-chlorophenyl)ethane (o,p'-DDT) and 1,1,-dichloro-2,2-bis (p-chlorophenyl)ethene (p,p'-DDE) are known to induce CYP3A4 activity through activation of nuclear receptors, such as the pregnane X receptor. However, this induction of CYP3A4 has not yet been investigated in humans. Thus, the aim of the study was to determine the variability of the CYP3A4 phenotype in regard to increased concentrations of PCBs and other persistent organohalogen pollutants (POPs) in healthy Faroese adults. In 310 randomly selected Faroese residents aged 18-60 years, the CYP3A4 activity was determined based on the urinary 6{beta}-hydroxycortisol/cortisol (6{beta}-OHC/FC) ratio. POP exposures were assessed by measuring their concentrations in serum lipid. The results showed a unimodal distribution of the 6{beta}-OHC/FC ratio with values ranging from 0.58 to 27.38. Women had a slightly higher 6{beta}-OHC/FC ratio than men (p = 0.07). Confounder-adjusted multiple regression analysis showed significant associations between 6{beta}-OHC/FC ratios and {sigma}PCB, PCB-TEQ and p,p'-DDE, o,p'-DDT and HCB, respectively, but the associations were statistically significant for men only.

  12. Fucoxanthin Attenuates Rifampin-Induced Cytochrome P450 3A4 (CYP3A4) and Multiple Drug Resistance 1 (MDR1) Gene Expression Through Pregnane X Receptor (PXR)-Mediated Pathways in Human Hepatoma HepG2 and Colon Adenocarcinoma LS174T Cells

    PubMed Central

    Liu, Cheng-Ling; Lim, Yun-Ping; Hu, Miao-Lin

    2012-01-01

    Pregnane X receptor (PXR) has been reported to regulate the expression of drug-metabolizing enzymes, such as the cytochrome P450 3A (CYP3A) family and transporters, such as multiple drug resistance 1 (MDR1). Fucoxanthin, the major carotenoid in brown sea algae, is a putative chemopreventive agent. In this study, we determined whether fucoxanthin could overcome drug resistance through attenuation of rifampin-induced CYP3A4 and MDR1 gene expression by PXR-mediated pathways in HepG2 hepatoma cells. We found that fucoxanthin (1–10 μM) significantly attenuated rifampin (20 μM)-induced CYP3A4, MDR1 mRNA and CYP3A4 protein expression at 24 h of incubation. Mechanistically, fucoxanthin strongly attenuated the PXR-mediated CYP3A4 promoter activity in HepG2 cells. In addition, fucoxanthin attenuated constitutive androstane receptor (CAR)- and rPXR-mediated CYP3A4 promoter activity in this cell line. Using the mammalian two-hybrid assay, we found that fucoxanthin significantly decreased the interaction between PXR and SRC-1, a PXR co-activator. Thus, fucoxanthin can decrease rifampin-induced CYP3A4 and MDR1 expression through attenuation of PXR-mediated CYP3A4 promoter activation and interaction between PXR and co-activator. These findings could lead to potentially important new therapeutic and dietary approaches to reduce the frequency of adverse drug reactions. PMID:22363234

  13. Spectroscopic studies and molecular docking on the interaction of organotin antitumor compound bis[2,4-difluoro-N-(hydroxy-⟨κ⟩O)benzamidato-⟨κ⟩O]diphenyltin(IV) with human cytochrome P450 3A4 protease

    NASA Astrophysics Data System (ADS)

    Wei, Ying; Niu, Lin; Liu, Xinxin; Zhou, Hongyan; Dong, Hongzhou; Kong, Depeng; Li, Yunlan; Li, Qingshan

    2016-06-01

    A novel organotin DFDPT was synthesized and characterized by elemental analysis, IR, 1H, 13C, 119Sn, NMR techniques,etc. In order to investigate profoundly the relationship between DFDPT with human CYP3A4 proteaset and anticancer molecular mechanism of DFDPT, the intercalative mode of binding of DFDPT with CYP3A4 under physiological conditions were comprehensively evaluated using steady state, synchronous, three-dimensional fluorescence spectroscopy,circular dichroism and molecular docking. Fluorescence emission data showed that CYP3A4 fluorescence affected by DFDPT was a static quenching procedure, which implied that DFDPT-CYP3A4 complex had been formed. Apparent binding constants Kb of CYP3A4 with compound at 298 and 310 K were 2.51 × 107 and 3.09 × 105, respectively. The binding sites number n was 1.64 and 1.22, respectively. The thermodynamic parameters ΔH and ΔS of the DFDPT-CYP3A4 complex were negative, which indicated that their interaction was driven mainly by hydrogen bonding and van der Waals force. The binding of DFDPT-CYP3A4 was spontaneous process in which ΔG was negative. The synchronous results showed DFDPT induced conformational changes of CYP3A4 protein. Three-dimensional fluorescence and circular dichroism spectra results also revealed conformation of CYP3A4 protein had been possible changed in the presence of DFDPT. Molecular docking was used to study the interaction orientation between DFDPT and CYP3A4 protease. The results indicated that DFDPT interacted with a panel of amino acids in the active sites of CYP3A4 protein mainly through formation of hydrogen bond. Furthermore, the predicted binding mode of DFDPT into CYP3A4 appeared to adopt an orientation with interactions among Arg105, Ser119 and Thr309.

  14. 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.

  15. Spectral Resolution of a Second Binding Site for Nile Red on Cytochrome P450 3A4

    PubMed Central

    Nath, Abhinav; Fernández, Cristina; Lampe, Jed N.; Atkins, William M.

    2008-01-01

    Nile Red is sequentially metabolized by Cytochrome P4503A4 to the N-monoethyl and N-desethyl products, which typifies the metabolism of many amine-containing drugs. Sequential metabolism of a single substrate results in complex kinetics that confound predictive models of drug clearance. As a fluorescent model for drugs which undergo sequential metabolism, Nile Red provides the opportunity to monitor drug-CYP interactions wherein the fluorescent properties of Nile Red could, in principle, be exploited to determine individual rate and equilibrium constants for the individual reactions. Previously, it was shown that Nile Red binds at the active site and fluoresces (KD = 50 nM) with maximum emission at ~620 nm, but it was unclear whether a red-shifted emission, at ~660 nm, consisted of only free Nile Red or Nile Red bound at a second site on the protein. Here, equilibrium binding studies, including ‘reverse titrations’ spanning low ratios of CYP3A4/Nile Red, indicate two binding sites for Nile Red with a contribution to the ‘red emission’ greater than can be accounted for by free Nile Red. Singular value decomposition affords basis spectra for both spectral components and fits well to the experimentally determined concentration dependence of Nile Red emission. In addition, the red spectral component, with an apparent KD = 2.2 µM, is selectively eliminated by titration with the known allosteric effectors of CYP3A4, α-napthoflavone and testosterone. Furthermore, the double mutant L2311F/D214E, previously demonstrated to perturb a peripheral allosteric site, lacks the red-emitting Nile Red binding site, but retains the blue-emitting site. Together these data indicate that a second Nile Red site competes with other effectors of CYP3A4 at a site that results in Nile Red emission at 660 nm. PMID:18395506

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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).

  9. 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

  10. In vitro metabolism of piperaquine is primarily mediated by CYP3A4

    PubMed Central

    Lee, Tina Ming-Na; Huang, Liusheng; Johnson, Marla K.; Lizak, Patricia; Kroetz, Deanna; Aweeka, Francesca; Parikh, Sunil

    2016-01-01

    Piperaquine (PQ) is part of a first-line treatment regimen for Plasmodium falciparum malaria recommended by the World Health Organization (WHO). We aimed to determine the major metabolic pathway(s) of PQ in vitro. A reliable, validated tandem mass spectrometry method was developed. Concentrations of PQ were measured after incubation with both human liver microsomes (HLMs) and expressed cytochrome P450 enzymes (P450s). In pooled HLMs, incubations with an initial PQ concentration of 0.3 µM resulted in a 34.8 ± 4.9% loss of substrate over 60 min, corresponding to a turnover rate of 0.009 min−1 (r2 = 0.9223). Miconazole, at nonspecific P450 inhibitory concentrations, resulted in almost complete inhibition of PQ metabolism. The greatest inhibition was demonstrated with selective CYP3A4 (100%) and CYP2C8 (66%) inhibitors. Using a mixture of recombinant P450 enzymes, turnover for PQ metabolism was estimated as 0.0099 min−1; recombinant CYP3A4 had a higher metabolic rate (0.017 min−1) than recombinant CYP2C8 (p < .0001). Inhibition of CYP3A4-mediated PQ loss was greatest using the selective inhibitor ketoconazole (9.1 ± 3.5% loss with ketoconazole vs 60.7 ± 5.9% with no inhibitor, p < .0001). In summary, the extent of inhibition of in vitro metabolism with ketoconazole (83%) denotes that PQ appears to be primarily catalyzed by CYP3A4. Further studies to support these findings through the identification and characterization of PQ metabolites are planned. PMID:22671777

  11. Fungal lactone ring opening of 6', 7'-dihydroxybergamottin diminishes cytochrome P450 3A4 inhibitory activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Furanocoumarins (FCs) are a class of aromatic compounds in grapefruit that inhibit human intestinal cytochrome P450 3A4 (CYP3A4). Since fungi metabolize polycyclic aromatic hydrocarbons, we hypothesized that certain fungi might also metabolize FCs into forms that may be inactive as CYP3A4 inhibitors...

  12. 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.

  13. 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.

  14. 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

  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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5.

    PubMed

    Yamazaki, H; Johnson, W W; Ueng, Y F; Shimada, T; Guengerich, F P

    1996-11-01

    Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates. The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex. We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6beta hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b5 could not substitute for b5 in a testosterone 6beta hydroxylation system composed of NADH-b5 reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min-1) to support testosterone 6beta hydroxylation ( approximately 14 min-1) unless b5 or apo-b5 was present, when rates of approximately 700 min-1 were measured. The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged ( approximately -310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b5. Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b5; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates. We conclude that b5 can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450. PMID:8910324

  2. 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

  3. 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.

  4. Augmented Inhibition of CYP3A4 in Human Primary Hepatocytes by Ritonavir Solid Drug Nanoparticles.

    PubMed

    Martin, Philip; Giardiello, Marco; McDonald, Tom O; Smith, Darren; Siccardi, Marco; Rannard, Steven P; Owen, Andrew

    2015-10-01

    Ritonavir is a protease inhibitor utilized primarily as a pharmaco-enhancer with concomitantly administered antiviral drugs including other protease inhibitors. However, poor tolerance, serious side effects, and toxicities associated with drug-drug interactions are common during exposure to ritonavir. The aim of this work was to investigate the impact of nanoformulation on ritonavir pharmacological properties. Emulsion-templated freeze-drying techniques were used to generate ritonavir (10 wt %) solid drug nanoparticle formulations. A total of 68 ritonavir formulations containing various mixtures of excipients were assessed for inhibition of CYP3A4 in baculosomes and primary human hepatocytes. Accumulation and cytotoxicity were assessed in HepG2 (hepatocytes), Caco-2 (intestinal), THP-1 (monocytes), A-THP-1 (macrophage), and CEM (lymphocytes). Transcellular permeation across Caco-2 cells was also assessed. From 68 solid drug nanoparticle formulations tested, 50 (73.5%) for baculosome and 44 (64.7%) for human primary hepatocytes exhibited enhanced CYP3A4 inhibition relative to an aqueous ritonavir solution. Sixty-one (89.7%) and 49 (72%) solid drug nanoformulations had higher apical to basal permeation across Caco-2 cells than aqueous solution of ritonavir after 60 and 120 min, respectively. No significant difference in cellular accumulation was observed for any solid drug nanoparticle for any cell type compared to aqueous ritonavir. However, incubation with the vast majority of solid drug nanoparticle formulations resulted in lower cytotoxicity of ritonavir than detected with an aqueous solution. These data provide in vitro proof of concept for improved inhibition of CYP3A4 by ritonavir through formation of solid drug nanoparticles. Nanodispersions also showed enhanced permeability across Caco-2 cells lower cytotoxicity across hepatic, intestinal, and immune cell types compared to an aqueous solution of ritonavir.

  5. 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.

  6. 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.

  7. Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

    SciTech Connect

    Grinkova, Yelena V.; Denisov, Ilia G.; McLean, Mark A.; Sligar, Stephen G.

    2013-01-25

    Highlights: ► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs. -- Abstract: The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. Prediction of CYP3A4 enzyme activity using haplotype tag SNPs in African Americans.

    PubMed

    Perera, M A; Thirumaran, R K; Cox, N J; Hanauer, S; Das, S; Brimer-Cline, C; Lamba, V; Schuetz, E G; Ratain, M J; Di Rienzo, A

    2009-02-01

    The CYP3A locus encodes hepatic enzymes that metabolize many clinically used drugs. However, there is marked interindividual variability in enzyme expression and clearance of drugs metabolized by these enzymes. We utilized comparative genomics and computational prediction of transcriptional factor binding sites to evaluate regions within CYP3A that were most likely to contribute to this variation. We then used a haplotype tagging single-nucleotide polymorphisms (htSNPs) approach to evaluate the entire locus with the fewest number of maximally informative SNPs. We investigated the association between these htSNPs and in vivo CYP3A enzyme activity using a single-point IV midazolam clearance assay. We found associations between the midazolam phenotype and age, diagnosis of hypertension and one htSNP (141689) located upstream of CYP3A4. 141689 lies near the xenobiotic responsive enhancer module (XREM) regulatory region of CYP3A4. Cell-based studies show increased transcriptional activation with the minor allele at 141689, in agreement with the in vivo association study findings. This study marks the first systematic evaluation of coding and noncoding variation that may contribute to CYP3A phenotypic variability.

  3. Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

    PubMed Central

    Liu, Wenqin; Shi, Jian; Zhu, Lijun; Dong, Lingna; Luo, Feifei; Zhao, Min; Wang, Ying; Hu, Ming; Lu, Linlin; Liu, Zhongqiu

    2015-01-01

    Oxymatrine (OMT) is a pharmacologically active primary quinolizidine alkaloid with various beneficial and toxic effects. It is confirmed that, after oral administration, OMT could be transformed to the more toxic metabolite matrine (MT), and this process may be through the reduction reaction, but the study on the characteristics of this transformation is limited. The aim of this study was to investigate the characteristics of this transformation of OMT in the human liver microsomes (HLMs) and human intestinal microsomes (HIMs) and the cytochrome P450 (CYP) isoforms involved in this transformation. The current studies demonstrated that OMT could be metabolized to MT rapidly in HLMs and HIMs and CYP3A4 greatly contributed to this transformation. All HLMs, HIMs, and CYP3A4 isoform mediated reduction reaction followed typical biphasic kinetic model, and Km, Vmax, and CL were significant higher in HLMs than those in HIMs. Importantly, different oxygen contents could significantly affect the metabolism of OMT, and with the oxygen content decreased, the formation of metabolite was increased, suggesting this transformation was very likely a reduction reaction. Results of this in vitro study elucidated the metabolic pathways and characteristics of metabolism of OMT to MT and would provide a theoretical basis and guidance for the safe application of OMT. PMID:26586934

  4. Trainable structure-activity relationship model for virtual screening of CYP3A4 inhibition.

    PubMed

    Didziapetris, Remigijus; Dapkunas, Justas; Sazonovas, Andrius; Japertas, Pranas

    2010-11-01

    A new structure-activity relationship model predicting the probability for a compound to inhibit human cytochrome P450 3A4 has been developed using data for >800 compounds from various literature sources and tested on PubChem screening data. Novel GALAS (Global, Adjusted Locally According to Similarity) modeling methodology has been used, which is a combination of baseline global QSAR model and local similarity based corrections. GALAS modeling method allows forecasting the reliability of prediction thus defining the model applicability domain. For compounds within this domain the statistical results of the final model approach the data consistency between experimental data from literature and PubChem datasets with the overall accuracy of 89%. However, the original model is applicable only for less than a half of PubChem database. Since the similarity correction procedure of GALAS modeling method allows straightforward model training, the possibility to expand the applicability domain has been investigated. Experimental data from PubChem dataset served as an example of in-house high-throughput screening data. The model successfully adapted itself to both data classified using the same and different IC₅₀ threshold compared with the training set. In addition, adjustment of the CYP3A4 inhibition model to compounds with a novel chemical scaffold has been demonstrated. The reported GALAS model is proposed as a useful tool for virtual screening of compounds for possible drug-drug interactions even prior to the actual synthesis. PMID:20814717

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase (POR)

    PubMed Central

    Agrawal, Vishal; Choi, Ji Ha; Giacomini, Kathleen M.; Miller, Walter L.

    2010-01-01

    Objectives CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis. Methods We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates. Results POR A287P and R457H had low activity with all substrates; Q153R had 76–94% of wild type (WT) activity with midazolam and erythromycin, but 129–150% activity with testosterone and quinidine. The A503V polymorphism reduced CYP3A4 activity to 61–77% of wild type with testosterone and midazolam, but had nearly wild type activity with quinidine and erythromycin. Conclusion POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably due to substrate-induced conformational changes in CYP3A4. PMID:20697309

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4.

    PubMed

    Denisov, Ilia G; Mak, Piotr J; Grinkova, Yelena V; Bastien, Dominic; Bérubé, Gervais; Sligar, Stephen G; Kincaid, James R

    2016-05-01

    Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4. PMID:26774838

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. Intestinal CYP3A4 protects against lithocholic acid-induced hepatotoxicity in intestine-specific VDR-deficient mice.

    PubMed

    Cheng, Jie; Fang, Zhong-Ze; Kim, Jung-Hwan; Krausz, Kristopher W; Tanaka, Naoki; Chiang, John Y L; Gonzalez, Frank J

    2014-03-01

    Vitamin D receptor (VDR) mediates vitamin D signaling involved in bone metabolism, cellular growth and differentiation, cardiovascular function, and bile acid regulation. Mice with an intestine-specific disruption of VDR (Vdr(ΔIEpC)) have abnormal body size, colon structure, and imbalance of bile acid metabolism. Lithocholic acid (LCA), a secondary bile acid that activates VDR, is among the most toxic of the bile acids that when overaccumulated in the liver causes hepatotoxicity. Because cytochrome P450 3A4 (CYP3A4) is a target gene of VDR-involved bile acid metabolism, the role of CYP3A4 in VDR biology and bile acid metabolism was investigated. The CYP3A4 gene was inserted into Vdr(ΔIEpC) mice to produce the Vdr(ΔIEpC)/3A4 line. LCA was administered to control, transgenic-CYP3A4, Vdr(ΔIEpC), and Vdr(ΔIEpC)/3A4 mice, and hepatic toxicity and bile acid levels in the liver, intestine, bile, and urine were measured. VDR deficiency in the intestine of the Vdr(ΔIEpC) mice exacerbates LCA-induced hepatotoxicity manifested by increased necrosis and inflammation, due in part to over-accumulation of hepatic bile acids including taurocholic acid and taurodeoxycholic acid. Intestinal expression of CYP3A4 in the Vdr(ΔIEpC)/3A4 mouse line reduces LCA-induced hepatotoxicity through elevation of LCA metabolism and detoxification, and suppression of bile acid transporter expression in the small intestine. This study reveals that intestinal CYP3A4 protects against LCA hepatotoxicity.

  10. Time-dependent inhibition of CYP3A4 by gallic acid in human liver microsomes and recombinant systems.

    PubMed

    Pu, Qiang-Hong; Shi, Liang; Yu, Chao

    2015-03-01

    1.Gallic acid is a main polyphenol in various fruits and plants. Inhibitory characteristics of gallic acid on CYP3A4 were still unclear. The objective of this work is hence to investigate inhibitory characteristics of gallic acid on CYP3A4 using testosterone as the probe substrate in human liver microsomes (HLMs) and recombinant CYP3A4 (rCYP3A4) systems. 2.Gallic acid caused concentration-dependent loss of CYP3A4 activity with IC50 values of 615.2 μM and 669.5 μM in HLM and rCYP3A4 systems, respectively. IC50-shift experiments showed that pre-incubation with gallic acid in the absence of NADPH contributed to 12- or 14-fold reduction of IC50 in HLM and rCYP3A4 systems, respectively, supporting a time-dependent inhibition. In HLM, time-dependent inactivation variables KI and Kinact were 485.8 μM and 0.05 min(-1), respectively. 3.Compared with the presence of NADPH, pre-incubation of gallic acid in the absence of NADPH markedly increased its inhibitory effects in HLM and rCYP3A4 systems. Those results indicate that CYP3A4 inactivation by gallic acid was independent on NADPH and was mainly mediated its oxidative products. 4.In conclusion, we showed that gallic acid weakly and time-dependently inactivated CYP3A4 via its oxidative products.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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…

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. CYP3A4*1B polymorphism and cancer risk: a HuGE review and meta-analysis.

    PubMed

    Zhou, Li-Ping; Yao, Fan; Luan, Hong; Wang, Yin-Ling; Dong, Xi-Hua; Zhou, Wen-Wen; Wang, Qi-Hui

    2013-04-01

    CYP450 3A4 (CYP3A4), encoded by the CYP3A4 gene, is a major enzyme catalyzing the metabolism of both endogenous and exogenous agents that may play a role in the etiology of carcinogenesis. Several potentially functional polymorphisms of the CYP3A4 gene have been implicated in cancer risk, but individually published studies have shown inconclusive results. The aim of this Human Genome Epidemiology (HuGE) review and meta-analysis was to investigate the association between CYP3A4*1B (rs2740574 A > G) polymorphism and cancer risk. Eleven studies were included with a total of 3,810 cancer patients and 3,173 healthy controls. We found that the G allele and GG genotype of CYP3A4*1B polymorphism were associated with increased risk of cancers using the fixed effects model (allele model: odds ratio (OR) = 1.24, 95 %CI: 1.09-1.42, P = 0.001; recessive model: OR = 1.77, 95 %CI: 1.30-2.41, P < 0.001; homozygous model: OR = 1.72, 95 %CI: 1.19-2.47, P = 0.004). Subgroup analyses by cancer type showed that the G allele and G carrier (AG + GG) of CYP3A4*1B polymorphism had significant associations with increased risk of prostate cancer, but not with breast cancer, leukemia, or other cancers. With further subgroup analysis based on different ethnicities, the results indicated that the GG genotype of CYP3A4*1B polymorphism might increase the risk of cancer among African populations. However, similar associations were not observed among Caucasian and Asian populations. Results from the current meta-analysis indicate that the G allele and GG genotype of CYP3A4*1B polymorphism might be associated with increased cancer risk, especially for prostate cancer among African populations.

  4. A Fibroblast Growth Factor 21-Pregnane X Receptor Pathway Downregulates Hepatic CYP3A4 in Nonalcoholic Fatty Liver Disease.

    PubMed

    Woolsey, Sarah J; Beaton, Melanie D; Mansell, Sara E; Leon-Ponte, Matilde; Yu, Janice; Pin, Christopher L; Adams, Paul C; Kim, Richard B; Tirona, Rommel G

    2016-10-01

    Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipid- and glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4 Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD. PMID:27482056

  5. A Fibroblast Growth Factor 21-Pregnane X Receptor Pathway Downregulates Hepatic CYP3A4 in Nonalcoholic Fatty Liver Disease.

    PubMed

    Woolsey, Sarah J; Beaton, Melanie D; Mansell, Sara E; Leon-Ponte, Matilde; Yu, Janice; Pin, Christopher L; Adams, Paul C; Kim, Richard B; Tirona, Rommel G

    2016-10-01

    Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipid- and glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4 Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD.

  6. A simultaneous assessment of CYP3A4 metabolism and induction in the DPX-2 cell line.

    PubMed

    Trubetskoy, Olga; Marks, Bryan; Zielinski, Thomas; Yueh, Mei-Fei; Raucy, Judy

    2005-03-04

    The DPX-2 cell line, a derivative of HepG2 cells, harbors human PXR and a luciferase-linked CYP3A4 promoter. These cells were used in a panel of cell-based assays for a parallel assessment of CYP3A4 induction, metabolism, and inhibition at the cellular level. CYP3A4 induction in the DPX-2 cell line by various agents was monitored in 96-well plates by a luciferase-based transcriptional activation assay. Of the prototypical CYP3A4 inducers examined, all exhibited elevated luciferase activity in DPX-2 cells. CYP3A4 enzyme activity in noninduced and rifampicin-induced DPX-2 cells was also assessed using Vivid fluorogenic substrates. Significantly elevated CYP3A4 activity levels (2.8-fold +/- 0.2-fold above DMSO-treated cells) were found in DPX-2 cells after 48 hours of exposure to rifampicin, but were undetectable in parental HepG2 cells. Rifampicin-induced activity levels were found to be suitable for assessing the inhibitory potential of new chemical entities in downstream CYP3A4 inhibition assays. The elevated CYP3A4 activity was inhibited 85% by 10 microM ketoconazole. In addition, a cytotoxicity assay to correct for possible toxic effects of compounds at the cellular level was applied. The comparative data obtained with a combination of the above assays suggests that the application of several independent in vitro technologies used in DPX-2 cells is the best possible strategy for the assessment of the complex phenomena of CYP3A4 induction and inhibition.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.}

  4. 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

  5. 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

  6. 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.

  7. Study Liver Cytochrome P450 3A4 Inhibition and Hepatotoxicity Using DMSO-Differentiated HuH-7 Cells.

    PubMed

    Liu, Yitong

    2016-01-01

    Metabolically competent, inexpensive, and robust in vitro cell models are needed for studying liver drug-metabolizing enzymes and hepatotoxicity. Human hepatoma HuH-7 cells develop into a differentiated in vitro model resembling primary human hepatocytes after a 2-week dimethyl sulfoxide (DMSO) treatment. DMSO-treated HuH-7 cells express elevated cytochrome P450 3A4 (CYP3A4) enzyme gene expression and activity compared to untreated HuH-7 cells. This cell model could be used to study CYP3A4 inhibition by reversible and time-dependent inhibitors, including drugs, food-related substances, and environmental chemicals. The DMSO-treated HuH-7 model is also a suitable tool for investigating hepatotoxicity. This chapter describes a detailed methodology for developing DMSO-treated HuH-7 cells, which are subsequently used for CYP3A4 inhibition and hepatotoxicity studies. PMID:27518624

  8. Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells.

    PubMed

    Liu, Yitong; Flynn, Thomas J; Xia, Menghang; Wiesenfeld, Paddy L; Ferguson, Martine S

    2015-10-01

    A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1 % dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 > HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC 50 within 2.5-fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity.

  9. Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells

    PubMed Central

    Liu, Yitong; Flynn, Thomas J.; Xia, Menghang; Wiesenfeld, Paddy L.; Ferguson, Martine S.

    2016-01-01

    A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1% dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 >> HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC50 within 2.5 fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7 cells, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity. PMID:26377104

  10. Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

    PubMed Central

    Ande, Anusha; Wang, Lei; Vaidya, Naveen K.; Li, Weihua; Kumar, Santosh; Kumar, Anil

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir

  11. Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4.

    PubMed

    Nookala, Anantha R; Li, Junhao; Ande, Anusha; Wang, Lei; Vaidya, Naveen K; Li, Weihua; Kumar, Santosh; Kumar, Anil

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir

  12. 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

  13. 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 phosphate ions. The cytotoxicity tests show that the as-prepared highly stable ACC nanospheres have excellent biocompatibility. The highly stable ACC nanospheres have high protein adsorption capacity, implying that they are promising for applications in biomedical fields such as drug delivery and protein adsorption.

  14. Effect of stabilizing additives on the structure and hydration of proteins: a study involving monoclinic lysozyme.

    PubMed

    Saraswathi, N T; Sankaranarayanan, R; Vijayan, M

    2002-07-01

    In pursuance of a long-range programme on the hydration, mobility and action of proteins, the structural basis of the stabilizing effect of sugars and polyols is being investigated. With two crystallographically independent molecules with slightly different packing environments in the crystal, monoclinic lysozyme constitutes an ideal system for exploring the problem. The differences in the structure and hydration of the two molecules provide a framework for examining the changes caused by stabilizing additives. Monoclinic crystals were grown under native conditions and also in the presence of 10% sucrose, 15% trehalose, 10% trehalose, 10% sorbitol and 5% glycerol. The crystal structures were refined at resolutions ranging from 1.8 to 2.1 A. The average B values, and hence the mobility of the structure, are lower in the presence of additives than in the native crystals. However, a comparison of the structures indicates that the effect of the additives on the structure and the hydration shell around the protein molecule is considerably less than that caused by differences in packing. It is also less than that caused by the replacement of NaNO(3) by NaCl as the precipitant in the crystallization experiments. This result is not in conformity with the commonly held belief that additives exert their stabilizing effect through the reorganization of the hydration shell, at least as far as the ordered water molecules are concerned.

  15. Unveiling the potential of novel yeast protein extracts in white wines clarification and stabilization

    PubMed Central

    Fernandes, Joana P.; Neto, Rodrigo; Centeno, Filipe; De Fátima Teixeira, Maria; Gomes, Ana Catarina

    2015-01-01

    Fining agents derived from animal and mineral sources are widely used to clarify and stabilize white wines. Nevertheless, health and environmental problems are being raised, concerning the allergenic and environmental impact of some of those fining products. In this study, our aim is to validate the potential of yeast protein extracts, obtained from an alternative and safe source, naturally present in wine: oenological yeasts. Three untreated white wines were used in this work in order to evaluate the impact of these novel yeast protein extracts (YPE) in terms of the wine clarification and stabilization improvement. Two separated fining trials were thus conducted at laboratory scale and the yeast alternatives were compared with reference fining agents, obtained from mineral, animal and vegetable origins. Our results indicate that YPE were capable to promote (i) brilliance/color improvement, (ii) turbidity reduction (76–89% comparing with the untreated wines), and (iii) production of compact and homogeneous lees (44% smaller volume than obtained with bentonite). Additionally, after submitting wines to natural and forced oxidations, YPE treatments revealed (iv) different forms of colloidal stabilization, by presenting comparable or superior effects when particularly compared to casein. Altogether, this study reveals that YPE represent a promising alternative for white wine fining, since they are resultant from a natural and more sustainable origin, at present not regarded as potential allergenic according to Regulation (EC) No. 1169/2011. PMID:25853122

  16. Capping motifs stabilize the leucine-rich repeat protein PP32 and rigidify adjacent repeats.

    PubMed

    Dao, Thuy P; Majumdar, Ananya; Barrick, Doug

    2014-06-01

    Capping motifs are found to flank most β-strand-containing repeat proteins. To better understand the roles of these capping motifs in organizing structure and stability, we carried out folding and solution NMR studies on the leucine-rich repeat (LRR) domain of PP32, which is composed of five tandem LRR, capped by α-helical and β-hairpin motifs on the N- and C-termini. We were able to purify PP32 constructs lacking either cap and containing destabilizing substitutions. Removing the C-cap results in complete unfolding of PP32. Removing the N-cap has a much less severe effect, decreasing stability but retaining much of its secondary structure. In contrast, the dynamics and tertiary structure of the first two repeats are significantly perturbed, based on (1)H-(15)N relaxation studies, chemical shift perturbations, and residual dipolar couplings. However, more distal repeats (3 to C-cap) retain their native tertiary structure. In this regard, the N-cap drives the folding of adjacent repeats from what appears to be a molten-globule-like state. This interpretation is supported by extensive analysis using core packing substitutions in the full-length and N-cap-truncated PP32. This work highlights the importance of caps to the stability and structural integrity of β-strand-containing LRR proteins, and emphasizes the different contributions of the N- and C-terminal caps. PMID:24659532

  17. Effects of monohydric alcohols and polyols on the thermal stability of a protein.

    PubMed

    Murakami, Shota; Kinoshita, Masahiro

    2016-03-28

    The thermal stability of a protein is lowered by the addition of a monohydric alcohol, and this effect becomes larger as the size of hydrophobic group in an alcohol molecule increases. By contrast, it is enhanced by the addition of a polyol possessing two or more hydroxyl groups per molecule, and this effect becomes larger as the number of hydroxyl groups increases. Here, we show that all of these experimental observations can be reproduced even in a quantitative sense by rigid-body models focused on the entropic effect originating from the translational displacement of solvent molecules. The solvent is either pure water or water-cosolvent solution. Three monohydric alcohols and five polyols are considered as cosolvents. In the rigid-body models, a protein is a fused hard spheres accounting for the polyatomic structure in the atomic detail, and the solvent is formed by hard spheres or a binary mixture of hard spheres with different diameters. The effective diameter of cosolvent molecules and the packing fractions of water and cosolvent, which are crucially important parameters, are carefully estimated using the experimental data of properties such as the density of solid crystal of cosolvent, parameters in the pertinent cosolvent-cosolvent interaction potential, and density of water-cosolvent solution. We employ the morphometric approach combined with the integral equation theory, which is best suited to the physical interpretation of the calculation result. It is argued that the degree of solvent crowding in the bulk is the key factor. When it is made more serious by the cosolvent addition, the solvent-entropy gain upon protein folding is magnified, leading to the enhanced thermal stability. When it is made less serious, the opposite is true. The mechanism of the effects of monohydric alcohols and polyols is physically the same as that of sugars. However, when the rigid-body models are employed for the effect of urea, its addition is predicted to enhance the

  18. Effects of monohydric alcohols and polyols on the thermal stability of a protein

    NASA Astrophysics Data System (ADS)

    Murakami, Shota; Kinoshita, Masahiro

    2016-03-01

    The thermal stability of a protein is lowered by the addition of a monohydric alcohol, and this effect becomes larger as the size of hydrophobic group in an alcohol molecule increases. By contrast, it is enhanced by the addition of a polyol possessing two or more hydroxyl groups per molecule, and this effect becomes larger as the number of hydroxyl groups increases. Here, we show that all of these experimental observations can be reproduced even in a quantitative sense by rigid-body models focused on the entropic effect originating from the translational displacement of solvent molecules. The solvent is either pure water or water-cosolvent solution. Three monohydric alcohols and five polyols are considered as cosolvents. In the rigid-body models, a protein is a fused hard spheres accounting for the polyatomic structure in the atomic detail, and the solvent is formed by hard spheres or a binary mixture of hard spheres with different diameters. The effective diameter of cosolvent molecules and the packing fractions of water and cosolvent, which are crucially important parameters, are carefully estimated using the experimental data of properties such as the density of solid crystal of cosolvent, parameters in the pertinent cosolvent-cosolvent interaction potential, and density of water-cosolvent solution. We employ the morphometric approach combined with the integral equation theory, which is best suited to the physical interpretation of the calculation result. It is argued that the degree of solvent crowding in the bulk is the key factor. When it is made more serious by the cosolvent addition, the solvent-entropy gain upon protein folding is magnified, leading to the enhanced thermal stability. When it is made less serious, the opposite is true. The mechanism of the effects of monohydric alcohols and polyols is physically the same as that of sugars. However, when the rigid-body models are employed for the effect of urea, its addition is predicted to enhance the

  19. Oral intake of curcumin markedly activated CYP 3A4: in vivo and ex-vivo studies.

    PubMed

    Hsieh, Yow-Wen; Huang, Ching-Ya; Yang, Shih-Ying; Peng, Yu-Hsuan; Yu, Chung-Ping; Chao, Pei-Dawn Lee; Hou, Yu-Chi

    2014-10-10

    Curcumin, a specific secondary metabolite of Curcuma species, has potentials for a variety of beneficial health effects. It is nowadays used as a dietary supplement. Everolimus (EVL) is an immunosuppressant indicated for allograft rejection and cancer therapy, but with narrow therapeutic window. EVL is a substrate of P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4). This study investigated the effect of coadministration of curcumin on the pharmacokinetics of EVL in rats and the underlying mechanisms. EVL (0.5 mg/kg) was orally administered without and with 50 and 100 mg/kg of curcumin, respectively, in rats. Blood samples were collected at specific time points and EVL concentrations in blood were determined by QMS immunoassay. The underlying mechanisms were evaluated using cell model and recombinant CYP 3A4 isozyme. The results indicated that 50 and 100 mg/kg of curcumin significantly decreased the AUC0-540 of EVL by 70.6% and 71.5%, respectively, and both dosages reduced the Cmax of EVL by 76.7%. Mechanism studies revealed that CYP3A4 was markedly activated by curcumin metabolites, which apparently overrode the inhibition effects of curcumin on P-gp. In conclusion, oral intake of curcumin significantly decreased the bioavailability of EVL, a probe substrate of P-gp/CYP 3A4, mainly through marked activation on CYP 3A4.

  20. Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

    SciTech Connect

    DuBois, Rebecca M.; Zaraket, Hassan; Reddivari, Muralidhar; Heath, Richard J.; White, Stephen W.; Russell, Charles J.

    2012-12-10

    Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 {angstrom} resolution and two structures of HP HA at 2.95 and 3.10 {angstrom} resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

  1. Alterations of Nonconserved Residues Affect Protein Stability and Folding Dynamics through Charge-Charge Interactions.

    PubMed

    Tripathi, Swarnendu; Garcìa, Angel E; Makhatadze, George I

    2015-10-15

    Charge-charge interactions play an important role in thermal stability of proteins. We employed an all-atom, native-topology-based model with non-native electrostatics to explore the interplay between folding dynamics and stability of TNfn3 (the third fibronectin type III domain from tenascin-C). Our study elucidates the role of charge-charge interactions in modulating the folding energy landscape. In particular, we found that incorporation of explicit charge-charge interactions in the WT TNfn3 induces energetic frustration due to the presence of residual structure in the unfolded state. Moreover, optimization of the surface charge-charge interactions by altering the evolutionarily nonconserved residues not only increases the thermal stability (in agreement with previous experimental study) but also reduces the formation of residual structure and hence minimizes the energetic frustration along the folding route. We concluded that charge-charge interaction in the rationally designed TNfn3 plays an important role not only in enhancing the stability but also in assisting folding. PMID:26413861

  2. Photochemical and Thermal Stability of Green and Blue Proteorhodopsins: Implications for Protein-Based Bioelectronic Devices

    PubMed Central

    Ranaghan, Matthew J.; Shima, Sumie; Ramos, Lavosier; Poulin, Daniel S.; Whited, Gregg; Rajasekaran, Sanguthevar; Stuart, Jeffery A.; Albert, Arlene D.; Birge, Robert R.

    2010-01-01

    The photochemical and thermal stability of the detergent solubilized blue- and green-absorbing proteorhodpsins, BPR and GPR respectively, are investigated to determine viability of these proteins for photonic device applications. Photochemical stability is studied by using pulsed laser excitation and differential uv-vis spectroscopy to assign the photocyclicity. GPR, with a cyclicity of 7×104 photocycles protein−1, is 4–5 times more stable than BPR (9×103 photocycles protein−1), but is less stable than native bacteriorhodopsin (9×105 photocycles protein−1) or the 4-keto-bacteriorhodopsin analog (1×105 photocycles protein−1). The thermal stabilities are assigned by using differential scanning calorimetry and thermal bleaching experiments. Both proteorhodopsins display excellent thermal stability, with melting temperatures above 85°C, and remain photochemically stable up to 75°C. The biological relevance of our results is also discussed. The lower cyclicity of BPR is found to be adequate for the long-term biological function of the host organism at ocean depths of 50 m or more. PMID:20964279

  3. Impact of glucose polymer chain length on heat and physical stability of milk protein-carbohydrate nutritional beverages.

    PubMed

    Chen, Biye; O'Mahony, James A

    2016-11-15

    This study investigated the impact of glucose polymer chain length on heat and physical stability of milk protein isolate (MPI)-carbohydrate nutritional beverages containing 8.5% w/w total protein and 5% w/w carbohydrate. The maltodextrin and corn syrup solids glucose polymers used had dextrose equivalent (DE) values of 17 or 38, respectively. Increasing DE value of the glucose polymers resulted in a greater increase in brown colour development, ionic calcium, protein particle size, apparent viscosity and pseudoplastic rheological behaviour, and greater reduction in pH, hydration and heat stability on sterilisation at 120°C. Incorporation of glucose polymers with MPI retarded sedimentation of protein during accelerated physical stability testing, with maltodextrin DE17 causing a greater reduction in sedimentation velocity and compressibility of sediment formed than corn syrup solids DE38. The results demonstrate that chain length of the glucose polymer used strongly impacts heat and physical stability of MPI-carbohydrate nutritional beverages.

  4. Subcritical Water Induced Complexation of Soy Protein and Rutin: Improved Interfacial Properties and Emulsion Stability.

    PubMed

    Chen, Xiao-Wei; Wang, Jin-Mei; Yang, Xiao-Quan; Qi, Jun-Ru; Hou, Jun-Jie

    2016-09-01

    Rutin is a common dietary flavonoid with important antioxidant and pharmacological activities. However, its application in the food industry is limited mainly because of its poor water solubility. The subcritical water (SW) treatment provides an efficient technique to solubilize and achieve the enrichment of rutin in soy protein isolate (SPI) by inducing their complexation. The physicochemical, interfacial, and emulsifying properties of the complex were investigated and compared to the mixtures. SW treatment had much enhanced rutin-combined capacity of SPI than that of conventional method, ascribing to the well-contacted for higher water solubility of rutin with stronger collision-induced hydrophobic interactions. Compared to the mixtures of rutin with proteins, the complex exhibited an excellent surface activity and improved the physical and oxidative stability of its stabilized emulsions. This improving effect could be attributed to the targeted accumulation of rutin at the oil-water interface accompanied by the adsorption of SPI resulting in the thicker interfacial layer, as evidenced by higher interfacial protein and rutin concentrations. This study provides a novel strategy for the design and enrichment of nanovehicle providing water-insoluble hydrophobic polyphenols for interfacial delivery in food emulsified systems.

  5. The Methionine-aromatic Motif Plays a Unique Role in Stabilizing Protein Structure*

    PubMed Central

    Valley, Christopher C.; Cembran, Alessandro; Perlmutter, Jason D.; Lewis, Andrew K.; Labello, Nicholas P.; Gao, Jiali; Sachs, Jonathan N.

    2012-01-01

    Of the 20 amino acids, the precise function of methionine (Met) remains among the least well understood. To establish a determining characteristic of methionine that fundamentally differentiates it from purely hydrophobic residues, we have used in vitro cellular experiments, molecular simulations, quantum calculations, and a bioinformatics screen of the Protein Data Bank. We show that approximately one-third of all known protein structures contain an energetically stabilizing Met-aromatic motif and, remarkably, that greater than 10,000 structures contain this motif more than 10 times. Critically, we show that as compared with a purely hydrophobic interaction, the Met-aromatic motif yields an additional stabilization of 1–1.5 kcal/mol. To highlight its importance and to dissect the energetic underpinnings of this motif, we have studied two clinically relevant TNF ligand-receptor complexes, namely TRAIL-DR5 and LTα-TNFR1. In both cases, we show that the motif is necessary for high affinity ligand binding as well as function. Additionally, we highlight previously overlooked instances of the motif in several disease-related Met mutations. Our results strongly suggest that the Met-aromatic motif should be exploited in the rational design of therapeutics targeting a range of proteins. PMID:22859300

  6. SDM—a server for predicting effects of mutations on protein stability and malfunction

    PubMed Central

    Worth, Catherine L.; Preissner, Robert; Blundell, Tom L.

    2011-01-01

    The sheer volume of non-synonymous single nucleotide polymorphisms that have been generated in recent years from projects such as the Human Genome Project, the HapMap Project and Genome-Wide Association Studies means that it is not possible to characterize all mutations experimentally on the gene products, i.e. elucidate the effects of mutations on protein structure and function. However, automatic methods that can predict the effects of mutations will allow a reduced set of mutations to be studied. Site Directed Mutator (SDM) is a statistical potential energy function that uses environment-specific amino-acid substitution frequencies within homologous protein families to calculate a stability score, which is analogous to the free energy difference between the wild-type and mutant protein. Here, we present a web server for SDM (http://www-cryst.bioc.cam.ac.uk/~sdm/sdm.php), which has obtained more than 10 000 submissions since being online in April 2008. To run SDM, users must upload a wild-type structure and the position and amino acid type of the mutation. The results returned include information about the local structural environment of the wild-type and mutant residues, a stability score prediction and prediction of disease association. Additionally, the wild-type and mutant structures are displayed in a Jmol applet with the relevant residues highlighted. PMID:21593128

  7. Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms.

    PubMed

    Stepanenko, Olga V; Roginskii, Denis O; Stepanenko, Olesya V; Kuznetsova, Irina M; Uversky, Vladimir N; Turoverov, Konstantin K

    2016-01-01

    In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located. PMID:27114857

  8. Fabrication of fibrillosomes from droplets stabilized by protein nanofibrils at all-aqueous interfaces

    PubMed Central

    Song, Yang; Shimanovich, Ulyana; Michaels, Thomas C. T.; Ma, Qingming; Li, Jingmei; Knowles, Tuomas P. J.; Shum, Ho Cheung

    2016-01-01

    All-aqueous emulsions exploit spontaneous liquid–liquid separation and due to their water-based nature are particular advantageous for the biocompatible storage and processing of biomacromolecules. However, the ultralow interfacial tensions characteristic of all-aqueous interfaces represent an inherent limitation to the use of thermally adsorbed particles to achieve emulsion stability. Here, we use protein nanofibrils to generate colloidosome-like two-dimensional crosslinked networks of nanostructures templated by all-aqueous emulsions, which we term fibrillosomes. We show that this approach not only allows us to operate below the thermal limit at ultra-low surface tensions but also yields structures that are stable even in the complete absence of an interface. Moreover, we show that the growth and multilayer deposition of fibrils allows us to control the thickness of the capsule shells. These results open up the possibility of stabilizing aqueous two-phase systems using natural proteins, and creating self-standing protein capsules without the requirement for three-phase emulsions or water/oil interfaces. PMID:27725629

  9. Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms

    PubMed Central

    Stepanenko, Olga V.; Roginskii, Denis O.; Stepanenko, Olesya V.; Kuznetsova, Irina M.

    2016-01-01

    In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located. PMID:27114857

  10. Can a pairwise contact potential stabilize native protein folds against decoys obtained by threading?

    PubMed

    Vendruscolo, M; Najmanovich, R; Domany, E

    2000-02-01

    We present a method to derive contact energy parameters from large sets of proteins. The basic requirement on which our method is based is that for each protein in the database the native contact map has lower energy than all its decoy conformations that are obtained by threading. Only when this condition is satisfied one can use the proposed energy function for fold identification. Such a set of parameters can be found (by perceptron learning) if Mp, the number of proteins in the database, is not too large. Other aspects that influence the existence of such a solution are the exact definition of contact and the value of the critical distance Rc, below which two residues are considered to be in contact. Another important novel feature of our approach is its ability to determine whether an energy function of some suitable proposed form can or cannot be parameterized in a way that satisfies our basic requirement. As a demonstration of this, we determine the region in the (Rc, Mp) plane in which the problem is solvable, i.e., we can find a set of contact parameters that stabilize simultaneously all the native conformations. We show that for large enough databases the contact approximation to the energy cannot stabilize all the native folds even against the decoys obtained by gapless threading. PMID:10656261

  11. Subcritical Water Induced Complexation of Soy Protein and Rutin: Improved Interfacial Properties and Emulsion Stability.

    PubMed

    Chen, Xiao-Wei; Wang, Jin-Mei; Yang, Xiao-Quan; Qi, Jun-Ru; Hou, Jun-Jie

    2016-09-01

    Rutin is a common dietary flavonoid with important antioxidant and pharmacological activities. However, its application in the food industry is limited mainly because of its poor water solubility. The subcritical water (SW) treatment provides an efficient technique to solubilize and achieve the enrichment of rutin in soy protein isolate (SPI) by inducing their complexation. The physicochemical, interfacial, and emulsifying properties of the complex were investigated and compared to the mixtures. SW treatment had much enhanced rutin-combined capacity of SPI than that of conventional method, ascribing to the well-contacted for higher water solubility of rutin with stronger collision-induced hydrophobic interactions. Compared to the mixtures of rutin with proteins, the complex exhibited an excellent surface activity and improved the physical and oxidative stability of its stabilized emulsions. This improving effect could be attributed to the targeted accumulation of rutin at the oil-water interface accompanied by the adsorption of SPI resulting in the thicker interfacial layer, as evidenced by higher interfacial protein and rutin concentrations. This study provides a novel strategy for the design and enrichment of nanovehicle providing water-insoluble hydrophobic polyphenols for interfacial delivery in food emulsified systems. PMID:27467966

  12. Fabrication of fibrillosomes from droplets stabilized by protein nanofibrils at all-aqueous interfaces

    NASA Astrophysics Data System (ADS)

    Song, Yang; Shimanovich, Ulyana; Michaels, Thomas C. T.; Ma, Qingming; Li, Jingmei; Knowles, Tuomas P. J.; Shum, Ho Cheung

    2016-10-01

    All-aqueous emulsions exploit spontaneous liquid-liquid separation and due to their water-based nature are particular advantageous for the biocompatible storage and processing of biomacromolecules. However, the ultralow interfacial tensions characteristic of all-aqueous interfaces represent an inherent limitation to the use of thermally adsorbed particles to achieve emulsion stability. Here, we use protein nanofibrils to generate colloidosome-like two-dimensional crosslinked networks of nanostructures templated by all-aqueous emulsions, which we term fibrillosomes. We show that this approach not only allows us to operate below the thermal limit at ultra-low surface tensions but also yields structures that are stable even in the complete absence of an interface. Moreover, we show that the growth and multilayer deposition of fibrils allows us to control the thickness of the capsule shells. These results open up the possibility of stabilizing aqueous two-phase systems using natural proteins, and creating self-standing protein capsules without the requirement for three-phase emulsions or water/oil interfaces.

  13. Interrelationship of Steric Stabilization and Self-Crowding of a Glycosylated Protein

    PubMed Central

    Høiberg-Nielsen, R.; Westh, P.; Skov, L.K.; Arleth, L.

    2009-01-01

    Abstract In the eukaryotic cell, protein glycosylation takes place in the crowded environment of the endoplasmatic reticulum. With the purpose of elucidating the impact of high concentration on the interactions of glycoproteins, we have conducted a series of small-angle x-ray scattering experiments on the heavily glycosylated enzyme Peniophora lycii phytase (Phy) and its deglycosylated counterpart (dgPhy). The small-angle x-ray scattering data were analyzed using an individual numerical form factor for each of the two glycoforms combined with two structure factors, a hard sphere and a screened coulomb potential structure factor, respectively, as determined by ab initio analysis. Based on this data analysis, three main conclusions could be drawn. First, at comparable protein concentrations (mg/ml), the relative excluded volume of Phy was ∼75% higher than that of dgPhy, showing that the glycans significantly increase excluded-volume interactions. Second, the relative excluded volume of dgPhy increased with concentration, as expected; however, the opposite effect was observed for Phy, where the relative excluded volume decreased in response to increasing protein concentration. Third, a clear difference in the effect of salinity on the excluded-volume interactions was observed between the two glycol forms. Although the relative excluded volume of dgPhy decreased with increasing ionic strength, the relative excluded volume of Phy was basically insensitive to increased salinity. We suggest that protrusion forces from the glycans contribute to steric stabilization of the protein, and that glycosylation helps to sustain repulsive electrostatic interactions under crowded conditions. In combination, this aids in stabilizing high concentrations of glycosylated proteins. PMID:19720033

  14. Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking.

    PubMed

    Wu, Jiande; Shi, Mengxuan; Li, Wei; Zhao, Luhai; Wang, Ze; Yan, Xinzhong; Norde, Willem; Li, Yuan

    2015-03-01

    A Pickering (o/w) emulsion was formed and stabilized by whey protein isolate nanoparticles (WPI NPs). Those WPI NPs were prepared by thermal cross-linking of denatured WPI proteins within w/o emulsion droplets at 80°C for 15 min. During heating of w/o emulsions containing 10% (w/v) WPI proteins in the water phase, the emulsions displayed turbid-transparent-turbid phase transitions, which is ascribed to the change in the size of the protein-containing water droplets caused by thermal cross-linking between denatured protein molecules. The transparent stage indicated the formation of WPI NPs. WPI NPs of different sizes were obtained by varying the mixing speed. WPI NPs of 200-500 nm were selected to prepare o/w Pickering emulsions because of their good stability against coalescence. By Confocal Laser Scanning Microscopy, it was observed that WPI NPs were closely packed and distributed at the surface of the emulsion droplets. By measuring water contact angles of WPI NPs films, it was found that under most conditions WPI NPs present good partial wetting properties, but that at the isoelectric point (pI) and high ionic strength the particles become more hydrophobic, resulting in less stable Pickering emulsion. Thus, at pH above and below the pI of WPI NPs and low to moderate ionic strengths (1-10 mM), and with a WPI NPs concentration of 2% (w/v), a stable Pickering emulsion can be obtained. The results may provide useful information for applications of WPI NPs in environmentally friendly and food grade applications, notably in food, pharmaceutical and cosmetic products.

  15. TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends*

    PubMed Central

    Izumi, Takashi; Shimizu, Shigeomi

    2016-01-01

    Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. PMID:27514743

  16. Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax.

    PubMed

    Friis, Dennis S; Johnsen, Johannes L; Kristiansen, Erlend; Westh, Peter; Ramløv, Hans

    2014-06-01

    The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, Tm , of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.

  17. Myc protein is stabilized by suppression of a novel E3 ligase complex in cancer cells

    PubMed Central

    Choi, Seung H.; Wright, Jason B.; Gerber, Scott A.; Cole, Michael D.

    2010-01-01

    Rapid Myc protein turnover is critical for maintaining basal levels of Myc activity in normal cells and a prompt response to changing growth signals. We characterize a new Myc-interacting factor, TRPC4AP (transient receptor potential cation channel, subfamily C, member 4-associated protein)/TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein), which is the receptor for a DDB1 (damage-specific DNA-binding protein 1)–CUL4 (Cullin 4) E3 ligase complex for selective Myc degradation through the proteasome. TRPC4AP/TRUSS binds specifically to the Myc C terminus and promotes its ubiquitination and destruction through the recognition of evolutionarily conserved domains in the Myc N terminus. TRPC4AP/TRUSS suppresses Myc-mediated transactivation and transformation in a dose-dependent manner. Finally, we found that TRPC4AP/TRUSS expression is strongly down-regulated in most cancer cell lines, leading to Myc protein stabilization. These studies identify a novel pathway targeting Myc degradation that is suppressed in cancer cells. PMID:20551172

  18. Molecular dynamics studies on the structural stability of wild-type dog prion protein.

    PubMed

    Zhang, Jiapu; Liu, David D W

    2011-06-01

    Prion diseases such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob diseases, Gerstmann-Sträussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (or 'mad-cow' disease) and chronic wasting disease in cattle are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches to treat all these prion diseases. In 2008, canine mammals including dogs (canis familials) were the first time academically reported to be resistant to prion diseases (Vaccine 26: 2601-2614 (2008)). Thus, it is very worth studying the molecular structures of dog prion protein to obtain insights into the immunity of dogs to prion diseases. This paper studies the molecular structural dynamics of wild-type dog prion protein. The comparison analyses with rabbit prion protein show that the dog prion protein has stable molecular structures whether under neutral or low pH environments. We also find that the salt bridges such as D177-R163 contribute to the structural stability of wild-type rabbit prion protein under neutral pH environment. PMID:21469747

  19. Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

    PubMed

    Goldenzweig, Adi; Goldsmith, Moshe; Hill, Shannon E; Gertman, Or; Laurino, Paola; Ashani, Yacov; Dym, Orly; Unger, Tamar; Albeck, Shira; Prilusky, Jaime; Lieberman, Raquel L; Aharoni, Amir; Silman, Israel; Sussman, Joel L; Tawfik, Dan S; Fleishman, Sarel J

    2016-07-21

    Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il. PMID:27425410

  20. Nanoscale assembly in biological systems: from neuronal cytoskeletal proteins to curvature stabilizing lipids.

    PubMed

    Safinya, Cyrus R; Raviv, Uri; Needleman, Daniel J; Zidovska, Alexandra; Choi, Myung Chul; Ojeda-Lopez, Miguel A; Ewert, Kai K; Li, Youli; Miller, Herbert P; Quispe, Joel; Carragher, Bridget; Potter, Clinton S; Kim, Mahn Won; Feinstein, Stuart C; Wilson, Leslie

    2011-05-24

    The review will describe experiments inspired by the rich variety of bundles and networks of interacting microtubules (MT), neurofilaments, and filamentous-actin in neurons where the nature of the interactions, structures, and structure-function correlations remain poorly understood. We describe how three-dimensional (3D) MT bundles and 2D MT bundles may assemble, in cell free systems in the presence of counter-ions, revealing structures not predicted by polyelectrolyte theories. Interestingly, experiments reveal that the neuronal protein tau, an abundant MT-associated-protein in axons, modulates the MT diameter providing insight for the control of geometric parameters in bio- nanotechnology. In another set of experiments we describe lipid-protein-nanotubes, and lipid nano-tubes and rods, resulting from membrane shape evolution processes involving protein templates and curvature stabilizing lipids. Similar membrane shape changes, occurring in cells for the purpose of specific functions, are induced by interactions between membranes and proteins. The biological materials systems described have applications in bio-nanotechnology.

  1. Thresholds, long delays and stability from generalized allosteric effect in protein networks

    NASA Astrophysics Data System (ADS)

    Chignola, Roberto; Dalla Pellegrina, Chiara; Fabbro, Alessio Del; Milotti, Edoardo

    2006-11-01

    Post-transductional modifications tune the functions of proteins and regulate the collective dynamics of biochemical networks that determine how cells respond to environmental signals. For example, protein phosphorylation and nitrosylation are well known to play a pivotal role in the intracellular transduction of activation and death signals. A protein can have multiple sites where chemical groups can reversibly attach in processes such as phosphorylation or nitrosylation. A microscopic description of these processes must take into account the intrinsic probabilistic nature of the underlying reactions. We apply combinatorial considerations to standard enzyme kinetics and in this way we extend to the dynamic regime a simplified version of the traditional models on the allosteric regulation of protein functions. We link a generic modification chain to a downstream Michaelis-Menten enzymatic reaction and we demonstrate numerically that this accounts both for thresholds and long time delays in the conversion of the substrate by the enzyme. The proposed mechanism is stable and robust and the higher the number of modification sites, the greater the stability. We show that a high number of modification sites converts a fast reaction into a slow process, and the slowing down depends on the number of sites and may span many orders of magnitude; in this way multisite modification of proteins stands out as a general mechanism that allows the transfer of information from the very short time scales of enzyme reactions (milliseconds) to the long time scale of cell response (hours).

  2. Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

    PubMed Central

    Friis, Dennis S; Johnsen, Johannes L; Kristiansen, Erlend; Westh, Peter; Ramløv, Hans

    2014-01-01

    The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, Tm, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed. PMID:24652821

  3. Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region*

    PubMed Central

    Cobb, Nathan J.; Apostol, Marcin I.; Chen, Shugui; Smirnovas, Vytautas; Surewicz, Witold K.

    2014-01-01

    Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrPSc. One key operational parameter used to define differences between strains has been conformational stability of PrPSc as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrPSc, especially because large strain-specific differences in PrPSc stability are often observed despite a similar size of the PrPSc core region. PMID:24338015

  4. Replica Exchange Molecular Dynamics Study of Dimerization in Prion Protein: Multiple Modes of Interaction and Stabilization.

    PubMed

    Chamachi, Neharika G; Chakrabarty, Suman

    2016-08-01

    The pathological forms of prions are known to be a result of misfolding, oligomerization, and aggregation of the cellular prion. While the mechanism of misfolding and aggregation in prions has been widely studied using both experimental and computational tools, the structural and energetic characterization of the dimer form have not garnered as much attention. On one hand dimerization can be the first step toward a nucleation-like pathway to aggregation, whereas on the other hand it may also increase the conformational stability preventing self-aggregation. In this work, we have used extensive all-atom replica exchange molecular dynamics simulations of both monomer and dimer forms of a mouse prion protein to understand the structural, dynamic, and thermodynamic stability of dimeric prion as compared to the monomeric form. We show that prion proteins can dimerize spontaneously being stabilized by hydrophobic interactions as well as intermolecular hydrogen bonding and salt bridge formation. We have computed the conformational free energy landscapes for both monomer and dimer forms to compare the thermodynamic stability and misfolding pathways. We observe large conformational heterogeneity among the various modes of interactions between the monomers and the strong intermolecular interactions may lead to as high as 20% β-content. The hydrophobic regions in helix-2, surrounding coil regions, terminal regions along with the natively present β-sheet region appear to actively participate in prion-prion intermolecular interactions. Dimerization seems to considerably suppress the inherent dynamic instability observed in monomeric prions, particularly because the regions of structural frustration constitute the dimer interface. Further, we demonstrate an interesting reversible coupling between the Q160-G131 interaction (which leads to inhibition of β-sheet extension) and the G131-V161 H-bond formation. PMID:27390876

  5. Investigating the binding interactions of the anti-Alzheimer's drug donepezil with CYP3A4 and P-glycoprotein.

    PubMed

    McEneny-King, Alanna; Edginton, Andrea N; Rao, Praveen P N

    2015-01-15

    The anti-Alzheimer's agent donepezil is known to bind to the hepatic enzyme CYP3A4, but its relationship with the efflux transporter P-glycoprotein (P-gp) is not as well elucidated. We conducted in vitro inhibition studies of donepezil using human recombinant CYP3A4 and P-gp. These studies show that donepezil is a weak inhibitor of CYP3A4 (IC50=54.68±1.00μM) whereas the reference agent ketoconazole exhibited potent inhibition (CYP3A4 IC50=0.20±0.01μM). P-gp inhibition studies indicate that donepezil exhibits better inhibition relative to CYP3A4 (P-gp EC50=34.85±4.63μM) although it was less potent compared to ketoconazole (P-gp EC50=9.74±1.23μM). At higher concentrations, donepezil exhibited significant inhibition of CYP3A4 (69%, 84% and 87% inhibition at 100, 250 and 500μM, respectively). This indicates its potential to cause drug-drug interactions with other CYP3A4 substrates upon co-administration; however, this scenario is unlikely in vivo due to the low therapeutic concentrations of donepezil. Similarly, donepezil co-administration with P-gp substrates or inhibitors is unlikely to result in beneficial or adverse drug interactions. The molecular docking studies show that the 5,6-dimethoxyindan-1-one moiety of donepezil was oriented closer to the heme center in CYP3A4 whereas in the P-gp binding site, the protonated benzylpiperidine pharmacophore of donepezil played a major role in its binding ability. Energy parameters indicate that donepezil complex with both CYP3A4 and P-gp was less stable (CDOCKER energies=-15.05 and -4.91kcal/mol, respectively) compared to the ketoconazole-CYP3A4 and P-gp complex (CDOCKER energies=-41.89 and -20.03kcal/mol, respectively).

  6. Modulation of human cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) in Caco-2 cell monolayers by selected commercial-source milk thistle and goldenseal products.

    PubMed

    Budzinski, Jason W; Trudeau, Vance L; Drouin, Cathy E; Panahi, Mitra; Arnason, J Thor; Foster, Brian C

    2007-09-01

    In this study, we used an in vitro Caco-2 cell monolayer model to evaluate aqueous extracts of commercial-source goldenseal (Hydrastis canadensis) and milk thistle (Silybum marianum) capsule formulations, their marker phytochemicals (berberine and silibinin, respectively), as well as dillapiol, vinblastine, and the HIV protease inhibitor saquinavir for their ability to modulate CYP3A4 and ABCB1 expression after short-term exposure (48 h). Both upregulation and downregulation of CYP3A4 expression was observed with extracts of varying concentrations of the two natural health products (NHPs). CYP3A4 was highly responsive in our system, showing a strong dose-dependent modulation by the CYP3A4 inhibitor dillapiol (upregulation) and the milk thistle flavonolignan silibinin (downregulation). ABCB1 was largely unresponsive in this cellular model and appears to be of little value as a biomarker under our experimental conditions. Therefore, the modulation of CYP3A4 gene expression can serve as an important marker for the in vitro assessment of NHP-drug interactions.

  7. 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.

  8. Protein thermal stability enhancement by designing salt bridges: a combined computational and experimental study.

    PubMed

    Lee, Chi-Wen; Wang, Hsiu-Jung; Hwang, Jenn-Kang; Tseng, Ching-Ping

    2014-01-01

    Protein thermal stability is an important factor considered in medical and industrial applications. Many structural characteristics related to protein thermal stability have been elucidated, and increasing salt bridges is considered as one of the most efficient strategies to increase protein thermal stability. However, the accurate simulation of salt bridges remains difficult. In this study, a novel method for salt-bridge design was proposed based on the statistical analysis of 10,556 surface salt bridges on 6,493 X-ray protein structures. These salt bridges were first categorized based on pairing residues, secondary structure locations, and Cα-Cα distances. Pairing preferences generalized from statistical analysis were used to construct a salt-bridge pair index and utilized in a weighted electrostatic attraction model to find the effective pairings for designing salt bridges. The model was also coupled with B-factor, weighted contact number, relative solvent accessibility, and conservation prescreening to determine the residues appropriate for the thermal adaptive design of salt bridges. According to our method, eight putative salt-bridges were designed on a mesophilic β-glucosidase and 24 variants were constructed to verify the predictions. Six putative salt-bridges leaded to the increase of the enzyme thermal stability. A significant increase in melting temperature of 8.8, 4.8, 3.7, 1.3, 1.2, and 0.7°C of the putative salt-bridges N437K-D49, E96R-D28, E96K-D28, S440K-E70, T231K-D388, and Q277E-D282 was detected, respectively. Reversing the polarity of T231K-D388 to T231D-D388K resulted in a further increase in melting temperatures by 3.6°C, which may be caused by the transformation of an intra-subunit electrostatic interaction into an inter-subunit one depending on the local environment. The combination of the thermostable variants (N437K, E96R, T231D and D388K) generated a melting temperature increase of 15.7°C. Thus, this study demonstrated a novel

  9. Autoclaving as a chemical-free process to stabilize recombinant silk-elastinlike protein polymer nanofibers

    NASA Astrophysics Data System (ADS)

    Qiu, Weiguo; Cappello, Joseph; Wu, Xiaoyi

    2011-06-01

    We report here that autoclaving is a chemical-free, physical crosslinking strategy capable of stabilizing electrospun recombinant silk-elastinlike protein (SELP) polymer nanofibers. Fourier transform infrared spectroscopy showed that the autoclaving of SELP nanofibers induced a conformational conversion of β-turns and unordered structures to ordered β-sheets. Tensile stress-strain analysis of the autoclaved SELP nanofibrous scaffolds in phosphate buffered saline at 37 °C revealed a Young's modulus of 1.02 ± 0.28 MPa, an ultimate tensile strength of 0.34 ± 0.04 MPa, and a strain at failure of 29% ± 3%.

  10. A method for evaluating nucleosome stability with a protein-binding fluorescent dye.

    PubMed

    Taguchi, Hiroyuki; Horikoshi, Naoki; Arimura, Yasuhiro; Kurumizaka, Hitoshi

    2014-12-01

    Nucleosomes are extremely stable histone-DNA complexes that form the building blocks of chromatin, which accommodates genomic DNA within the nucleus. The dynamic properties of chromatin play essential roles in regulating genomic DNA functions, such as DNA replication, recombination, repair, and transcription. Histones are the protein components of nucleosomes, and their diverse modifications and variants increase the versatility of nucleosome structures and their dynamics in chromatin. Therefore, a technique to evaluate the physical properties of nucleosomes would facilitate functional studies of the various nucleosomes. In this report, we describe a convenient assay for evaluating the thermal stability of nucleosomes in vitro.

  11. Small-Protein-Stabilized Semiconductor Nanoprobe for Targeted Imaging of Cancer Cells.

    PubMed

    Zhao, Ning; Liu, Siyu; Jiang, Qike; Lan, Tian; Cheng, Zhen; Liu, Hongguang

    2016-07-01

    Recently, semiconductor nanoparticles such as quantum dots (QDs) have attracted significant attention for bioimaging. Complex chemical functionalization, surface modification, and bioconjugation chemistry are generally required to tag biomolecules to QDs for imaging of different biomarkers. In this study, we report a simple method for production of QDs stabilized by the small protein, Affibody (AF-QDs) for fluorescent imaging of the human epidermal growth factor receptor type 2 (HER2) in human A549 lung cancer cells. This one-pot synthesis of AF-QDs avoids complex chemical conjugation procedures and demonstrates a promising approach for the preparation of fluorescent nanoprobes for imaging of cancer targets. PMID:27123671

  12. Protein Thermal Stability Enhancement by Designing Salt Bridges: A Combined Computational and Experimental Study

    PubMed Central

    Hwang, Jenn-Kang; Tseng, Ching-Ping

    2014-01-01

    Protein thermal stability is an important factor considered in medical and industrial applications. Many structural characteristics related to protein thermal stability have been elucidated, and increasing salt bridges is considered as one of the most efficient strategies to increase protein thermal stability. However, the accurate simulation of salt bridges remains difficult. In this study, a novel method for salt-bridge design was proposed based on the statistical analysis of 10,556 surface salt bridges on 6,493 X-ray protein structures. These salt bridges were first categorized based on pairing residues, secondary structure locations, and Cα–Cα distances. Pairing preferences generalized from statistical analysis were used to construct a salt-bridge pair index and utilized in a weighted electrostatic attraction model to find the effective pairings for designing salt bridges. The model was also coupled with B-factor, weighted contact number, relative solvent accessibility, and conservation prescreening to determine the residues appropriate for the thermal adaptive design of salt bridges. According to our method, eight putative salt-bridges were designed on a mesophilic β-glucosidase and 24 variants were constructed to verify the predictions. Six putative salt-bridges leaded to the increase of the enzyme thermal stability. A significant increase in melting temperature of 8.8, 4.8, 3.7, 1.3, 1.2, and 0.7°C of the putative salt-bridges N437K–D49, E96R–D28, E96K–D28, S440K–E70, T231K–D388, and Q277E–D282 was detected, respectively. Reversing the polarity of T231K–D388 to T231D–D388K resulted in a further increase in melting temperatures by 3.6°C, which may be caused by the transformation of an intra-subunit electrostatic interaction into an inter-subunit one depending on the local environment. The combination of the thermostable variants (N437K, E96R, T231D and D388K) generated a melting temperature increase of 15.7°C. Thus, this study

  13. Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

    PubMed Central

    Lee, Yang; Willers, Chrissie; Kunji, Edmund R. S.; Crichton, Paul G.

    2015-01-01

    Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein. Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria. PMID:26038550

  14. Rifampicin-activated human pregnane X receptor and CYP3A4 induction enhance acetaminophen-induced toxicity.

    PubMed

    Cheng, Jie; Ma, Xiaochao; Krausz, Kristopher W; Idle, Jeffrey R; Gonzalez, Frank J

    2009-08-01

    Acetaminophen (APAP) is safe at therapeutic levels but causes hepatotoxicity via N-acetyl-p-benzoquinone imine-induced oxidative stress upon overdose. To determine the effect of human (h) pregnane X receptor (PXR) activation and CYP3A4 induction on APAP-induced hepatotoxicity, mice humanized for PXR and CYP3A4 (TgCYP3A4/hPXR) were treated with APAP and rifampicin. Human PXR activation and CYP3A4 induction enhanced APAP-induced hepatotoxicity as revealed by hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated in serum, and hepatic necrosis after coadministration of rifampicin and APAP, compared with APAP administration alone. In contrast, hPXR mice, wild-type mice, and Pxr-null mice exhibited significantly lower ALT/AST levels compared with TgCYP3A4/hPXR mice after APAP administration. Toxicity was coincident with depletion of hepatic glutathione and increased production of hydrogen peroxide, suggesting increased oxidative stress upon hPXR activation. Moreover, mRNA analysis demonstrated that CYP3A4 and other PXR target genes were significantly induced by rifampicin treatment. Urinary metabolomic analysis indicated that cysteine-APAP and its metabolite S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid were the major contributors to the toxic phenotype. Quantification of plasma APAP metabolites indicated that the APAP dimer formed coincident with increased oxidative stress. In addition, serum metabolomics revealed reduction of lysophosphatidylcholine in the APAP-treated groups. These findings demonstrated that human PXR is involved in regulation of APAP-induced toxicity through CYP3A4-mediated hepatic metabolism of APAP in the presence of PXR ligands.

  15. Milk thistle's active components silybin and isosilybin: novel inhibitors of PXR-mediated CYP3A4 induction.

    PubMed

    Mooiman, Kim D; Maas-Bakker, Roel F; Moret, Ed E; Beijnen, Jos H; Schellens, Jan H M; Meijerman, Irma

    2013-08-01

    Because cancer is often treated with combination therapy, unexpected pharmacological effects can occur because of drug-drug interactions. Several drugs are able to cause upregulation or downregulation of drug transporters or cytochrome P450 enzymes, particularly CYP3A4. Induction of CYP3A4 may result in decreased plasma levels and therapeutic efficacy of anticancer drugs. Since the pregnane X receptor (PXR) is one of the major transcriptional regulators of CYP3A4, PXR antagonists can possibly prevent CYP3A4 induction. Currently, a limited number of PXR antagonists are available. Some of these antagonists, such as sulphoraphane and coumestrol, belong to the so-called complementary and alternative medicines (CAM). Therefore, the aim was to determine the potential of selected CAM (β-carotene, Echinacea purpurea, garlic, Ginkgo biloba, ginseng, grape seed, green tea, milk thistle, saw palmetto, valerian, St. John's Wort, and vitamins B6, B12, and C) to inhibit PXR-mediated CYP3A4 induction at the transcriptional level, using a reporter gene assay and a real-time polymerase chain reaction assay in LS180 colon adenocarcinoma cells. Furthermore, computational molecular docking and a LanthaScreen time-resolved fluorescence resonance energy transfer (TR-FRET) PXR competitive binding assay were performed to explore whether the inhibiting CAM components interact with PXR. The results demonstrated that milk thistle is a strong inhibitor of PXR-mediated CYP3A4 induction. The components of milk thistle responsible for this effect were identified as silybin and isosilybin. Furthermore, computational molecular docking revealed a strong interaction between both silybin and isosilybin and PXR, which was confirmed in the TR-FRET PXR assay. In conclusion, silybin and isosilybin might be suitable candidates to design potent PXR antagonists to prevent drug-drug interactions via CYP3A4 in cancer patients.

  16. Co-medication of statins and CYP3A4 inhibitors before and after introduction of new reimbursement policy

    PubMed Central

    Devold, Helene M; Molden, Espen; Skurtveit, Svetlana; Furu, Kari

    2009-01-01

    AIMS To assess the prevalence of co-medication of statins and CYP3A4 inhibitors before and after introduction of a new Norwegian reimbursement policy, which states that all patients should be prescribed simvastatin as first-line lipid-lowering therapy. METHODS Data from patients receiving simvastatin, lovastatin, pravastatin, fluvastatin or atorvastatin in 2004 and 2006, including co-medication of potent CYP3A4 inhibitors, were retrieved from the Norwegian Prescription Database covering the total population of Norway. Key measurements were prevalence of continuous statin use (two or more prescriptions on one statin) and proportions of different statin types among all patients and those co-medicated with CYP3A4 inhibitors. RESULTS In 2004, 5.9% (n = 272 342) of the Norwegian population received two or more prescriptions on one statin compared with 7.0% (n = 324 267) in 2006. The relative number of simvastatin users increased from 39.7% (n = 112 122) in 2004 to 63.1% (n = 226 672) in 2006. A parallel increase was observed within the subpopulation co-medicated with statins and CYP3A4 inhibitors, i.e. from 42.9% (n = 7706) in 2004 to 63.6% (n = 13 367) in 2006. For all other statins the number of overall users decreased to a similar extent to those co-medicated with CYP3A4 inhibitors. CONCLUSIONS In both 2004 and 2006, the choice of statin type did not depend on whether the patient used a CYP3A4 inhibitor or not. Considering the pronounced interaction potential of simvastatin with CYP3A4 inhibitors, a negative influence of the new policy on overall statin safety seems likely. PMID:19220274

  17. A Combined Molecular Docking/Dynamics Approach to Probe the Binding Mode of Cancer Drugs with Cytochrome P450 3A4.

    PubMed

    Panneerselvam, Suresh; Yesudhas, Dhanusha; Durai, Prasannavenkatesh; Anwar, Muhammad Ayaz; Gosu, Vijayakumar; Choi, Sangdun

    2015-08-14

    Cytarabine, daunorubicin, doxorubicin and vincristine are clinically used for combinatorial therapies of cancers in different combinations. However, the knowledge about the interaction of these drugs with the metabolizing enzyme cytochrome P450 is limited. Therefore, we utilized computational methods to predict and assess the drug-binding modes. In this study, we performed docking, MD simulations and free energy landscape analysis to understand the drug-enzyme interactions, protein domain motions and the most populated free energy minimum conformations of the docked protein-drug complexes, respectively. The outcome of docking and MD simulations predicted the productive, as well as the non-productive binding modes of the selected drugs. Based on these interaction studies, we observed that S119, R212 and R372 are the major drug-binding residues in CYP3A4. The molecular mechanics Poisson-Boltzmann surface area analysis revealed the dominance of hydrophobic forces in the CYP3A4-drug association. Further analyses predicted the residues that may contain favorable drug-specific interactions. The probable binding modes of the cancer drugs from this study may extend the knowledge of the protein-drug interaction and pave the way to design analogs with reduced toxicity. In addition, they also provide valuable insights into the metabolism of the cancer drugs.

  18. Centlein, a novel microtubule-associated protein stabilizing microtubules and involved in neurite formation.

    PubMed

    Jing, Zhenli; Yin, Huilong; Wang, Pan; Gao, Juntao; Yuan, Li

    2016-04-01

    We have previously reported that the centriolar protein centlein functions as a molecular link between C-Nap1 and Cep68 to maintain centrosome cohesion [1]. In this study, we identified centlein as a novel microtubule-associated protein (MAP), directly binding to purified microtubules (MTs) via its longest coiled-coil domain. Overexpression of centlein caused profound nocodazole- and cold-resistant MT bundles, which also relied on its MT-binding domain. siRNA-mediated centlein depletion resulted in a significant reduction in tubulin acetylation level and overall fluorescence intensity of cytoplasmic MT acetylation. Centlein was further characterized in neurons. We found that centlein overexpression inhibited neurite formation in retinoic acid (RA)-induced SH-SY5Y and N2a cells. Taken together, we propose that centlein is involved in MT stability and neuritogenesis in vivo. PMID:26915804

  19. Excluded volume entropic effects on protein unfolding times and intermediary stability

    NASA Astrophysics Data System (ADS)

    Chapagain, Prem P.; Gerstman, Bernard S.

    2004-02-01

    The dynamics of protein folding result from both enthalpic and entropic contributions to the free energy. In this paper we focus on entropic volume exclusion effects. We carry out computer simulations using a model that allows us to independently change the size or biochemical properties of amino acid residues. To determine the importance of excluded volume effects, we investigate the effects of changing the size of side chains on the unfolding dynamics of a model four-helix bundle protein. In addition, we also investigate the effects of changing the thickness of the chain's backbone. This has relevance to the behavior of synthetic polymers where the size of the constituent units can be varied. We find that entropic excluded volume effects are crucially important for stabilizing the organized native state relative to the molten globule.

  20. Excluded volume entropic effects on protein unfolding times and intermediary stability

    NASA Astrophysics Data System (ADS)

    Chapagain, Prem

    2005-03-01

    The dynamics of protein folding result from both enthalpic and entropic contributions to the free energy. In this paper we focus on entropic volume exclusion effects. We carry out computer simulations using a model that allows us to independently change the size or biochemical properties of amino acid residues. To determine the importance of excluded volume effects, we investigate the effects of changing the size of side chains on the unfolding