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Sample records for 3a4 protein stability

  1. 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

  2. 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

  3. 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.

  4. 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

  5. Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1.

    PubMed

    Bakari, Sana; Lembrouk, Mehdi; Sourd, Laura; Ousalem, Fares; André, François; Orlowski, Stéphane; Delaforge, Marcel; Frelet-Barrand, Annie

    2016-04-01

    Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies. PMID:26961909

  6. 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.

  7. Performance of protein stability predictors.

    PubMed

    Khan, Sofia; Vihinen, Mauno

    2010-06-01

    Stability is a fundamental property affecting function, activity, and regulation of biomolecules. Stability changes are often found for mutated proteins involved in diseases. Stability predictors computationally predict protein-stability changes caused by mutations. We performed a systematic analysis of 11 online stability predictors' performances. These predictors are CUPSAT, Dmutant, FoldX, I-Mutant2.0, two versions of I-Mutant3.0 (sequence and structure versions), MultiMutate, MUpro, SCide, Scpred, and SRide. As input, 1,784 single mutations found in 80 proteins were used, and these mutations did not include those used for training. The programs' performances were also assessed according to where the mutations were found in the proteins, that is, in secondary structures and on the surface or in the core of a protein, and according to protein structure type. The extents to which the mutations altered the occupied volumes at the residue sites and the charge interactions were also characterized. The predictions of all programs were in line with the experimental data. I-Mutant3.0 (utilizing structural information), Dmutant, and FoldX were the most reliable predictors. The stability-center predictors performed with similar accuracy. However, at best, the predictions were only moderately accurate ( approximately 60%) and significantly better tools would be needed for routine analysis of mutation effects. PMID:20232415

  8. Protein Fibrils Induce Emulsion Stabilization.

    PubMed

    Peng, Jinfeng; Simon, Joana Ralfas; Venema, Paul; van der Linden, Erik

    2016-03-01

    The behavior of an oil-in-water emulsion was studied in the presence of protein fibrils for a wide range of fibril concentrations by using rheology, diffusing wave spectroscopy, and confocal laser scanning microscopy. Results showed that above a minimum fibril concentration depletion flocculation occurred, leading to oil droplet aggregation and enhanced creaming of the emulsion. Upon further increasing the concentration of the protein fibrils, the emulsions were stabilized. In this stable regime both aggregates of droplets and single droplets are present, and these aggregates are smaller than the aggregates in the flocculated emulsion samples at the lower fibril concentrations. The size of the droplet aggregates in the stabilized emulsions is independent of fibril concentration. In addition, the droplet aggregation was reversible upon dilution both by a pH 2 HCl solution and by a fibril solution at the same concentration. The viscosity of the emulsions containing fibrils was comparable to that of the pure fibril solution. Neither fibril networks nor droplet gel networks were observed in our study. The stabilization mechanism of emulsions containing long protein fibrils at high protein fibril concentrations points toward the mechanism of a kinetic stabilization. PMID:26882086

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. Computational approaches for predicting mutant protein stability.

    PubMed

    Kulshreshtha, Shweta; Chaudhary, Vigi; Goswami, Girish K; Mathur, Nidhi

    2016-05-01

    Mutations in the protein affect not only the structure of protein, but also its function and stability. Prediction of mutant protein stability with accuracy is desired for uncovering the molecular aspects of diseases and design of novel proteins. Many advanced computational approaches have been developed over the years, to predict the stability and function of a mutated protein. These approaches based on structure, sequence features and combined features (both structure and sequence features) provide reasonably accurate estimation of the impact of amino acid substitution on stability and function of protein. Recently, consensus tools have been developed by incorporating many tools together, which provide single window results for comparison purpose. In this review, a useful guide for the selection of tools that can be employed in predicting mutated proteins' stability and disease causing capability is provided. PMID:27160393

  14. Contribution of hydrogen bonds to protein stability.

    PubMed

    Pace, C Nick; Fu, Hailong; Lee Fryar, Katrina; Landua, John; Trevino, Saul R; Schell, David; Thurlkill, Richard L; Imura, Satoshi; Scholtz, J Martin; Gajiwala, Ketan; Sevcik, Jozef; Urbanikova, Lubica; Myers, Jeffery K; Takano, Kazufumi; Hebert, Eric J; Shirley, Bret A; Grimsley, Gerald R

    2014-05-01

    Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein. PMID:24591301

  15. Contribution of hydrogen bonds to protein stability

    PubMed Central

    Pace, C Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R; Schell, David; Thurlkill, Richard L; Imura, Satoshi; Scholtz, J Martin; Gajiwala, Ketan; Sevcik, Jozef; Urbanikova, Lubica; Myers, Jeffery K; Takano, Kazufumi; Hebert, Eric J; Shirley, Bret A; Grimsley, Gerald R

    2014-01-01

    Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein. PMID:24591301

  16. Contribution of Hydrogen Bonds to Protein Stability

    NASA Astrophysics Data System (ADS)

    Pace, Nick

    2014-03-01

    I will discuss the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(Δ G), for a series of hydrogen bonding mutants in four proteins: villin head piece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A (1.1Å), Y51F(1.5Å), and T95A(1.3Å). The structures are very similar to wild type RNase Sa and the hydrogen bonding partners always form intermolecular hydrogen bonds to water in the mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions: 1) Hydrogen bonds contribute favorably to protein stability. 2) The contribution of hydrogen bonds to protein stability is strongly context dependent. 3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. 5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

  17. Kinetics of electron transfer in the complex of cytochrome P450 3A4 with the flavin domain of cytochrome P450BM-3 as evidence of functional heterogeneity of the heme protein

    PubMed Central

    Fernando, Harshica; Halpert, James R.; Davydov, Dmitri R.

    2008-01-01

    We used a rapid scanning stop-flow technique to study the kinetics of reduction of cytochrome P450 3A4 (CYP3A4) by the flavin domain of cytochrome P450-BM3 (BMR), which was shown to form a stoichiometric complex (KD = 0.48 µM) with CYP3A4. In the absence of substrates only about 50% of CYP3A4 was able to accept electrons from BMR. Whereas the high-spin fraction was completely reducible, the reducibility of the low-spin fraction did not exceed 42%. Among four substrates tested (testosterone, 1-pyrenebutanol, bromocriptine, or α-naphthoflavone (ANF)) only ANF is capable of increasing the reducibility of the low-spin fraction to 75%. Our results demonstrate that the pool of CYP3A4 is heterogeneous, and not all P450 is competent for electron transfer in the complex with reductase. The increase in the reducibility of the enzyme in the presence of ANF may represent an important element of the mechanism of action of this activator. PMID:18086551

  18. Contribution of Hydrophobic Interactions to Protein Stability

    PubMed Central

    Pace, C. Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R.; Shirley, Bret A.; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J. Martin; Grimsley, Gerald R.

    2011-01-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin head piece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compare our results with previous studies and reach the following conclusions. 1. Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mole per –CH2– group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per –CH2– group). 2. Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kcal/mol): Phe 18 (3.9), Met 13 (3.1), Phe 7 (2.9), Phe 11 (2.7), and Leu 21 (2.7). 3. Based on Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a –CH2– group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. 4. The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔGtr values from water to cyclohexane. 5. For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds 40 ± 4% to protein stability. 6. Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominately by hydrophobic interactions. PMID:21377472

  19. Contribution of hydrophobic interactions to protein stability.

    PubMed

    Pace, C Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R; Shirley, Bret A; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J Martin; Grimsley, Gerald R

    2011-05-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6±0.3 kcal/mol per -CH(2)- group), than to the stability of a large protein, VlsE (1.6±0.3 kcal/mol per -CH(2)- group). (2) Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kilocalories per mole) Phe18 (3.9), Met13 (3.1), Phe7 (2.9), Phe11 (2.7), and Leu21 (2.7). (3) Based on the Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a -CH(2)- group on folding contributes, on average, 1.1±0.5 kcal/mol to protein stability. (4) The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔG(tr) values from water to cyclohexane. (5) For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60±4% and hydrogen bonds contribute 40±4% to protein stability. (6) Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominantly by hydrophobic interactions. PMID:21377472

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Stabilizing effect of knots on proteins

    PubMed Central

    Sułkowska, Joanna I.; Sułkowski, Piotr; Szymczak, P.; Cieplak, Marek

    2008-01-01

    Molecular dynamics studies within a coarse-grained, structure-based model were used on two similar proteins belonging to the transcarbamylase family to probe the effects of the knot in the native structure of a protein. The first protein, N-acetylornithine transcarbamylase, contains no knot, whereas human ormithine transcarbamylase contains a trefoil knot located deep within the sequence. In addition, we also analyzed a modified transferase with the knot removed by the appropriate change of a knot-making crossing of the protein chain. The studies of thermally and mechanically induced unfolding processes suggest a larger intrinsic stability of the protein with the knot. PMID:19064918

  6. Stabilizing effect of knots on proteins.

    PubMed

    Sułkowska, Joanna I; Sulkowski, Piotr; Szymczak, P; Cieplak, Marek

    2008-12-16

    Molecular dynamics studies within a coarse-grained, structure-based model were used on two similar proteins belonging to the transcarbamylase family to probe the effects of the knot in the native structure of a protein. The first protein, N-acetylornithine transcarbamylase, contains no knot, whereas human ormithine transcarbamylase contains a trefoil knot located deep within the sequence. In addition, we also analyzed a modified transferase with the knot removed by the appropriate change of a knot-making crossing of the protein chain. The studies of thermally and mechanically induced unfolding processes suggest a larger intrinsic stability of the protein with the knot. PMID:19064918

  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. Redox Potential Control by Drug Binding to Cytochrome P450 3A4

    PubMed Central

    Das, Aditi; Grinkova, Yelena V.; Sligar, Stephen G.

    2008-01-01

    The cytochrome P450s are ubiquitous heme proteins that utilize two reducing equivalents to cleave a ferrous iron - dioxygen complex to produce a single water molecule with the insertion of one oxygen atom into a bound substrate. For the case of soluble cytochrome P450 CYP101, it has been shown that there is a linear free energy relationship between heme redox potential and the spin state of the ferric protein. However, the universality of this relationship has been challenged in the case of mammalian enzymes. Most cytochrome P450s are integral membrane proteins, and detailed redox potential measurements have proved difficult due protein aggregation or the necessary presence of detergent. In this communication we utilize a soluble nanometer scale membrane bilayer disc (Nanodisc) to stabilize monomeric human cytochrome P450 CYP3A4. The Nanodisc system allows facile redox potential measurements to be made on substrate-free CYP3A4 as well as with several drug molecules bound at the active site. We show that substrate binding can dramatically effect the redox potential of the heme protein through modulation of the ferric spin state. A linear free energy relationship is observed, analogous to that noted for the soluble P450s, indicating a common mechanism for this linkage and providing a means for control of electron input in response to the presence of a metabolizable substrate, this potentially limiting the unwanted production of reduced oxygen species. PMID:17948999

  9. Protein stability at a carbon nanotube interface

    NASA Astrophysics Data System (ADS)

    Vaitheeswaran, S.; Garcia, A. E.

    2011-03-01

    The interactions of proteins with solid surfaces occur in a variety of situations. Motivated by the many nanoengineering applications of protein-carbon nanotube hybrids, we investigate the conformational transitions of hen egg white lysozyme adsorbed on a carbon nanotube. Using a Cα structure-based model and replica exchange molecular dynamics, we show how the folding/unfolding equilibrium of the adsorbed protein varies with the strength of its coupling to the surface. The stability of the native state depends on the balance between the favorable entropy and unfavorable enthalpy change on adsorption. In the case of a weakly attractive surface when the former dominates, the protein is stabilized. In this regime, the protein can fold and unfold while maintaining the same binding fraction. With increasing surface attraction, the unfavorable enthalpic effect dominates, the native state is destabilized, and the protein has to extensively unbind before changing states from unfolded to folded. At the highest surface coupling, the entropic penalty of folding vanishes, and a folding intermediate is strongly stabilized. In this intermediate state, the α-domain of lysozyme is disrupted, while the β-sheet remains fully structured. We rationalize the relative stability of the two domains on the basis of the residue contact order.

  10. Dipeptide Prodrug Approach to Evade Efflux Pumps and CYP3A4 Metabolism of Lopinavir

    PubMed Central

    Patel, Mitesh; Sheng, Ye; Mandava, Nanda K.; Pal, Dhananjay; Mitra, Ashim K.

    2014-01-01

    Oral absorption of lopinavir (LPV) is limited due to P-glycoprotein (P-gp) and multidrug resistance-associated protein2 (MRP2) mediated efflux by intestinal epithelial cells. Moreover, LPV is extensively metabolized by CYP3A4 enzymes. In the present study, dipeptide prodrug approach was employed to circumvent efflux pumps (P-gp and MRP2) and CYP3A4 mediated metabolism of LPV. Valine-isoleucine-LPV (Val-Ile-LPV) was synthesized and identified by LCMS and NMR techniques. The extent of LPV and Val-Ile-LPV interactions with P-gp and MRP2 was studied by uptake and transport studies across MDCK-MDR1 and MDCK-MRP2 cells. To determine the metabolic stability, time and concentration dependent degradation study was performed in liver microsomes. Val-Ile-LPV exhibited significantly higher aqueous solubility relative to LPV. This prodrug generated higher stability under acidic pH. Val-Ile-LPV demonstrated significantly lower affinity towards P-gp and MRP2 relative to LPV. Transepithelial transport of Val-Ile-LPV was significantly higher in the absorptive direction (apical to basolateral) relative to LPV. Importantly, Val-Ile-LPV was recognized as an excellent substrate by peptide transporter. Moreover, Val-Ile-LPV displayed significantly higher metabolic stability relative to LPV. Results obtained from this study suggested that dipeptide prodrug approach is a viable option to elevate systemic levels of LPV following oral administration PMID:25261710

  11. 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

  12. Protein stability: the value of 'old literature'.

    PubMed

    Franks, Felix

    2002-05-01

    The concepts of protein structure and function have been subjects of intensive study throughout the 20th century; they continue to fascinate present-day scientists. Our understanding received a major boost when it was realised during the 1960s, that the physical properties of water play a major role in determining the stability of native proteins in vitro. This recognition changed the emphasis of physicochemical studies towards 'hydration', i.e. protein-water interactions. A rigorous quantitative description of 'hydration' still escapes us, but several semi-quantitative treatments, some with predictive potential, are now available and can account for the marginal stabilities of native proteins in aqueous solvent environments. This article charts the progress achieved during the latter half of the 20th century, which in present day parlance is termed 'old literature'. The thesis is advanced that the common practice of uncritically equating 'recent literature' with 'progress' is of dubious value. In the general area of in vitro protein stability some recent developments seem questionable and have yet to stand the test of time before their usefulness or validity can be accepted. PMID:12034434

  13. Effects of confinement on protein folding and protein stability

    NASA Astrophysics Data System (ADS)

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

    2003-05-01

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

  14. Enhancing protein stability with extended disulfide bonds.

    PubMed

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

    2016-05-24

    Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes. PMID:27162342

  15. Trehalose glycopolymers as excipients for protein stabilization.

    PubMed

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

    2013-08-12

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

  16. 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

  17. 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.

  18. 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

  19. 22 CFR 3a.4 - Procedure for requesting approval.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 22 Foreign Relations 1 2012-04-01 2012-04-01 false Procedure for requesting approval. 3a.4 Section 3a.4 Foreign Relations DEPARTMENT OF STATE GENERAL ACCEPTANCE OF EMPLOYMENT FROM FOREIGN GOVERNMENTS BY MEMBERS OF THE UNIFORMED SERVICES § 3a.4 Procedure for requesting approval. (a) An applicant...

  20. 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

  1. 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

  2. Sequence-based protein stabilization in the absence of glycosylation.

    PubMed

    Tan, Nikki Y; Bailey, Ulla-Maja; Jamaluddin, M Fairuz; Mahmud, S Halimah Binte; Raman, Suresh C; Schulz, Benjamin L

    2014-01-01

    Asparagine-linked N-glycosylation is a common modification of proteins that promotes productive protein folding and increases protein stability. Although N-glycosylation is important for glycoprotein folding, the precise sites of glycosylation are often not conserved between protein homologues. Here we show that, in Saccharomyces cerevisiae, proteins upregulated during sporulation under nutrient deprivation have few N-glycosylation sequons and in their place tend to contain clusters of like-charged amino-acid residues. Incorporation of such sequences complements loss of in vivo protein function in the absence of glycosylation. Targeted point mutation to create such sequence stretches at glycosylation sequons in model glycoproteins increases in vitro protein stability and activity. A dependence on glycosylation for protein stability or activity can therefore be rescued with a small number of local point mutations, providing evolutionary flexibility in the precise location of N-glycans, allowing protein expression under nutrient-limiting conditions, and improving recombinant protein production. PMID:24434425

  3. 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

  4. Induction of human CYP3A4 by huperzine A, ligustrazine and oridonin through pregnane X receptor-mediated pathways.

    PubMed

    Zhang, Yi-Wen; Bao, Mei-Hua; Wang, Guo; Qu, Qiang; Zhou, Hong-Hao

    2014-07-01

    The pregnane X receptor (PXR) is a key regulator of CYP3A4, which is involved in catalyzing the metabolic conversion of a number of endogenous substrates. In this study, we screened 22 compounds isolated from traditional Chinese herbal medicines using luciferase reporter gene assays for inspecting their capabilities in inducing PXR-mediated transactivation of CYP3A4 expression. In addition, the mRNA and protein expressions of CYP3A4 and PXR as well as the enzymatic activites of CYP3A4 were analyzed by real-time PCR, Western blot analysis and UPLC-MS/MS-based metabolite assay in LS174T cells. Huperzine A, ligustrazine and oridonin were identified to be the inducers of CYP3A4. These compounds induced the CYP3A4 reporter luciferase activity, and up-regulated CYP3A4 mRNA and protein levels significantly. Besides, huperzine A, ligustrazine and oridonin significantly up-regulated enzymatic activities of CYP3A4. However, the three compounds showed no effects on PXR mRNA and protein expression. To our knowledge, it is the first identification of these three compounds as PXR activators to induce CYP3A4. These results indicate that huperzine A, ligustrazine and oridonin induced CYP3A4 expression and activation via PXR dependent pathways, and might contribute to drug-drug interactions. PMID:25073399

  5. 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.

  6. Protein thermal stabilization in aqueous solutions of osmolytes.

    PubMed

    Bruździak, Piotr; Panuszko, Aneta; Jourdan, Muriel; Stangret, Janusz

    2016-01-01

    Proteins' thermal stabilization is a significant problem in various biomedical, biotechnological, and technological applications. We investigated thermal stability of hen egg white lysozyme in aqueous solutions of the following stabilizing osmolytes: Glycine (GLY), N-methylglycine (NMG), N,N-dimethylglycine (DMG), N,N,N-trimethylglycine (TMG), and trimethyl-N-oxide (TMAO). Results of CD-UV spectroscopic investigation were compared with FTIR hydration studies' results. Selected osmolytes increased lysozyme's thermal stability in the following order: Gly>NMG>TMAO≈DMG>TMG. Theoretical calculations (DFT) showed clearly that osmolytes' amino group protons and water molecules interacting with them played a distinctive role in protein thermal stabilization. The results brought us a step closer to the exact mechanism of protein stabilization by osmolytes. PMID:26495438

  7. Cold denaturation as a tool to measure protein stability

    PubMed Central

    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. PMID:26026885

  8. 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. PMID:26026885

  9. 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. PMID:26934171

  10. Electrodynamic pressure modulation of protein stability in cosolvents.

    PubMed

    Damodaran, Srinivasan

    2013-11-19

    Cosolvents affect structural stability of proteins in aqueous solutions. A clear understanding of the mechanism by which cosolvents impact protein stability is critical to understanding protein folding in a biological milieu. In this study, we investigated the Lifshitz-van der Waals dispersion interaction of seven different solutes with nine globular proteins and report that in an aqueous medium the structure-stabilizing solutes exert a positive electrodynamic pressure, whereas the structure-destabilizing solutes exert a negative electrodynamic pressure on the proteins. The net increase in the thermal denaturation temperature (ΔTd) of a protein in 1 M solution of various solutes was linearly related to the electrodynamic pressure (PvdW) between the solutes and the protein. The slope of the PvdW versus ΔTd plots was protein-dependent. However, we find a positive linear relationship (r(2) = 0.79) between the slope (i.e., d(ΔTd)/dPvdW) and the adiabatic compressibility (βs) of the proteins. Together, these results clearly indicate that the Lifshitz's dispersion forces are inextricably involved in solute-induced stabilization/destabilization of globular proteins. The positive and/or negative electrodynamic pressure generated by the solute-protein interaction across the water medium seems to be the fundamental mechanism by which solutes affect protein stability. This is at variance with the existing preferential hydration concept. The implication of these results is significant in the sense that, in addition to the hydrophobic effect that drives protein folding, the electrodynamic forces between the proteins and solutes in the biological milieu also might play a role in the folding process as well as in the stability of the folded state. PMID:24156352

  11. 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

  12. Mutation analysis of barley malt protein Z4 and protein Z7 on beer foam stability.

    PubMed

    Iimure, Takashi; Kimura, Tatsuji; Araki, Shigeki; Kihara, Makoto; Sato, Masahide; Yamada, Shinji; Shigyou, Tatsuro; Sato, Kazuhiro

    2012-02-15

    Beer foam stability is an important characteristic. It has been suggested that isoforms of protein Z, that is, protein Z4 and protein Z7, contribute to beer foam stability. We investigated the relationship between beer foam stability and protein Z4 and protein Z7 using their deficient mutants. As a protein Z4-deficient mutant, cv. Pirkka was used. Protein Z7 deficiency was screened in 1564 barley accessions in the world collection of Okayama University, Japan. The barley samples from normal, protein Z4-deficient, protein Z7-deficient, and double-deficient were genotyped in F(2) populations and then pooled based on the DNA marker genotypes of protein Z4 and protein Z7. For a brewing trial, F(5) pooled subpopulations were used. After malting and brewing, the foam stability was determined, and the results showed that the levels of foam stability in the four samples were comparable. Two-dimensional gel electrophoresis was used to investigate the proteome in these beer samples. The results showed that low molecular weight proteins, including lipid transfer protein (LTP2), in the deficient mutants were higher than those in the normal sample. Our results suggest that the contribution of protein Z4 and protein Z7 to beer foam stability was not greater than that of other beer proteins. PMID:22251057

  13. 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

  14. 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

  15. Energetics of Heterotropic Cooperativity between α-Naphthoflavone and Testosterone Binding to CYP3A4

    PubMed Central

    Roberts, Arthur G.; Atkins, William M.

    2007-01-01

    Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of a majority of drugs. Heterotropic cooperativity of drug binding to CYP3A4 was examined with the flavanoid, α-naphthoflavone (ANF) and the steroid, testosterone (TST). UV-vis and EPR spectroscopy of CYP3A4 show that ANF binding to CYP3A4 occurs with apparent negative cooperativity and that there are at least two binding sites: 1) a relatively tight spin-state insensitive binding site (CYP●ANF) and 2) a relatively low affinity spin-state sensitive binding site (CYP●ANF●ANF). Since binding to the spin-state insensitive binding site is considerably tighter for ANF than TST, the spin-state insensitive binding site could be occupied by ANF, while titrating TST at the other site(s). The spin-state insensitive binding site of ANF appears to compete with the spin-state insensitive binding site of TST. The formation of the spin-state insensitive CYP●ANF complex is strongly temperature dependent, when compared to the formation of the CYP●TST complex, suggesting that the formation of the CYP3A4●ANF complex leads to long-range conformational changes within the protein. When the CYP●ANF complex is titrated with TST, the formation of CYP●ANF●TST is favored by 3:1 over the formation of CYP●TST●TST, suggesting that there is an allosteric interaction between ANF and TST. A model of heterotropic cooperativity of CYP3A4 is presented, where the spin-state insensitive binding of ANF occurs at the same peripheral binding site of CYP3A4 as TST. PMID:17459328

  16. 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

  17. Tandem Facial Amphiphiles for Membrane Protein Stabilization

    PubMed Central

    Chae, Pil Seok; Gotfryd, Kamil; Pacyna, Jennifer; Miercke, Larry J. W.; Rasmussen, Søren G. F.; Robbins, Rebecca A.; Rana, Rohini R.; Loland, Claus J.; Kobilka, Brian; Stroud, Robert; Byrne, Bernadette; Gether, Ulrik; Gellman, Samuel H.

    2010-01-01

    We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles. PMID:21049926

  18. 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

  19. 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

  20. SPR and electrochemical analyses of interactions between CYP3A4 or 3A5 and cytochrome b5

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

    The combination of SPR biosensor with electrochemical analysis was used for the study of protein-protein interaction between cytochromes CYP3A4 or 3А5 and cytochromes b5: the microsomal, mitochondrial forms of this protein, and 2 ≪chimeric≫ proteins. Kinetic constants of CYP3A4 and CYP3А5 complex formation with cytochromes b5 were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was observed upon their interactions with mitochondrial cytochrome b5. The electrochemical analysis of CYP3A4, CYP3A5, and cytochromes b5 immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 to -0.350 V (vs. Ag/AgCl).

  1. Unexpected effects of macromolecular crowding on protein stability.

    PubMed

    Benton, Laura A; Smith, Austin E; Young, Gregory B; Pielak, Gary J

    2012-12-11

    Most theories about macromolecular crowding focus on two ideas: the macromolecular nature of the crowder and entropy. For proteins, the volume excluded by the crowder favors compact native states over expanded denatured states, enhancing protein stability by decreasing the entropy of unfolding. We tested these ideas with the widely used crowding agent Ficoll-70 and its monomer, sucrose. Contrary to expectations, Ficoll and sucrose have approximately the same stabilizing effect on chymotrypsin inhibitor 2. Furthermore, the stabilization is driven by enthalpy, not entropy. These results point to the need for carefully controlled studies and more sophisticated theories for understanding crowding effects. PMID:23167542

  2. Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation.

    PubMed Central

    Neet, K. E.; Timm, D. E.

    1994-01-01

    The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed. PMID:7756976

  3. Energetics-Based Methods for Protein Folding and Stability Measurements

    NASA Astrophysics Data System (ADS)

    Geer, M. Ariel; Fitzgerald, Michael C.

    2014-06-01

    Over the past 15 years, a series of energetics-based techniques have been developed for the thermodynamic analysis of protein folding and stability. These techniques include Stability of Unpurified Proteins from Rates of amide H/D Exchange (SUPREX), pulse proteolysis, Stability of Proteins from Rates of Oxidation (SPROX), slow histidine H/D exchange, lysine amidination, and quantitative cysteine reactivity (QCR). The above techniques, which are the subject of this review, all utilize chemical or enzymatic modification reactions to probe the chemical denaturant- or temperature-induced equilibrium unfolding properties of proteins and protein-ligand complexes. They employ various mass spectrometry-, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-, and optical spectroscopy-based readouts that are particularly advantageous for high-throughput and in some cases multiplexed analyses. This has created the opportunity to use protein folding and stability measurements in new applications such as in high-throughput screening projects to identify novel protein ligands and in mode-of-action studies to identify protein targets of a particular ligand.

  4. Sperm Motility Requires Wnt/GSK3 Stabilization of Proteins.

    PubMed

    De Robertis, Edward M; Ploper, Diego

    2015-11-23

    Inhibition of GSK3 by Wnt signaling stabilizes many cellular proteins, but proof that this effect is independent of β-catenin-mediated transcription is lacking. Koch, Acebron, and colleagues (2015) now demonstrate that transcriptionally silent mammalian sperm require Wnt signaling via exosomes to prevent protein degradation during their lengthy travels through the epididymis. PMID:26609954

  5. Probing protein stabilization by glycerol using electrospray mass spectrometry.

    PubMed

    Grandori, R; Matecko, I; Mayr, P; Müller, N

    2001-08-01

    This study shows that electrospray ionization mass spectrometry (ESI-MS), combined with a heated turbo ion-spray interface, allows monitoring protein stabilization by glycerol in solution. Measurements obtained with the two proteins lysozyme and cytochrome c are presented. The observed mass-to-charge (m/z) distributions reveal the stabilizing effect of the additive on the protein conformations against temperature and acid-induced unfolding, as well as against denaturation by acetonitrile. The data obtained with lysozyme allow detection of minor conformational changes upon glycerol addition to the native protein, and suggest that the protein structure in the presence of the additive is slightly compressed compared with its state in water. This result corroborates previous evidence obtained by nuclear magnetic resonance. It is also shown that analysis of the m/z distributions obtained by ESI-MS can lead to detection of partially folded and partially populated states in protein samples. PMID:11523091

  6. Predicting stability of Arc repressor mutants with protein stochastic moments.

    PubMed

    González-Díaz, Humberto; Uriarte, Eugenio; Ramos de Armas, Ronal

    2005-01-17

    As more and more protein structures are determined and applied to drug manufacture, there is increasing interest in studying their stability. In this study, the stochastic moments ((SR)pi(k)) of 53 Arc repressor mutants were introduced as molecular descriptors modeling protein stability. The Linear Discriminant Analysis model developed correctly classified 43 out of 53, 81.13% of proteins according to their thermal stability. More specifically, the model classified 20/28 (71.4%) proteins with near wild-type stability and 23/25 (92%) proteins with reduced stability. Moreover, validation of the model was carried out by re-substitution procedures (81.0%). In addition, the stochastic moments based model compared favorably with respect to others based on physicochemical and geometric parameters such as D-Fire potential, surface area, volume, partition coefficient, and molar refractivity, which presented less than 77% of accuracy. This result illustrates the possibilities of the stochastic moments' method for the study of bioorganic and medicinal chemistry relevant proteins. PMID:15598555

  7. Dual degradation signals control Gli protein stability and tumor formation

    PubMed Central

    Huntzicker, Erik G.; Estay, Ivette S.; Zhen, Hanson; Lokteva, Ludmila A.; Jackson, Peter K.; Oro, Anthony E.

    2006-01-01

    Regulated protein destruction controls many key cellular processes with aberrant regulation increasingly found during carcinogenesis. Gli proteins mediate the transcriptional effects of the Sonic hedgehog pathway, which is implicated in up to 25% of human tumors. Here we show that Gli is rapidly destroyed by the proteasome and that mouse basal cell carcinoma induction correlates with Gli protein accumulation. We identify two independent destruction signals in Gli1, DN and DC, and show that removal of these signals stabilizes Gli1 protein and rapidly accelerates tumor formation in transgenic animals. These data argue that control of Gli protein accumulation underlies tumorigenesis and suggest a new avenue for antitumor therapy. PMID:16421275

  8. 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

  9. Fluorinated proteins: from design and synthesis to structure and stability.

    PubMed

    Marsh, E Neil G

    2014-10-21

    Fluorine is all but absent from biology; however, it has proved to be a remarkably useful element with which to modulate the activity of biological molecules and to study their mechanism of action. Our laboratory's interest in incorporating fluorine into proteins was stimulated by the unusual physicochemical properties exhibited by perfluorinated small molecules. These include extreme chemical inertness and thermal stability, properties that have made them valuable as nonstick coatings and fire retardants. Fluorocarbons also exhibit an unusual propensity to phase segregation. This phenomenon, which has been termed the "fluorous effect", has been effectively exploited in organic synthesis to purify compounds from reaction mixtures by extracting fluorocarbon-tagged molecules into fluorocarbon solvents. As biochemists, we were curious to explore whether the unusual physicochemical properties of perfluorocarbons could be engineered into proteins. To do this, we developed a synthesis of a highly fluorinated amino acid, hexafluoroleucine, and designed a model 4-helix bundle protein, α4H, in which the hydrophobic core was packed exclusively with leucine. We then investigated the effects of repacking the hydrophobic core of α4H with various combinations of leucine and hexafluoroleucine. These initial studies demonstrated that fluorination is a general and effective strategy for enhancing the stability of proteins against chemical and thermal denaturation and proteolytic degradation. We had originally envisaged that the "fluorous interactions", postulated from the self-segregating properties of fluorous solvents, might be used to mediate specific protein-protein interactions orthogonal to those of natural proteins. However, various lines of evidence indicate that no special, favorable fluorine-fluorine interactions occur in the core of the fluorinated α4 protein. This makes it unlikely that fluorinated amino acids can be used to direct protein-protein interactions. More

  10. 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

  11. Reprint of: Impact of alcohols on the formation and stability of protein-stabilized nanoemulsions.

    PubMed

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

    2015-07-01

    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<100 nm) were obtained when 10%w/w 1-butanol was added to sodium caseinate-stabilized nanoemulsions, but relatively small droplets (d<150 nm) 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:25865241

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

    PubMed Central

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

    2005-01-01

    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 . PMID:15980477

  13. 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. PMID:15980477

  14. 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

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

    PubMed

    Oshima, Hiraku; Kinoshita, Masahiro

    2013-06-28

    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

  16. 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

  17. 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.

  18. Mechanism of protein precipitation and stabilization by co-solvents

    NASA Astrophysics Data System (ADS)

    Timasheff, Serge N.; Arakawa, Tsutomu

    1988-07-01

    The interactions between proteins and a number of substances which, when present at high concentration, stabilize or precipitate proteins, have been analyzed in terms of the preferential interactions of these co-solvents with proteins. In all cases, stabilization or precipitation was accompanied by preferential exclusion of the co-solvent from the immediate domain of the protein, i.e., preferential hydration of the protein. This means that addition of the co-solvent to the aqueous protein solution increased the chemical potentials of both components. The thermodynamic interaction parameters derived from such data make it possible to calculate the salting out constant, Ks, as well as to construct a phase isotherm for any given solvent mixture which indicates the limiting protein solubility. The salting-out effect can be decomposed into contributions from non-specific preferential exclusion and specific binding of the ligand to the protein, the balance leading to solubilization or precipitation. In reactions, such as denaturation, the effect of co-solvent on the reaction depends on the difference in the preferential interactions of the two end states of the protein. Principal sources of preferential exclusion have been identified as steric exclusion, increase of the surface tension of water by the co-solvent, repulsion by charged loci on the protein and solvophobicity.

  19. Magic-Angle Spinning Solid-State NMR Spectroscopy of Nanodisc– Embedded Human CYP3A4

    PubMed Central

    Kijac, Aleksandra; Li, Ying; Sligar, Stephen G.; Rienstra, Chad M.

    2008-01-01

    Cytochrome P450 (CYP) 3A4 contributes to the metabolism of approximately 50% of commercial drugs by oxidizing a large number of structurally diverse substrates. Like other endoplasmic reticulum-localized P450s, CYP3A4 contains a membrane-anchoring N-terminal helix and a significant number of hydrophobic domains, important for the interaction between CYP3A4 and the membrane. Although the membrane affects specificity of CYP3A4 ligand binding, the structural details of the interaction have not been revealed so far because x-ray crystallography studies are available only for the soluble domain of CYP3A4. Here we report sample preparation and initial magic-angle spinning (MAS) solid-state NMR (SSNMR) of CYP3A4 (Δ3−12) embedded in a nanoscale membrane bilayer, or Nanodisc. The growth protocol yields ∼2.5 mg of the enzymatically active, uniformly 13C, 15N-enriched CYP3A4 from a liter of growth medium. Polyethylene glycol 3350-precipitated CYP3A4 in Nanodiscs yields spectra of high resolution and sensitivity, consistent with a folded, homogeneous protein. CYP3A4 in Nanodiscs remains enzymatically active throughout the precipitation protocol as monitored by bromocriptine binding. The 13C line widths measured from 13C-13C 2D chemical shift correlation spectra are ∼0.5 ppm. The secondary structure distribution within several amino acid types determined from 13C chemical shifts is consistent with the ligand-free x-ray structures. These results demonstrate that MAS SSNMR can be performed on Nanodisc-embedded membrane proteins in a folded, active state. The combination of SSNMR and Nanodisc methodologies opens up new possibilities for obtaining structural information on CYP3A4 and other integral membrane proteins with full retention of functionality. PMID:17985934

  20. 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

  1. 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.

  2. 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

  3. 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. PMID:24915379

  4. 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.

  5. 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

  6. 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

  7. The Role of Trehalose for the Stabilization of Proteins.

    PubMed

    Olsson, Christoffer; Jansson, Helén; Swenson, Jan

    2016-05-26

    Understanding of how the stabilization mechanism of trehalose operates on biological molecules against different types of environmental stress could prove to gain great advancements in many different types of conservation techniques, such as cryopreservation or freeze-drying. Many theories exist that aim to explain why trehalose possesses an extraordinary ability to stabilize biomolecules. However, all of them just explain parts of its mechanism and a comprehensive picture is still lacking. In this study, we have used differential scanning calorimetry (DSC) and viscometry measurements to determine how the glass transition temperature Tg, the protein denaturation temperature Tden, and the dynamic viscosity depend on both the trehalose and the protein concentration in myoglobin-trehalose-water systems. The aim has been to determine whether these physical properties are related and to gain indirect structural insights from the limits of water crystallization at different concentration ratios. The results show that for systems without partial crystallization of water the addition of protein increases Tg, most likely due to the fact that the protein adsorbs water and thereby reduces the water content in the trehalose-water matrix. Furthermore, these systems are generally decreasing in Tden with an increasing protein concentration, and thereby also an increasing viscosity, showing that the dynamics of the trehalose-water matrix and the stability of the native structure of the protein are not necessarily coupled. We also infer, by analyzing the maximum amount of water for which ice formation is avoided, that the preferential hydration model is consistent with our experimental data. PMID:27135987

  8. 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

  9. Mutational effects on stability are largely conserved during protein evolution.

    PubMed

    Ashenberg, Orr; Gong, L Ian; Bloom, Jesse D

    2013-12-24

    Protein stability and folding are the result of cooperative interactions among many residues, yet phylogenetic approaches assume that sites are independent. This discrepancy has engendered concerns about large evolutionary shifts in mutational effects that might confound phylogenetic approaches. Here we experimentally investigate this issue by introducing the same mutations into a set of diverged homologs of the influenza nucleoprotein and measuring the effects on stability. We find that mutational effects on stability are largely conserved across the homologs. We reach qualitatively similar conclusions when we simulate protein evolution with molecular-mechanics force fields. Our results do not mean that proteins evolve without epistasis, which can still arise even when mutational stability effects are conserved. However, our findings indicate that large evolutionary shifts in mutational effects on stability are rare, at least among homologs with similar structures and functions. We suggest that properly describing the clearly observable and highly conserved amino acid preferences at individual sites is likely to be far more important for phylogenetic analyses than accounting for rare shifts in amino acid propensities due to site covariation. PMID:24324165

  10. 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.

  11. Graph theory and stability analysis of protein complex interaction networks.

    PubMed

    Huang, Chien-Hung; Chen, Teng-Hung; Ng, Ka-Lok

    2016-04-01

    Protein complexes play an essential role in many biological processes. Complexes can interact with other complexes to form protein complex interaction network (PCIN) that involves in important cellular processes. There are relatively few studies on examining the interaction topology among protein complexes; and little is known about the stability of PCIN under perturbations. We employed graph theoretical approach to reveal hidden properties and features of four species PCINs. Two main issues are addressed, (i) the global and local network topological properties, and (ii) the stability of the networks under 12 types of perturbations. According to the topological parameter classification, we identified some critical protein complexes and validated that the topological analysis approach could provide meaningful biological interpretations of the protein complex systems. Through the Kolmogorov-Smimov test, we showed that local topological parameters are good indicators to characterise the structure of PCINs. We further demonstrated the effectiveness of the current approach by performing the scalability and data normalization tests. To measure the robustness of PCINs, we proposed to consider eight topological-based perturbations, which are specifically applicable in scenarios of targeted, sustained attacks. We found that the degree-based, betweenness-based and brokering-coefficient-based perturbations have the largest effect on network stability. PMID:26997661

  12. 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

  13. Designed protein reveals structural determinants of extreme kinetic stability

    PubMed Central

    Broom, Aron; Ma, S. Martha; Xia, Ke; Rafalia, Hitesh; Trainor, Kyle; Colón, Wilfredo; Gosavi, Shachi; Meiering, Elizabeth M.

    2015-01-01

    The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge. PMID:26554002

  14. 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.

  15. [Protein Folding and Stability in the Presence of Osmolytes].

    PubMed

    Fonin, A V; Uversky, V N; Kuznetsova, I M; Turoverov, K K

    2016-01-01

    Osmolytes are molecules with the function among others to align hydrostatic pressure between intracellular and extracellular spaces. Accumulation of osmolytes occurs in the cell in response to stress caused by pressure change, change in temperature, pH, and concentration of inorganic salts. Osmolytes can prevent native proteins denaturation and promote folding of unfolding proteins. Investigation of the osmolytes effect on these processes is essential for understanding the mechanisms of folding and functioning of proteins in vivo. A score of works, devoted to the effect of osmolytes on proteins, are not always consistent with each other. In this review an attempt was made to systemize available array of data on the subject and consider the problem of folding and stability of proteins in solutions in the presence of osmolytes from the single viewpoint. PMID:27192822

  16. 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. PMID:27139003

  17. 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. PMID:24888734

  18. The yeast ubiquitin protease, Ubp3p, promotes protein stability.

    PubMed Central

    Brew, Christine T; Huffaker, Tim C

    2002-01-01

    Stu1p is a microtubule-associated protein required for spindle assembly. In this article we show that the temperature-sensitive stu1-5 allele is synthetically lethal in combination with ubp3, gim1-gim5, and kem1 mutations. The primary focus of this article is on the stu1-5 ubp3 interaction. Ubp3 is a deubiquitination enzyme and a member of a large family of cysteine proteases that cleave ubiquitin moieties from protein substrates. UBP3 is the only one of 16 UBP genes in yeast whose loss is synthetically lethal with stu1-5. Stu1p levels in stu1-5 cells are several-fold lower than the levels in wild-type cells and the stu1-5 temperature sensitivity can be rescued by additional copies of stu1-5. These results indicate that the primary effect of the stu1-5 mutation is to make the protein less stable. The levels of Stu1p are even lower in ubp3Delta stu1-5 cells, suggesting that Ubp3p plays a role in promoting protein stability. We also found that ubp3Delta produces growth defects in combination with mutations in other genes that decrease protein stability. Overall, these data support the idea that Ubp3p has a general role in the reversal of protein ubiquitination. PMID:12454057

  19. Arsenite and its metabolites, MMA(III) and DMA(III), modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice.

    PubMed

    Medina-Díaz, I M; Estrada-Muñiz, E; Reyes-Hernández, O D; Ramírez, 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(III)) and dimethylarsinous acid (DMA(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(III) induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA(III) increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA(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. PMID:19084030

  20. 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.

  1. 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

  2. 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

  3. Nanobody stabilization of G protein coupled receptor conformational states

    PubMed Central

    Steyaert, Jan; K Kobilka, Brian

    2011-01-01

    Remarkable progress has been made in the field of G protein coupled receptor (GPCR) structural biology during the past four years. Several obstacles to generating diffraction quality crystals of GPCRs have been overcome by combining innovative methods ranging from protein engineering to lipid-based screens and microdiffraction technology. The initial GPCR structures represent energetically stable inactive-state conformations. However, GPCRs signal through different G protein isoforms or G protein-independent effectors upon ligand binding suggesting the existence of multiple ligand-specific active states. These active-state conformations are unstable in the absence of specific cytosolic signaling partners representing new challenges for structural biology. Camelid single chain antibody fragments (nanobodies) show promise for stabilizing active GPCR conformations and as chaperones for crystallogenesis. PMID:21782416

  4. Hill Parameters and Heterogeneity of alpha-Naphthoflavone Binding to Human Cytochrome P450 3A4 by Fluorescence Spectroscopic Analysis

    NASA Astrophysics Data System (ADS)

    Carlson, Benjamin; Marsch, Glenn; Martin, Martha; Guengerich, F. Peter

    2009-03-01

    Human cytochrome P450 3A4 (CYP 3A4) is an alpha-helical membrane-bound protein that metabolizes approximately 50% of all drugs. The interaction between CYP450 3A4 and alpha-naphthoflavone (ANF) was characterized using fluorescence methods. ANF quenched fluorescence from tryptophan residues in CYP 3A4, and CYP 3A4 quenched bound ANF. The ANF emission energy was unchanged upon binding to CYP 3A4, implying that enzyme-bound 3A4 is completely quenched. Fluorescence difference spectra were fit to the Hill equation by varying the parameters Kd and n. For quenching of tryptophan fluorescence by ANF, no significant sigmoidal behavior was observed with n=1, and the spectral dissociation constant revealed a strong ANF-CYP 3A4 interaction with Kd=27nM. Modest cooperativity and very tight binding was observed in the quenching of ANF by CYP 3A4, with n=1.4 and Kd= 4.9nM. Fluorescence polarization anisotropy decreased at low ANF/CYP 3A4 molar ratios; then increased at higher ratios. Compared to substrate-free CYP 3A4, adding substrate at low molar ratios increases the CYP 3A4 rotation, suggesting the molecular volume decreases.

  5. Redox control of iron regulatory protein 2 stability.

    PubMed

    Hausmann, Anja; Lee, Julie; Pantopoulos, Kostas

    2011-02-18

    Iron regulatory protein 2 (IRP2) is a critical switch for cellular and systemic iron homeostasis. In iron-deficient or hypoxic cells, IRP2 binds to mRNAs containing iron responsive elements (IREs) and regulates their expression. Iron promotes proteasomal degradation of IRP2 via the F-box protein FBXL5. Here, we explored the effects of oxygen and cellular redox status on IRP2 stability. We show that iron-dependent decay of tetracycline-inducible IRP2 proceeds efficiently under mild hypoxic conditions (3% oxygen) but is compromised in severe hypoxia (0.1% oxygen). A treatment of cells with exogenous H(2)O(2) protects IRP2 against iron and increases its IRE-binding activity. IRP2 is also stabilized during menadione-induced oxidative stress. These data demonstrate that the degradation of IRP2 in iron-replete cells is not only oxygen-dependent but also sensitive to redox perturbations. PMID:21281640

  6. 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. PMID:26948597

  7. The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator.

    PubMed

    Baturin, Simon; Galka, Jamie J; Piyadasa, Hadeesha; Gajjeraman, S; O'Neil, Joe D

    2014-12-01

    Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric α-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10 °C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein-protein interactions are discussed. PMID:25387032

  8. Quantitation of protein-protein interactions by thermal stability shift analysis.

    PubMed

    Layton, Curtis J; Hellinga, Homme W

    2011-08-01

    Thermal stability shift analysis is a powerful method for examining binding interactions in proteins. We demonstrate that under certain circumstances, protein-protein interactions can be quantitated by monitoring shifts in thermal stability using thermodynamic models and data analysis methods presented in this work. This method relies on the determination of protein stabilities from thermal unfolding experiments using fluorescent dyes such as SYPRO Orange that report on protein denaturation. Data collection is rapid and straightforward using readily available real-time polymerase chain reaction instrumentation. We present an approach for the analysis of the unfolding transitions corresponding to each partner to extract the affinity of the interaction between the proteins. This method does not require the construction of a titration series that brackets the dissociation constant. In thermal shift experiments, protein stability data are obtained at different temperatures according to the affinity- and concentration-dependent shifts in unfolding transition midpoints. Treatment of the temperature dependence of affinity is, therefore, intrinsic to this method and is developed in this study. We used the interaction between maltose-binding protein (MBP) and a thermostable synthetic ankyrin repeat protein (Off7) as an experimental test case because their unfolding transitions overlap minimally. We found that MBP is significantly stabilized by Off7. High experimental throughput is enabled by sample parallelization, and the ability to extract quantitative binding information at a single partner concentration. In a single experiment, we were able to quantify the affinities of a series of alanine mutants, covering a wide range of affinities (∼ 100 nM to ∼ 100 μM). PMID:21674662

  9. Machine learning algorithms for predicting protein folding rates and stability of mutant proteins: comparison with statistical methods.

    PubMed

    Gromiha, M Michael; Huang, Liang-Tsung

    2011-09-01

    Machine learning algorithms have wide range of applications in bioinformatics and computational biology such as prediction of protein secondary structures, solvent accessibility, binding site residues in protein complexes, protein folding rates, stability of mutant proteins, and discrimination of proteins based on their structure and function. In this work, we focus on two aspects of predictions: (i) protein folding rates and (ii) stability of proteins upon mutations. We briefly introduce the concepts of protein folding rates and stability along with available databases, features for prediction methods and measures for prediction performance. Subsequently, the development of structure based parameters and their relationship with protein folding rates will be outlined. The structure based parameters are helpful to understand the physical basis for protein folding and stability. Further, basic principles of major machine learning techniques will be mentioned and their applications for predicting protein folding rates and stability of mutant proteins will be illustrated. The machine learning techniques could achieve the highest accuracy of predicting protein folding rates and stability. In essence, statistical methods and machine learning algorithms are complimenting each other for understanding and predicting protein folding rates and the stability of protein mutants. The available online resources on protein folding rates and stability will be listed. PMID:21787301

  10. 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.

  11. 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

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

    PubMed

    Wu, Yuanzhong; Kang, Tiebang

    2016-01-01

    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. PMID:27357860

  13. A useful model capable of predicting the clearance of cytochrome 3A4 (CYP3A4) substrates in humans: validity of CYP3A4 transgenic mice lacking their own Cyp3a enzymes.

    PubMed

    Mitsui, Tetsuya; Nemoto, Takayuki; Miyake, Taiji; Nagao, Shunsuke; Ogawa, Kotaro; Kato, Motohiro; Ishigai, Masaki; Yamada, Hideyuki

    2014-09-01

    The accurate prediction for the body clearance of a novel drug candidate by humans during the preclinical stage contributes to its successful development. To improve the predictability of human hepatic clearance, we focused on CYP3A4, which is involved in the metabolism of more than 50% of all currently marketed drugs. In this study, we investigated the validity of the in vivo model using transgenic mice carrying the human CYP3A4 gene and lacking their own Cyp3a genes (CYP3A4-Tg mice). The CYP3A4 activity toward its substrates in liver microsomes was similar in CYP3A4-Tg mice and humans. As for the clearance, six CYP3A4 substrates (alprazolam, felodipine, midazolam, nifedipine, nitrendipine, and quinidine) were given intravenously to CYP3A4-Tg mice, and their hepatic intrinsic clearance (CLint,h) was evaluated. A regression analysis of the data obtained indicated that the CLint,h values of six substrates in CYP3A4-Tg mice were highly correlated with those in humans (R(2) = 0.95). This correlation could be improved by correcting the CLint,h values by the relative contribution of artificially expressed CYP3A4 to the overall metabolism in the mice. From these findings, it is reasonable to expect that the CLint,h of a particular drug in humans is predictable by applying the CLint,h obtained in CYP3A4-Tg mice to a regression line prepared in advance. The variance of the CLint,h prediction by this method was evaluated and found to be within a range of 2-fold of the regression value. These results suggest that the CYP3A4-Tg mouse model has the potential to accurately predict the human hepatic clearance of CYP3A4 substrates. PMID:25005602

  14. 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.

  15. Pharmacokinetics of ruboxistaurin are significantly altered by rifampicin-mediated CYP3A4 induction

    PubMed Central

    Yeo, Kwee Poo; Lowe, Stephen L; Lim, Ming Tung; Voelker, James R; Burkey, Jennifer L; Wise, Stephen D

    2006-01-01

    Aims The aim of this study was to evaluate the effect of rifampicin co-administration on the pharmacokinetics of ruboxistaurin and its active metabolite, N-desmethyl ruboxistaurin and, in addition, to compare the changes in pharmacokinetics of ruboxistaurin and N-desmethyl ruboxistaurin with the urinary 6β-hydroxycortisol : cortisol ratio. Ruboxistaurin is a specific protein-kinase-C β inhibitor in clinical development for the treatment of diabetic microvascular complications. Methods This was a two-period, one-sequence study. Sixteen healthy male subjects completed both study periods. In period one, a single 64 mg oral dose of ruboxistaurin was administered. In period two, 600 mg rifampicin was administered daily for 9 days, during which another single 64 mg ruboxistaurin dose was administered on day 7. Blood samples were collected and assayed for ruboxistaurin and N-desmethyl ruboxistaurin. CYP3A4 induction was assessed by ratios of urinary 6β-hydroxycortisol : cortisol (6β-OHC : C) obtained via 24 h and morning-spot sampling techniques. Results Following repeated doses of rifampicin, both the mean Cmax and AUC(0,∞) of ruboxistaurin were significantly reduced by approximately 95% (P ≤ 0.001). For the metabolite, the mean Cmax decreased by 68% (P ≤ 0.001), and AUC(0,∞) decreased by 77% (P ≤ 0.001). The tmax values did not appear affected. The 6β-OHC : C ratios from both 24 h and morning spot methods increased significantly, consistent with CYP3A4 induction. Conclusions The effect of rifampicin co-administration on the exposure of ruboxistaurin is consistent with ruboxistaurin being a substrate of CYP3A4. Therefore, co-administration with known CYP3A4 inducing agents (rifampicin, carbamazepine, phenobarbital, etc.) may decrease the concentrations of ruboxistaurin and N-desmethyl-ruboxistaurin. In this study, 6β OHC : C ratios substantially underestimated the impact of rifampicin on ruboxistaurin. PMID:16433874

  16. 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.

  17. Electrostatic Interactions in the Denatured State Ensemble: Their Effect Upon Protein Folding and Protein Stability

    PubMed Central

    Sato, Satoshi; Horng, Jia-Cherng; Anil, Burcu

    2009-01-01

    It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol−1 to the stability of the DSE. PMID:17900519

  18. AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins.

    PubMed Central

    Shen, W F; Montgomery, J C; Rozenfeld, S; Moskow, J J; Lawrence, H J; Buchberg, A M; Largman, C

    1997-01-01

    Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets. PMID:9343407

  19. AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins.

    PubMed

    Shen, W F; Montgomery, J C; Rozenfeld, S; Moskow, J J; Lawrence, H J; Buchberg, A M; Largman, C

    1997-11-01

    Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets. PMID:9343407

  20. Elucidation of distinct ligand binding sites for cytochrome P450 3A4.

    PubMed

    Hosea, N A; Miller, G P; Guengerich, F P

    2000-05-23

    Cytochrome P450 (P450) 3A4 is the most abundant human P450 enzyme and has broad selectivity for substrates. The enzyme can show marked catalytic regioselectivity and unusual patterns of homotropic and heterotropic cooperativity, for which several models have been proposed. Spectral titration studies indicated one binding site for the drug indinavir (M(r) 614), a known substrate and inhibitor. Several C-terminal aminated peptides, including the model morphiceptin (YPFP-NH(2)), bind with spectral changes indicative of Fe-NH(2) bonding. The binding of the YPFP-NH(2) N-terminal amine and the influence of C-terminal modification on binding argue that the entire molecule (M(r) 521) fits within P450 3A4. YPFP-NH(2) was not oxidized by P450 3A4 but blocked binding of the substrates testosterone and midazolam, with K(i) values similar to the spectral binding constant (K(s)) for YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of several typical P450 substrates with K(i) values 10-fold greater than the K(s) for binding YPFP-NH(2) and its K(i) for inhibiting substrate binding. The n values for cooperativity of these oxidations were not altered by YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of midazolam at two different positions (1'- and 4-) with 20-fold different K(i) values. The differences in the K(i) values for blocking the binding to ferric P450 3A4 and the oxidation of several substrates may be attributed to weaker binding of YPFP-NH(2) to ferrous P450 3A4 than to the ferric form. The ferrous protein can be considered a distinct form of the enzyme in binding and catalysis because many substrates (but not YPFP-NH(2)) facilitate reduction of the ferric to ferrous enzyme. Our results with these peptides are considered in the context of several proposed models. A P450 3A4 model based on these peptide studies contains at least two and probably three distinct ligand sites, with testosterone and alpha-naphthoflavone occupying distinct sites. Midazolam appears to be able to

  1. Heat shock protein 90 stabilizes nucleolin to increase mRNA stability in mitosis.

    PubMed

    Wang, Shao-An; Li, Hao-Yi; Hsu, Tsung-I; Chen, Shu-Hui; Wu, Chin-Jen; Chang, Wen-Chang; Hung, Jan-Jong

    2011-12-23

    Most studies on heat shock protein 90 (Hsp90) have focused on the involvement of Hsp90 in the interphase, whereas the role of this protein in the nucleus during mitosis remains largely unclear. In this study, we found that the level of the acetylated form of Hsp90 decreased dramatically during mitosis, which indicates more chaperone activity during mitosis. We thus probed proteins that interacted with Hsp90 by liquid chromatography/mass spectrometry (LC/MS) and found that nucleolin was one of those interacting proteins during mitosis. The nucleolin level decreased upon geldanamycin treatment, and Hsp90 maintained the cyclin-dependent kinase 1 (CDK1) activity to phosphorylate nucleolin at Thr-641/707. Mutation of Thr-641/707 resulted in the destabilization of nucleolin in mitosis. We globally screened the level of mitotic mRNAs and found that 229 mRNAs decreased during mitosis in the presence of geldanamycin. Furthermore, a bioinformatics tool and an RNA immunoprecipitation assay found that 16 mRNAs, including cadherin and Bcl-xl, were stabilized through the recruitment of nucleolin to the 3'-untranslated regions (3'-UTRs) of those genes. Overall, strong correlations exist between the up-regulation of Hsp90, nucleolin, and the mRNAs related to tumorigenesis of the lung. Our findings thus indicate that nucleolin stabilized by Hsp90 contributes to the lung tumorigenesis by increasing the level of many tumor-related mRNAs during mitosis. PMID:21998300

  2. What makes a protein a protein? Hydrophobic core designs that specify stability and structural properties.

    PubMed Central

    Munson, M.; Balasubramanian, S.; Fleming, K. G.; Nagi, A. D.; O'Brien, R.; Sturtevant, J. M.; Regan, L.

    1996-01-01

    Here we describe how the systematic redesign of a protein's hydrophobic core alters its structure and stability. We have repacked the hydrophobic core of the four-helix-bundle protein, Rop, with altered packing patterns and various side chain shapes and sizes. Several designs reproduce the structure and native-like properties of the wild-type, while increasing the thermal stability. Other designs, either with similar sizes but different shapes, or with decreased sizes of the packing residues, destabilize the protein. Finally, overpacking the core with the larger side chains causes a loss of native-like structure. These results allow us to further define the roles of tight residue packing and the burial of hydrophobic surface area in the construction of native-like proteins. PMID:8844848

  3. 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. PMID:25172695

  4. 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. PMID:27353330

  5. Molecular docking of chemotherapeutic agents to CYP3A4 in non-small cell lung cancer.

    PubMed

    Subhani, Syed; Jamil, Kaiser

    2015-07-01

    CYP3A4, a "heme" containing isoform, abundantly found in the liver, gastro-intestinal tract, lungs and renal cells, also known as drug metabolising enzyme (DME) may be responsible for the disease progression in cancers such as lung cancer. Hence, we have targeted this protein for improving drug selection and in preventing adverse reactions. The aim of this study was to examine chemotherapeutic drug binding to CYP3A4 and the interactions therein. We have used Schrödinger suite 2014, to perform molecular docking of human CYP3A4, by Induced Fit Docking using gemcitabine, cisplatin, carboplatin, docetaxel and paclitaxel drugs. We evaluated drug-binding affinities using Prime/MMGBSA and using these scores we compared the affinities of combination therapies against CYP3A4. Analysis of the docking results showed gemcitabine>carboplatin>cisplatin as the order of binding affinities, with gemcitabine having the best docking score. Interestingly, docetaxel and paclitaxel did not bind to CYP3A4*1B. The combination drug-binding affinity analysis showed gemcitabine+carboplatin to have the best docking score and hence, efficacy. Our investigation has identified the residue Arg 105 to be more frequently involved in drug binding to CYP3A4. Our results suggest that gemcitabine or combination of gemcitabine+carboplatin could serve as an excellent therapy against CYP3A4 in NSCLC patients. Thus, our study depicts binding of chemotherapeutic drugs to CYP3A4 and has identified the residues that may be targeted for therapy in NSCLC patients. PMID:26211584

  6. 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.

  7. Stability and folding of the tumour suppressor protein p16.

    PubMed

    Tang, K S; Guralnick, B J; Wang, W K; Fersht, A R; Itzhaki, L S

    1999-01-29

    The tumour suppressor p16 is a member of the INK4 family of inhibi tors of the cyclin D-dependent kinases, CDK4 and CDK6, that are involved in the key growth control pathway of the eukaryotic cell cycle. The 156 amino acid residue protein is composed of four ankyrin repeats (a helix-turn-helix motif) that stack linearly as two four-helix bundles resulting in a non-globular, elongated molecule. The thermodynamic and kinetic properties of the folding of p16 are unusual. The protein has a very low free energy of unfolding, Delta GH-2O/D-N, of 3.1 kcal mol-1 at 25 degreesC. The rate-determining transition state of folding/unfolding is very compact (89% as compact as the native state). The other unusual feature is the very rapid rate of unfolding in the absence of denaturant of 0.8 s-1 at 25 degreesC. Thus, p16 has both thermodynamic and kinetic instability. These features may be essential for the regulatory function of the INK4 proteins and of other ankyrin-repeat-containing proteins that mediate a wide range of protein-protein interactions. The mechanisms of inactivation of p16 by eight cancer-associated mutations were dissected using a systematic method designed to probe the integrity of the secondary structure and the global fold. The structure and folding of p16 appear to be highly vulnerable to single point mutations, probably as a result of the protein's low stability. This vulnerability provides one explanation for the striking frequency of p16 mutations in tumours and in immortalised cell lines. PMID:9917418

  8. Discovery of Manassantin A Protein Targets Using Large-Scale Protein Folding and Stability Measurements.

    PubMed

    Geer Wallace, M Ariel; Kwon, Do-Yeon; Weitzel, Douglas H; Lee, Chen-Ting; Stephenson, Tesia N; Chi, Jen-Tsan; Mook, Robert A; Dewhirst, Mark W; Hong, Jiyong; Fitzgerald, Michael C

    2016-08-01

    Manassantin A is a natural product that has been shown to have anticancer activity in cell-based assays, but has a largely unknown mode-of-action. Described here is the use of two different energetics-based approaches to identify protein targets of manassantin A. Using the stability of proteins from rates of oxidation technique with an isobaric mass tagging strategy (iTRAQ-SPROX) and the pulse proteolysis technique with a stable isotope labeling with amino acids in cell culture strategy (SILAC-PP), over 1000 proteins in a MDA-MB-231 cell lysate grown under hypoxic conditions were assayed for manassantin A interactions (both direct and indirect). A total of 28 protein hits were identified with manassantin A-induced thermodynamic stability changes. Two of the protein hits (filamin A and elongation factor 1α) were identified using both experimental approaches. The remaining 26 hit proteins were only assayed in either the iTRAQ-SPROX or the SILAC-PP experiment. The 28 potential protein targets of manassantin A identified here provide new experimental avenues along which to explore the molecular basis of manassantin A's mode of action. The current work also represents the first application iTRAQ-SPROX and SILAC-PP to the large-scale analysis of protein-ligand binding interactions involving a potential anticancer drug with an unknown mode-of-action. PMID:27322910

  9. Regulation of the protein stability of EMT transcription factors

    PubMed Central

    Díaz, VM; Viñas-Castells, R; García de Herreros, A

    2014-01-01

    The epithelial to mesenchymal transition (EMT) consists of a rapid change of cell phenotype, characterized by the loss of epithelial characteristics and the acquisition of a more invasive phenotype. Transcription factors regulating EMT (Snail, Twist and Zeb) are extremely labile proteins, rapidly degraded by the proteasome system. In this review we analyze the current mechanisms controlling degradation of EMT transcription factors, focusing on the role of new E3 ubiquitin-ligases involved in EMT. We also summarize the regulation of the stability of these EMT transcription factors, specially observed in different stress conditions, such as hypoxia, chemotherapeutic drugs, oxidative stress or γ-irradiation. PMID:25482633

  10. Protein accumulation and rumen stability of wheat γ-gliadin fusion proteins in tobacco and alfalfa.

    PubMed

    Sun, Xiaodong; Chi-Ham, Cecilia L; Cohen-Davidyan, Tamar; DeBen, Christopher; Getachow, Girma; DePeters, Edward; Putnam, Daniel; Bennett, Alan

    2015-09-01

    The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine-rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine-rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ-gliadin-δ-zein and γ-δ-zein, as well as δ-zein co-expressed with β-zein, all formed protein bodies. However, the γ-gliadin-δ-zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ-gliadin-δ-zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ-gliadin-δ-zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ-gliadin-GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ-gliadin-δ-zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ-gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants. PMID:25659597

  11. Stabilization of collagen through bioconversion: An insight in protein-protein interaction.

    PubMed

    Usharani, Nagarajan; Jayakumar, Gladstone Christopher; Kanth, Swarna Vinodh; Rao, Jonnalagadda Raghava

    2014-08-01

    Collagen is a natural protein, which is used as a vital biomaterial in tissue engineering. The major concern about native collagen is lack of its thermal stability and weak resistance to proteolytic degradation. In this scenario, the crosslinking compounds used for stabilization of collagen are mostly of chemical nature and exhibit toxicity. The enzyme mediated crosslinking of collagen provides a novel alternative, nontoxic method for stabilization. In this study, aldehyde forming enzyme (AFE) is used in the bioconversion of hydroxylmethyl groups of collagen to formyl groups that results in the formation of peptidyl aldehyde. The resulted peptidyl aldehyde interacts with bipolar ions of basic amino acid residues of collagen. Further interaction leads to the formation of conjugated double bonds (aldol condensation involving the aldehyde group of peptidyl aldehyde) within the collagen. The enzyme modified collagen matrices have shown an increase in the denaturation temperature, when compared with native collagen. Enzyme modified collagen membranes exhibit resistance toward collagenolytic activity. Moreover, they exhibited a nontoxic nature. The catalytic activity of AFE on collagen as a substrate establishes an efficient modification, which enhances the structural stability of collagen. This finds new avenues in the context of protein-protein stabilization and discovers paramount application in tissue engineering. PMID:25098180

  12. Gene stability in mammalian cells and protein consistency.

    PubMed

    Berthold, W

    1994-01-01

    The safety of a patient who is the recipient of protein drugs has to be assured. A "wrong" protein is thought to represent a great risk. The philosophy of testing strategies related to gene stability with product safety will be discussed in the light of experimental data available today. Although all mammalian cell lines used in the production of biologicals including recombinant DNA-derived lines have been produced from individual clones (functional monoclonality) they have been found to be heterogenous with regard to the genomic content (number of chromosomes, characteristics of identifiable chromosomes and position and number of integrated recombinant sequences). The verification of the presence of correct gene in a production cell line constitutes a well accepted and useful test, especially if derived by "population sequencing". A batch not related repeated confirmation of this fact cannot lead to any additional assurance for the correctness of all proteins constituting a given product beyond the level provided by cheminal testing. In contrast to this obvious and unavoidable heterogeneity in cellular genomes, the coding regions of genes have not been shown to change. Evidence is available to demonstrate the consistency of protein products originating from recombinant (and hybridoma) cell lines, e.g. more than 500,000 patients have received and tolerated rtPA well. PMID:7883100

  13. Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

    PubMed

    Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

    2013-12-13

    Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

  14. Structural Assessment of the Effects of Amino Acid Substitutions on Protein Stability and Protein-Protein Interaction

    PubMed Central

    Teng, Shaolei; Wang, Liangjiang; Srivastava, Anand K.; Schwartz, Charles E.; Alexov, Emil

    2012-01-01

    A structure-based approach is described for predicting the effects of amino acid substitutions on protein function. Structures were predicted using a homology modelling method. Folding and binding energy differences between wild-type and mutant structures were computed to quantitatively assess the effects of amino acid substitutions on protein stability and protein–protein interaction, respectively. We demonstrated that pathogenic mutations at the interaction interface could affect binding energy and destabilise protein complex, whereas mutations at the non-interface might reduce folding energy and destabilise monomer structure. The results suggest that the structure-based analysis can provide useful information for understanding the molecular mechanisms of diseases. PMID:21297231

  15. 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.

  16. Enhancing protein stability by adsorption onto raftlike lipid domains.

    PubMed

    Litt, Jeffrey; Padala, Chakradhar; Asuri, Prashanth; Vutukuru, Srinavya; Athmakuri, Krishna; Kumar, Sanat; Dordick, Jonathan; Kane, Ravi S

    2009-05-27

    We demonstrate that the stability of adsorbed proteins can be enhanced by controlling the heterogeneity of the surfaceby creating raftlike domains in a soft liposomal membrane. Recent work has shown that enzymes adsorbed onto highly curved nanoscale supports can be more stable than those adsorbed on flat surfaces with nominally the same chemical structure. This effect has been attributed to a decrease in lateral interenzyme interactions on a curved surface. Exploiting this idea, we asked if adsorbing enzymes onto "patchy" surfaces composed of adsorbing and nonadsorbing regions can be used to reduce lateral interactions even on relatively flat surfaces. We demonstrate that creating domains on which an enzyme can adsorb enhances the stability of that enzyme under denaturing conditions. Furthermore, we demonstrate that the size of these domains has a considerable effect on the degree of stability imparted by adsorption. Such biomimetic raft-inspired systems may find use in applications ranging from biorecognition to the design of novel strategies for the separation of biomolecules and controlling the interaction of multicomponent membrane-bound enzymes. PMID:19385631

  17. Cementing proteins provide extra mechanical stabilization to viral cages.

    PubMed

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

    2014-01-01

    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. PMID:25072871

  18. 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

  19. Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy.

    PubMed

    Davydov, Dmitri R; Yang, Zhongyu; Davydova, Nadezhda; Halpert, James R; Hubbell, Wayne L

    2016-04-12

    We used high hydrostatic pressure as a tool for exploring the conformational landscape of human cytochrome P450 3A4 (CYP3A4) by electron paramagnetic resonance and fluorescence spectroscopy. Site-directed incorporation of a luminescence resonance energy transfer donor-acceptor pair allowed us to identify a pressure-dependent equilibrium between two states of the enzyme, where an increase in pressure increased the spatial separation between the two distantly located fluorophores. This transition is characterized by volume change (ΔV°) and P1/2 values of -36.8 ± 5.0 mL/mol and 1.45 ± 0.33 kbar, respectively, which corresponds to a Keq° of 0.13 ± 0.06, so that only 15% of the enzyme adopts the pressure-promoted conformation at ambient pressure. This pressure-promoted displacement of the equilibrium is eliminated by the addition of testosterone, an allosteric activator. Using site-directed spin labeling, we demonstrated that the pressure- and testosterone-sensitive transition is also revealed by pressure-induced changes in the electron paramagnetic resonance spectra of a nitroxide side chain placed at position 85 or 409 of the enzyme. Furthermore, we observed a pressure-induced displacement of the emission maxima of a solvatochromic fluorophore (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin) placed at the same positions, which suggests a relocation to a more polar environment. Taken together, the results reveal an effector-dependent conformational equilibrium between open and closed states of CYP3A4 that involves a pronounced change at the interface between the region of α-helices A/A' and the meander loop of the enzyme, where residues 85 and 409 are located. Our study demonstrates the high potential of pressure-perturbation strategies for studying protein conformational landscapes. PMID:27074675

  20. Significantly reduced cytochrome P450 3A4 expression and activity in liver from humans with diabetes mellitus

    PubMed Central

    Dostalek, Miroslav; Court, Michael H; Yan, Bingfang; Akhlaghi, Fatemeh

    2011-01-01

    BACKGROUND AND PURPOSE Patients with diabetes mellitus require pharmacotherapy with numerous medications. However, the effect of diabetes on drug biotransformation is not well understood. Our goal was to investigate the effect of diabetes on liver cytochrome P450 3As, the most abundant phase I drug-metabolizing enzymes in humans. EXPERIMENTAL APPROACH Human liver microsomal fractions (HLMs) were prepared from diabetic (n = 12) and demographically matched nondiabetic (n = 12) donors, genotyped for CYP3A4*1B and CYP3A5*3 polymorphisms. Cytochrome P450 3A4, 3A5 and 2E1 mRNA expression, protein level and enzymatic activity were compared between the two groups. KEY RESULTS Midazolam 1′- or 4-hydroxylation and testosterone 6β-hydroxylation, catalyzed by P450 3A, were markedly reduced in diabetic HLMs, irrespective of genotype. Significantly lower P450 3A4 protein and comparable mRNA levels were observed in diabetic HLMs. In contrast, neither P450 3A5 protein level nor mRNA expression differed significantly between the two groups. Concurrently, we have observed increased P450 2E1 protein level and higher chlorzoxazone 6-hydroxylation activity in diabetic HLMs. CONCLUSIONS AND IMPLICATIONS These studies indicate that diabetes is associated with a significant decrease in hepatic P450 3A4 enzymatic activity and protein level. This finding could be clinically relevant for diabetic patients who have additional comorbidities and are receiving multiple medications. To further characterize the effect of diabetes on P450 3A4 activity, a well-controlled clinical study in diabetic patients is warranted. PMID:21323901

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

    PubMed

    Brogan, Alex P S; Hallett, Jason P

    2016-04-01

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

  2. Excluded volume effects upon protein stability in covalently crosslinked proteins with variable linker lengths†

    PubMed Central

    Kim, Yun Ho; Stites, Wesley E.

    2008-01-01

    To explore the effects of molecular crowding and excluded volume upon protein stability a series of crosslinking reagents have been used with nine different single cysteine mutants of staphylococcal nuclease to make covalently linked dimers. These crosslinkers ranged in length from 10.5 Å to 21.3 Å, compelling separations which would normally be found only in the most concentrated protein solutions. The stabilities of the dimeric proteins and monomeric controls were determined by guanidine hydrochloride and thermal denaturation. Dimers with short linkers tend to show pronounced three state denaturation behavior, as opposed to the two state behavior of the monomeric controls. Increasing linker length leads to less pronounced three state behavior. The three state behavior is interpreted in a three state model where crosslinked native protein dimer, N-N, interconverts in a two state transition with a dimer where one protein subunit is denatured, N-D. The remaining native protein in turn can denature in another two state transition to a state, D-D, where both tethered proteins are denatured. Three state behavior is best explained by excluded volume effects in the denatured state. For many dimers, linkers longer than 17 Å removed most three state character. This sets a limit on the flexibility and size of the denatured state. Notably, in contradiction to theoretical predictions, these crosslinked dimers were not stabilized. The failure of these predictions is possibly due to neglect of the alteration in hydrophobic exposure that accompanies any significant reduction in the conformational space of the denatured state. PMID:18656955

  3. Characterizing the Membrane-Bound State of Cytochrome P450 3A4: Structure, Depth of Insertion, and Orientation

    PubMed Central

    2013-01-01

    Cytochrome P450 3A4 (CYP3A4) is the most abundant membrane-associated isoform of the P450 family in humans and is responsible for biotransformation of more than 50% of drugs metabolized in the body. Despite the large number of crystallographic structures available for CYP3A4, no structural information for its membrane-bound state at an atomic level is available. In order to characterize binding, depth of insertion, membrane orientation, and lipid interactions of CYP3A4, we have employed a combined experimental and simulation approach in this study. Taking advantage of a novel membrane representation, highly mobile membrane mimetic (HMMM), with enhanced lipid mobility and dynamics, we have been able to capture spontaneous binding and insertion of the globular domain of the enzyme into the membrane in multiple independent, unbiased simulations. Despite different initial orientations and positions of the protein in solution, all the simulations converged into the same membrane-bound configuration with regard to both the depth of membrane insertion and the orientation of the enzyme on the surface of the membrane. In tandem, linear dichroism measurements performed on CYP3A4 bound to Nanodisc membranes were used to characterize the orientation of the enzyme in its membrane-bound form experimentally. The heme tilt angles measured experimentally are in close agreement with those calculated for the membrane-bound structures resulted from the simulations, thereby verifying the validity of the developed model. Membrane binding of the globular domain in CYP3A4, which appears to be independent of the presence of the transmembrane helix of the full-length enzyme, significantly reshapes the protein at the membrane interface, causing conformational changes relevant to access tunnels leading to the active site of the enzyme. PMID:23697766

  4. 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.

  5. 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. PMID:26476082

  6. 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…

  7. Phosphorylation in protein-protein binding: effect on stability and function

    PubMed Central

    Nishi, Hafumi; Hashimoto, Kosuke; Panchenko, Anna R.

    2011-01-01

    Summary Post-translational modifications offer a dynamic way to regulate protein activity, subcellular localization and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites have a tendency to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. The analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and binding hotspots have a tendency to be phosphorylated in heterooligomers. Although majority of phosphosites do not show significant estimated stability differences upon attaching the phosphate groups, for about one third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are not only more likely to be evolutionary conserved than surface residues but even more so than other interfacial residues. PMID:22153503

  8. 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

  9. 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

  10. Osmolytes stabilize ribonuclease S by stabilizing its fragments S protein and S peptide to compact folding-competent states.

    PubMed

    Ratnaparkhi, G S; Varadarajan, R

    2001-08-01

    Osmolytes stabilize proteins to thermal and chemical denaturation. We have studied the effects of the osmolytes sarcosine, betaine, trimethylamine-N-oxide, and taurine on the structure and stability of the protein.peptide complex RNase S using x-ray crystallography and titration calorimetry, respectively. The largest degree of stabilization is achieved with 6 m sarcosine, which increases the denaturation temperatures of RNase S and S pro by 24.6 and 17.4 degrees C, respectively, at pH 5 and protects both proteins against tryptic cleavage. Four crystal structures of RNase S in the presence of different osmolytes do not offer any evidence for osmolyte binding to the folded state of the protein or any perturbation in the water structure surrounding the protein. The degree of stabilization in 6 m sarcosine increases with temperature, ranging from -0.52 kcal mol(-1) at 20 degrees C to -5.4 kcal mol(-1) at 60 degrees C. The data support the thesis that osmolytes that stabilize proteins, do so by perturbing unfolded states, which change conformation to a compact, folding competent state in the presence of osmolyte. The increased stabilization thus results from a decrease in conformational entropy of the unfolded state. PMID:11373282

  11. Unique CYP3A4 genetic variant in Brazilian tuberculosis patients with/without HIV.

    PubMed

    Jeovanio-Silva, André L; Monteiro, Thaís P; El-Jaick, Kênia B; do Brasil, Pedro E A A; Rolla, Valéria C; de Castro, Liane

    2012-01-01

    CYP3A4 is involved in tuberculosis (TB) and human immunodeficiency virus (HIV) drug metabolism. Transcriptional activation by rifampicin involves the CYP3A4 gene 5'-upstream region. Consequently, variation may interfere with transcription and enzymatic activity and even drug response. However, genetic polymorphisms and distribution of CYP3A4 allelic frequencies in individuals from Rio de Janeiro remain unknown. The aim of this study was to conduct research into sequencing the CYP3A4 5'-upstream region in Brazilian patients with and without HIV. This follow-up study involved 106 individuals undergoing treatment for TB and/or HIV. The CYP3A4 5'-upstream region was analyzed using PCR, sequencing and clinical data. Male patients revealed a higher HIV frequency (p=0.021). The TB forms observed were pulmonary (48.1%), extrapulmonary (22.64%) and disseminated (27.36%). Lymph node form was the most frequent (70.83%) extrapulmonary form of TB. The only single nucleotide polymorphism detected in the population was c.-392A>G. Genotypes observed were CYP3A4*1A/CYP3A4*1A (45.3%), CYP3A4*1A/CYP3A4*1B (40.6%) and CYP3A4*1B/CYP3A4*1B (14.2%), revealing a different distribution with extrapulmonary TB cases (17.6% CYP3A4*1A/CYP3A4*1B and 23.5% CYP3A4*1B/CYP3A4*1B). The CYP3A4*1A allele was found to be associated with tobacco use. The CYP3A4*1B mutant allele occurred in 34% of patients. This study revealed that the CYP3A4 5'-upstream regulatory region was highly conserved with the exception of the -392 position. Genotype association with tobacco suggests that CYP3A4 may participate in tobacco metabolism. Genotype distribution inversion in extrapulmonary TB cases suggests that CYP3A4 may be involved in TB prognosis. PMID:21964586

  12. Phenotype-genotype variability in the human CYP3A locus as assessed by the probe drug quinine and analyses of variant CYP3A4 alleles

    SciTech Connect

    Rodriguez-Antona, Cristina . E-mail: cristina.rodriguez-antona@cnio.es; Sayi, Jane G.; Gustafsson, Lars L.; Bertilsson, Leif; Ingelman-Sundberg, Magnus

    2005-12-09

    The human cytochrome P450 3A (CYP3A) enzymes, which metabolize 50% of currently used therapeutic drugs, exhibit great interindividual differences in activity that have a major impact on drug treatment outcome, but hitherto no genetic background importantly contributing to this variation has been identified. In this study we show that CYP3A4 mRNA and hnRNA contents with a few exceptions vary in parallel in human liver, suggesting that mechanisms affecting CYP3A4 transcription, such as promoter polymorphisms, are relevant for interindividual differences in CYP3A4 expression. Tanzanian (n = 143) healthy volunteers were phenotyped using quinine as a CYP3A probe and the results were used for association studies with CYP3A4 genotypes. Carriers of CYP3A4*1B had a significantly lower activity than those with CYP3A4*1 whereas no differences were seen for five other SNPs investigated. Nuclear proteins from the B16A2 hepatoma cells were found to bind with less affinity to the CYP3A4*1B element around -392 bp as compared to CYP3A4*1. The data indicate the existence of a genetic CYP3A4 polymorphism with functional importance for interindividual differences in enzyme expression.

  13. 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

  14. 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

  15. Effect of cosolvent on protein stability: a theoretical investigation.

    PubMed

    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°/∂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

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

    NASA Astrophysics Data System (ADS)

    Chalikian, Tigran V.

    2014-12-01

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

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

    SciTech Connect

    Chalikian, Tigran V.

    2014-12-14

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

  18. Stability and Immunogenicity of Hypoallergenic Peanut Protein-Polyphenol Complexes during In Vitro Pepsin Diges

    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...

  19. 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...

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

    PubMed Central

    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.

    2011-01-01

    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. Spy is unlike the structure 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. PMID:21317898

  1. Salting the Charged Surface: pH and Salt Dependence of Protein G B1 Stability

    PubMed Central

    Lindman, Stina; Xue, Wei-Feng; Szczepankiewicz, Olga; Bauer, Mikael C.; Nilsson, Hanna; Linse, Sara

    2006-01-01

    This study shows significant effects of protein surface charges on stability and these effects are not eliminated by salt screening. The stability for a variant of protein G B1 domain was studied in the pH-range of 1.5–11 at low, 0.15 M, and 2 M salt. The variant has three mutations, T2Q, N8D, and N37D, to guarantee an intact covalent chain at all pH values. The stability of the protein shows distinct pH dependence with the highest stability close to the isoelectric point. The stability is pH-dependent at all three NaCl concentrations, indicating that interactions involving charged residues are important at all three conditions. We find that 2 M salt stabilizes the protein at low pH (protein net charge is +6 and total number of charges is 6) but not at high pH (net charge is ≤−6 and total number of charges is ≥18). Furthermore, 0.15 M salt slightly decreases the stability of the protein over the pH range. The results show that a net charge of the protein is destabilizing and indicate that proteins contain charges for reasons other than improved stability. Salt seems to reduce the electrostatic contributions to stability under conditions with few total charges, but cannot eliminate electrostatic effects in highly charged systems. PMID:16443658

  2. CYP3A4-based drug-drug interaction: CYP3A4 substrates' pharmacokinetic properties and ketoconazole dose regimen effect.

    PubMed

    Boulenc, Xavier; Nicolas, Olivier; Hermabessière, Stéphanie; Zobouyan, Isabelle; Martin, Valérie; Donazzolo, Yves; Ollier, Céline

    2016-02-01

    The aim of the study was to assess the magnitude of the CYP3A4 inhibitory effect of 2 dosing regimens of ketoconazole and the influence of the pharmacokinetic properties of the CYP3A4 substrate on the extent of the substrate exposure increase. For this purpose, a clinical study was conducted and PBPK modeling simulations were performed. A crossover study was conducted in healthy subjects. The study was designed to compare the effects of different regimens of reversible CYP3A4 inhibitors, i.e., ketoconazole 400 mg OD, ketoconazole 200 mg BID, on two CYP3A4 substrates, alprazolam and midazolam, reflecting different pharmacokinetic properties in terms of first-pass effect and elimination. In parallel, time-based simulations were performed using the Simcyp population-based Simulator to address the usefulness of modeling to assess interaction clinical study design with CYP3A4 substrates. Comparison of the OD versus BID regimens for ketoconazole showed an opposite trend for the 2 substrates: BID (200 mg) dosing regimen provided the maximal clearance inhibition for alprazolam, while it was OD (400 mg) dosing regimen for midazolam. However, these effects are moderate despite the well-known pharmacokinetic differences between these substrates, suggesting that these differences are not enough. In the other way round, these investigations show how two CYP3A4 substrates can be different without leading to a major impact of the ketoconazole dosing regimen. The clinical findings are consistent with the Simcyp predictions, in particular the opposite trend observed with midazolam and alprazolam and the ketoconazole dosing regimen. These clinical investigations showed the influence of the CYP3A4 substrates' pharmacokinetic properties and the relevance of ketoconazole dose regimen on the magnitude of the interaction ratios. In addition, PBPK Simcyp simulations demonstrated how they can be used to help clinical study design assessment to capture the maximum effect. PMID:25374256

  3. 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. PMID:23250807

  4. 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

  5. [3a,4]-Dihydropyrazolo[1,5a]pyrimidines: Novel, Potent, and Selective Phosphatidylinositol-3-kinase β Inhibitors.

    PubMed

    Yu, Hongyi; Moore, Michael L; Erhard, Karl; Hardwicke, Mary Ann; Lin, Hong; Luengo, Juan I; McSurdy-Freed, Jeanelle; Plant, Ramona; Qu, Junya; Raha, Kaushik; Rominger, Cynthia M; Schaber, Michael D; Spengler, Michael D; Rivero, Ralph A

    2013-02-14

    A series of novel [3a,4]dihydropyrazolo[1,5a]pyrimidines were identified, which were highly potent and selective inhibitors of PI3Kβ. The template afforded the opportunity to develop novel SAR for both the hinge-binding (R3) and back-pocket (R4) substitutents. While cellular potency was relatively modest due to high protein binding, the series displayed low clearance in rat, mouse, and monkey. PMID:24900655

  6. [3a,4]-Dihydropyrazolo[1,5a]pyrimidines: Novel, Potent, and Selective Phosphatidylinositol-3-kinase β Inhibitors

    PubMed Central

    2013-01-01

    A series of novel [3a,4]dihydropyrazolo[1,5a]pyrimidines were identified, which were highly potent and selective inhibitors of PI3Kβ. The template afforded the opportunity to develop novel SAR for both the hinge-binding (R3) and back-pocket (R4) substitutents. While cellular potency was relatively modest due to high protein binding, the series displayed low clearance in rat, mouse, and monkey. PMID:24900655

  7. Another Role of Proline: Stabilization Interactions in Proteins and Protein Complexes Concerning Proline and Tryptophane

    SciTech Connect

    Biedermannova, Lada; Riley, Kevin E.; Berka, Karel; Hobza, Pavel; Vondrasek, Jiri

    2008-09-11

    Proline–tryptophan complexes derived from experimental structures are investigated by quantum chemical procedures known to properly describe the London dispersion energy. We study two geometrical arrangements: the “L-shaped”, stabilized by an H-bond, and the “stacked-like”, where the two residues are in parallel orientation without any H-bond. Interestingly, the interaction energies in both cases are comparable and very large (~7 kcal mol⁻¹). The strength of stabilization in the stacked arrangement is rather surprising considering the fact that only one partner has an aromatic character. The interaction energy decomposition using the SAPT method further demonstrates the very important role of dispersion energy in such arrangement. To elucidate the structural features responsible for this unexpectedly large stabilization we examined the role of the nitrogen heteroatom and the importance of the cyclicity of the proline residue. We show that the electrostatic interaction due to the presence of the dipole, caused by the nitrogen heteroatom, contributes largely to the strength of the interaction. Nevertheless, the cyclic arrangement of proline, which allows for the largest amount of dispersive contact with the aromatic partner, also has a notable-effect. Geometry optimizations carried out for the “stackedlike” complexes show that the arrangements derived from protein structure are close to their gas phase optimum geometry, suggesting that the environment has only a minor effect on the geometry of the interaction. We conclude that the strength of proline non-covalent interactions, combined with this residue’s rigidity, might be the explanation for its prominent role in protein stabilization and recognition processes.

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

    PubMed

    Planchais, Séverine; Camborde, Laurent; Jupin, Isabelle

    2016-01-01

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

  9. In vitro inhibition of CYP3A4 by herbal remedies frequently used by cancer patients.

    PubMed

    Engdal, Silje; Nilsen, Odd Georg

    2009-07-01

    The herbal remedies Natto K2, Agaricus, mistletoe, noni juice, green tea and garlic, frequently used by cancer patients, were investigated for their in vitro inhibition potential of cytochrome P-450 3A4 (CYP3A4) metabolism. To our knowledge, only garlic and green tea had available data on the possible inhibition of CYP3A4 metabolism. Metabolic studies were performed with human c-DNA baculovirus expressed CYP3A4. Testosterone was used as a substrate and ketoconazole as a positive quantitative inhibition control. The formation of 6-beta-OH-testosterone was quantified by a validated HPLC methodology. Green tea was the most potent inhibitor of CYP3A4 metabolism (IC(50): 73 microg/mL), followed by Agaricus, mistletoe and noni juice (1324, 3594, >10 000 microg/mL, respectively). All IC(50) values were high compared with those determined for crude extracts of other herbal remedies. The IC(50)/IC(25) ratios for the inhibiting herbal remedies ranged from 2.15 to 2.67, indicating similar inhibition profiles of the herbal inhibitors of CYP3A4. Garlic and Natto K2 were classified as non-inhibitors. Although Agaricus, noni juice, mistletoe and green tea inhibited CYP3A4 metabolism in vitro, clinically relevant systemic or intestinal interactions with CYP3A4 were considered unlikely, except for a probable inhibition of intestinal CYP3A4 by the green tea product. PMID:19170155

  10. 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

  11. 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. PMID:25880701

  12. Ultra-High Pressure Homogenization improves oxidative stability and interfacial properties of soy protein isolate-stabilized emulsions.

    PubMed

    Fernandez-Avila, C; Trujillo, A J

    2016-10-15

    Ultra-High Pressure Homogenization (100-300MPa) has great potential for technological, microbiological and nutritional aspects of fluid processing. Its effect on the oxidative stability and interfacial properties of oil-in-water emulsions prepared with 4% (w/v) of soy protein isolate and soybean oil (10 and 20%, v/v) were studied and compared to emulsions treated by conventional homogenization (15MPa). Emulsions were characterized by particle size, emulsifying activity index, surface protein concentration at the interface and by transmission electron microscopy. Primary and secondary lipid oxidation products were evaluated in emulsions upon storage. Emulsions with 20% oil treated at 100 and 200MPa exhibited the most oxidative stability due to higher amount of oil and protein surface load at the interface. This manuscript addresses the improvement in oxidative stability in emulsions treated by UHPH when compared to conventional emulsions. PMID:27173541

  13. Denatured state aggregation parameters derived from concentration dependence of protein stability.

    PubMed

    Schön, Arne; Clarkson, Benjamin R; Siles, Rogelio; Ross, Patrick; Brown, Richard K; Freire, Ernesto

    2015-11-01

    Protein aggregation is a major issue affecting the long-term stability of protein preparations. Proteins exist in equilibrium between the native and denatured or partially denatured conformations. Often denatured or partially denatured conformations are prone to aggregate because they expose to solvent the hydrophobic core of the protein. The aggregation of denatured protein gradually shifts the protein equilibrium toward increasing amounts of denatured and ultimately aggregated protein. Recognizing and quantitating the presence of denatured protein and its aggregation at the earliest possible time will bring enormous benefits to the identification and selection of optimal solvent conditions or the engineering of proteins with the best stability/aggregation profile. In this article, a new approach that allows simultaneous determination of structural stability and the amount of denatured and aggregated protein is presented. This approach is based on the analysis of the concentration dependence of the Gibbs energy (ΔG) of protein stability. It is shown that three important quantities can be evaluated simultaneously: (i) the population of denatured protein, (ii) the population of aggregated protein, and (iii) the fraction of denatured protein that is aggregated. PMID:26239214

  14. Coordination contributions to protein stability in metal-substituted carbonic anhydrase.

    PubMed

    Lisi, George P; Hughes, Russell P; Wilcox, Dean E

    2016-09-01

    Contributions of the active site metal to the stability of carbonic anhydrase (CA) were quantified by differential scanning calorimetry and complementary unfolding measurements of CA substituted with Co(2+), Cd(2+), Cu(2+), Ni(2+) and Mn(2+). The metal ions stabilize the protein to different extent, with the highest stability provided by the native Zn(2+). This additional stability does not correlate with the enthalpy of the three metal-imidazole (His) bonds at the active site or other properties of the metal ions (charge density, hydration enthalpy). However, DFT calculations reveal an energetic penalty associated with metal coordination at the active site, and the magnitude of this penalty correlates inversely with metal contributions to the stability of the protein. While the affinity of CA for metal ions generally reflects the Irving-Williams series, the additional thermal stability provided by metal ions is modulated by the rigid His3 coordination that is imposed at the protein site. PMID:27350155

  15. 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.

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

    PubMed Central

    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

  17. Measuring the interaction of urea and protein stabilizing osmolytes with the nonpolar surface of hydroxypropyl cellulose†

    PubMed Central

    Stanley, Christopher; Rau, Donald C.

    2008-01-01

    The interaction of urea and several naturally occurring protein stabilizing osmolytes, glycerol, sorbitol, glycine betaine, trimethylamine oxide (TMAO), and proline, with condensed arrays of a hydrophobically modified polysaccharide, hydroxypropylcellulose (HPC), has been inferred from the effect of these solutes on the forces acting between HPC polymers. Urea interacts only very weakly. The protein stabilizing osmolytes are strongly excluded. The observed energies indicate that the exclusion of the protein stabilizing osmolytes from protein hydrophobic side chains would add significantly to protein stability. The temperature dependence of exclusion indicates a significant enthalpy contribution to the interaction energy in contrast to expectations from ‘molecular crowding’ theories based on steric repulsion. The dependence of exclusion on the distance between HPC polymers rather indicates that perturbations of water structuring or hydration forces underlie exclusion. PMID:18512956

  18. 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

  19. 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

  20. Modeling of drug-mediated CYP3A4 induction by using human iPS cell-derived enterocyte-like cells.

    PubMed

    Negoro, Ryosuke; Takayama, Kazuo; Nagamoto, Yasuhito; Sakurai, Fuminori; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-04-15

    Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test. PMID:26966071

  1. Structure-Based Inhibitor Design for Evaluation of a CYP3A4 Pharmacophore Model.

    PubMed

    Kaur, Parminder; Chamberlin, A Richard; Poulos, Thomas L; Sevrioukova, Irina F

    2016-05-12

    Human cytochrome P450 3A4 (CYP3A4) is a key xenobiotic-metabolizing enzyme that oxidizes and clears the majority of drugs. CYP3A4 inhibition may lead to drug-drug interactions, toxicity, and other adverse effects but, in some cases, could be beneficial and enhance therapeutic efficiency of coadministered pharmaceuticals that are metabolized by CYP3A4. On the basis of our investigations of analogs of ritonavir, a potent CYP3A4 inactivator and pharmacoenhancer, we have built a pharmacophore model for a CYP3A4-specific inhibitor. This study is the first attempt to test this model using a set of rationally designed compounds. The functional and structural data presented here agree well with the proposed pharmacophore. In particular, we confirmed the importance of a flexible backbone, the H-bond donor/acceptor moiety, and aromaticity of the side group analogous to Phe-2 of ritonavir and demonstrated the leading role of hydrophobic interactions at the sites adjacent to the heme and phenylalanine cluster in the ligand binding process. The X-ray structures of CYP3A4 bound to the rationally designed inhibitors provide deeper insights into the mechanism of the CYP3A4-ligand interaction. Most importantly, two of our compounds (15a and 15b) that are less complex than ritonavir have comparable submicromolar affinity and inhibitory potency for CYP3A4 and, thus, could serve as templates for synthesis of second generation inhibitors for further evaluation and optimization of the pharmacophore model. PMID:26371436

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

    PubMed

    Nie, Kun; An, Qi; Zhang, Yihe

    2016-04-21

    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. PMID:27064353

  3. Unraveling protein stabilization mechanisms: vitrification and water replacement in a glass transition temperature controlled system.

    PubMed

    Grasmeijer, N; Stankovic, M; de Waard, H; Frijlink, H W; Hinrichs, W L J

    2013-04-01

    The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (<50°C), the enzymatic activity of the protein strongly decreased during storage at 60°C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20°C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominantly determines stability at higher glass transition temperatures. PMID:23360765

  4. Identification of VPS13C as a Galectin-12-Binding Protein That Regulates Galectin-12 Protein Stability and Adipogenesis

    PubMed Central

    Yang, Ri-Yao; Xue, Huiting; Yu, Lan; Velayos-Baeza, Antonio; Monaco, Anthony P.; Liu, Fu-Tong

    2016-01-01

    Galectin-12, a member of the galectin family of β-galactoside-binding animal lectins, is preferentially expressed in adipocytes and required for adipocyte differentiation in vitro. This protein was recently found to regulate lipolysis, whole body adiposity, and glucose homeostasis in vivo. Here we identify VPS13C, a member of the VPS13 family of vacuolar protein sorting-associated proteins highly conserved throughout eukaryotic evolution, as a major galectin-12-binding protein. VPS13C is upregulated during adipocyte differentiation, and is required for galectin-12 protein stability. Knockdown of Vps13c markedly reduces the steady-state levels of galectin-12 by promoting its degradation through primarily the lysosomal pathway, and impairs adipocyte differentiation. Our studies also suggest that VPS13C may have a broader role in protein quality control. The regulation of galectin-12 stability by VPS13C could potentially be exploited for therapeutic intervention of obesity and related metabolic diseases. PMID:27073999

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

    PubMed

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

    2015-09-29

    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

  6. 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

  7. 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

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

    PubMed

    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

  9. 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

  10. Repeat-protein folding: new insights into origins of cooperativity, stability, and topology

    PubMed Central

    Kloss, Ellen; Courtemanche, Naomi; Barrick, Doug

    2008-01-01

    Although our understanding of globular protein folding continues to advance, the irregular tertiary structures and high cooperativity of globular proteins complicates energetic dissection. Recently, proteins with regular, repetitive tertiary structures have been identified that sidestep limitations imposed by globular protein architecture. Here we review recent studies of repeat-protein folding. These studies uniquely advance our understanding of both the energetics and kinetics of protein folding. Equilibrium studies provide detailed maps of local stabilities, access to energy landscapes, insights into cooperativity, determination of nearest-neighbor interaction parameters using statistical thermodynamics, relationships between consensus sequences and repeat-protein stability. Kinetic studies provide insight into the influence of short-range topology on folding rates, the degree to which folding proceeds by parallel (versus localized) pathways, and the factors that select among multiple potential pathways. The recent application of force spectroscopy to repeat-protein unfolding is providing a unique route to test and extend many of these findings. PMID:17963718

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

    PubMed

    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 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, 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 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/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. PMID:20361990

  12. 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.

  13. 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.

  14. 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

  15. Association of CYP3A4 genotype with treatment-related leukemia.

    PubMed

    Felix, C A; Walker, A H; Lange, B J; Williams, T M; Winick, N J; Cheung, N K; Lovett, B D; Nowell, P C; Blair, I A; Rebbeck, T R

    1998-10-27

    Epipodophyllotoxins are associated with leukemias characterized by translocations of the MLL gene at chromosome band 11q23 and other translocations. Cytochrome P450 (CYP) 3A metabolizes epipodophyllotoxins and other chemotherapeutic agents. CYP3A metabolism generates epipodophyllotoxin catechol and quinone metabolites, which could damage DNA. There is a polymorphism in the 5' promoter region of the CYP3A4 gene (CYP3A4-V) that might alter the metabolism of anticancer drugs. We examined 99 de novo and 30 treatment-related leukemias with a conformation-sensitive gel electrophoresis assay for the presence of the CYP3A4-V. In all treatment-related cases, there was prior exposure to one or more anticancer drugs metabolized by CYP3A. Nineteen of 99 de novo (19%) and 1 of 30 treatment-related (3%) leukemias carried the CYP3A4-V (P = 0.026; Fisher's Exact Test, FET). Nine of 42 de novo leukemias with MLL gene translocations (21%), and 0 of 22 treatment-related leukemias with MLL gene translocations carried the CYP3A4-V (P = 0. 016, FET). This relationship remained significant when 19 treatment-related leukemias with MLL gene translocations that followed epipodophyllotoxin exposure were compared with the same 42 de novo cases (P = 0.026, FET). These data suggest that individuals with CYP3A4-W genotype may be at increased risk for treatment-related leukemia and that epipodophyllotoxin metabolism by CYP3A4 may contribute to the secondary cancer risk. The CYP3A4-W genotype may increase production of potentially DNA-damaging reactive intermediates. The variant may decrease production of the epipodophyllotoxin catechol metabolite, which is the precursor of the potentially DNA-damaging quinone. PMID:9789061

  16. Association of CYP3A4 genotype with treatment-related leukemia

    PubMed Central

    Felix, Carolyn A.; Walker, Amy H.; Lange, Beverly J.; Williams, Terence M.; Winick, Naomi J.; Cheung, Nai-Kong V.; Lovett, Brian D.; Nowell, Peter C.; Blair, Ian A.; Rebbeck, Timothy R.

    1998-01-01

    Epipodophyllotoxins are associated with leukemias characterized by translocations of the MLL gene at chromosome band 11q23 and other translocations. Cytochrome P450 (CYP) 3A metabolizes epipodophyllotoxins and other chemotherapeutic agents. CYP3A metabolism generates epipodophyllotoxin catechol and quinone metabolites, which could damage DNA. There is a polymorphism in the 5′ promoter region of the CYP3A4 gene (CYP3A4-V) that might alter the metabolism of anticancer drugs. We examined 99 de novo and 30 treatment-related leukemias with a conformation-sensitive gel electrophoresis assay for the presence of the CYP3A4-V. In all treatment-related cases, there was prior exposure to one or more anticancer drugs metabolized by CYP3A. Nineteen of 99 de novo (19%) and 1 of 30 treatment-related (3%) leukemias carried the CYP3A4-V (P = 0.026; Fisher’s Exact Test, FET). Nine of 42 de novo leukemias with MLL gene translocations (21%), and 0 of 22 treatment-related leukemias with MLL gene translocations carried the CYP3A4-V (P = 0.016, FET). This relationship remained significant when 19 treatment-related leukemias with MLL gene translocations that followed epipodophyllotoxin exposure were compared with the same 42 de novo cases (P = 0.026, FET). These data suggest that individuals with CYP3A4-W genotype may be at increased risk for treatment-related leukemia and that epipodophyllotoxin metabolism by CYP3A4 may contribute to the secondary cancer risk. The CYP3A4-W genotype may increase production of potentially DNA-damaging reactive intermediates. The variant may decrease production of the epipodophyllotoxin catechol metabolite, which is the precursor of the potentially DNA-damaging quinone. PMID:9789061

  17. Beta-turn propensities as paradigms for the analysis of structural motifs to engineer protein stability.

    PubMed Central

    Ohage, E. C.; Graml, W.; Walter, M. M.; Steinbacher, S.; Steipe, B.

    1997-01-01

    The thermodynamic stability of a protein provides an experimental metric for the relationship of protein sequence and native structure. We have investigated an approach based on an analysis of the structural database for stability engineering of an immunoglobulin variable domain. The most frequently occurring residues in specific positions of beta-turn motifs were predicted to increase the folding stability of mutants that were constructed by site-directed mutagenesis. Even in positions in which different residues are conserved in immunoglobulin sequences, the predictions were confirmed. Frequently, mutants with increased beta-turn propensities display increased folding cooperativities, suggesting pronounced effects on the unfolded state independent of the expected effect on conformational entropy. We conclude that structural motifs with predominantly local interactions can serve as templates with which patterns of sequence preferences can be extracted from the database of protein structures. Such preferences can predict the stability effects of mutations for protein engineering and design. PMID:9007995

  18. ONTOGENIC EXPRESSION OF HUMAN CARBOXYLESTERASE-2 AND CYTOCHROME P450 3A4 IN LIVER AND DUODENUM: POSTNATAL SURGE AND ORGAN-DEPENDENT REGULATION1

    PubMed Central

    Chen, Yi-Tzai; Trzoss, Lynnie; Yang, Dongfang; Yan, Bingfang

    2015-01-01

    Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner. PMID:25724353

  19. Inhibition of Human Cytochrome P450 3A4 by Cholesterol*

    PubMed Central

    Shinkyo, Raku; Guengerich, F. Peter

    2011-01-01

    Cholesterol has been shown to be hydroxylated at the 4β-position by cytochrome P450 3A4, and the reaction occurs in vivo (Bodin, K., Andersson, U., Rystedt, E., Ellis, E., Norlin, M., Pikuleva, I., Eggertsen, G., Björkhem, I., and Diczfalusy, U. (2002) J. Biol. Chem. 277, 31534–31540). If cholesterol is a substrate of P450 3A4, then it follows that it should also be an inhibitor, particularly in light of the high concentrations found in liver. Heme perturbation spectra indicated a Kd value of 8 μm for the P450 3A4-cholesterol complex. Cholesterol inhibited the P450 3A4-catalyzed oxidations of nifedipine and quinidine, two prototypic substrates, in liver microsomes and a reconstituted enzyme system with Ki ∼ 10 μm in an apparently non-competitive manner. The concentration of cholesterol could be elevated 4–6-fold in cultured human hepatocytes by incubation with cholesterol; the level of P450 3A4 and cell viability were not altered under the conditions used. Nifedipine oxidation was inhibited when the cholesterol level was increased. We conclude that cholesterol is both a substrate and an inhibitor of P450 3A4, and a model is presented to explain the kinetic behavior. We propose that the endogenous cholesterol in hepatocytes should be considered in models of prediction of metabolism of drugs and steroids, even in the absence of changes in the concentrations of free cholesterol. PMID:21471209

  20. 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

  1. Contribution of Charged Groups to the Enthalpic Stabilization of the Folded States of Globular Proteins

    PubMed Central

    Dadarlat, Voichita M.; Post, Carol Beth

    2016-01-01

    In this paper we use the results from all atom MD simulations of proteins and peptides to assess individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the “unfolded state”) and charged amino acids in globular proteins (the “folded state”). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO−) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while NH3+ groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously. PMID:18303881

  2. 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. PMID:26716569

  3. 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.

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

    PubMed

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

    2010-06-14

    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 A), and polyol molecules cluster around the protein at a distance of about 4 A. 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. PMID:20550422

  5. 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

  6. Stereospecific Metabolism of Itraconazole by CYP3A4: Dioxolane Ring Scission of Azole Antifungals

    PubMed Central

    Peng, Chi-Chi; Shi, Wei; Lutz, Justin D.; Kunze, Kent L.; Liu, Jun O.; Nelson, Wendel L.

    2012-01-01

    Itraconazole (ITZ) is a mixture of four cis-stereoisomers that inhibit CYP3A4 potently and coordinate CYP3A4 heme via the triazole nitrogen. However, (2R,4S,2′R)-ITZ and (2R,4S,2′S)-ITZ also undergo stereoselective sequential metabolism by CYP3A4 at a site distant from the triazole ring to 3′-OH-ITZ, keto-ITZ, and N-desalkyl-ITZ. This stereoselective metabolism demonstrates specific interactions of ITZ within the CYP3A4 active site. To further investigate this process, the binding and metabolism of the four trans-ITZ stereoisomers by CYP3A4 were characterized. All four trans-ITZ stereoisomers were tight binding inhibitors of CYP3A4-mediated midazolam hydroxylation (IC50 16–26 nM), and each gave a type II spectrum upon binding to CYP3A4. However, instead of formation of 3′-OH-ITZ, they were oxidized at the dioxolane ring, leading to ring scission and formation of two new metabolites of ITZ. These two metabolites were also formed from the four cis-ITZ stereoisomers, although not as efficiently. The catalytic rates of dioxolane ring scission were similar to the dissociation rates of ITZ stereoisomers from CYP3A4, suggesting that the heme iron is reduced while the triazole moiety coordinates to it and no dissociation of ITZ is necessary before catalysis. The triazole containing metabolite [1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone] also inhibited CYP3A4 (IC50 >15 μM) and showed type II binding with CYP3A4. The dioxolane ring scission appears to be clinically relevant because this metabolite was detected in urine samples from subjects that had been administered the mixture of cis-ITZ isomers. These data suggest that the dioxolane ring scission is a metabolic pathway for drugs that contain this moiety. PMID:22106171

  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. Correlations between internal mobility and stability of globular proteins.

    PubMed

    Wüthrich, K; Wagner, G; Richarz, R; Braun, W

    1980-10-01

    The recent work is surveyed which leads to the suggestions that the conformation of globular proteins in solution corresponds to a dynamic ensemble of rapidly interconverting spatial structures, that clusters of hydrophobic amino acid side chains have an important role in the architecture of protein molecules, and that mechanistic aspects of protein denaturation can be correlated with internal mobility seen in the native conformation. These conclusions resulted originally from high resolution 1H nuclear magnetic resonance (NMR) studies of aromatic ring mobility, exchange of interior amide protons and thermal denaturation of the basic pancreatic trypsin inhibitor and a group of related proteins. Various new approaches to further characterize proteins in solution have now been taken and preliminary data are presented. These include computer graphics to outline hydrophobic clusters in globular protein structures, high resolution 1H-NMR experiments at variable hydrostatic pressure and 13C-NMR relaxation measurements. At the present early stage of these new investigations it appears that the hydrophobic cluster model for globular proteins is compatible with the data obtained. PMID:7248460

  9. 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

  10. 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

  11. 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.

  12. Influence of osmolytes on protein and water structure: a step to understanding the mechanism of protein stabilization.

    PubMed

    Bruździak, Piotr; Panuszko, Aneta; Stangret, Janusz

    2013-10-01

    Results concerning the thermostability of hen egg white lysozyme in aqueous solutions with stabilizing osmolytes, trimethylamine-N-oxide (TMAO), glycine (Gly), and its N-methyl derivatives, N-methylglycine (NMG), N,N-dimethylglycine (DMG), and N,N,N-trimethylglycine (betaine, TMG), have been presented. The combination of spectroscopic (IR) and calorimetric (DSC) data allowed us to establish a link between osmolytes' influence on water structure and their ability to thermally stabilize protein molecule. Structural and energetic characteristics of stabilizing osmolytes' and lysozyme's hydration water appear to be very similar. The osmolytes increase lysozyme stabilization in the order bulk water < TMAO < TMG < Gly < DMG < NMG, which is consistent with the order corresponding to the value of the most probable oxygen-oxygen distance of water molecules affected by osmolytes in their surrounding. Obtained results verified the hypothesis concerning the role of water molecules in protein stabilization, explained the osmophobic effect, and finally helped to bring us nearer to the exact mechanism of protein stabilization by osmolytes. PMID:23992436

  13. Small-Molecule Stabilization of the 14-3-3/Gab2 Protein-Protein Interaction (PPI) Interface.

    PubMed

    Bier, David; Bartel, Maria; Sies, Katharina; Halbach, Sebastian; Higuchi, Yusuke; Haranosono, Yu; Brummer, Tilman; Kato, Nobuo; Ottmann, Christian

    2016-04-19

    Small-molecule modulation of protein-protein interactions (PPIs) is one of the most promising new areas in drug discovery. In the vast majority of cases only inhibition or disruption of PPIs is realized, whereas the complementary strategy of targeted stabilization of PPIs is clearly under-represented. Here, we report the example of a semi-synthetic natural product derivative-ISIR-005-that stabilizes the cancer-relevant interaction of the adaptor protein 14-3-3 and Gab2. The crystal structure of ISIR-005 in complex with 14-3-3 and the binding motif of Gab2 comprising two phosphorylation sites (Gab2pS210pT391) showed how the stabilizing molecule binds to the rim-of-the-interface of the protein complex. Only in the direct vicinity of 14-3-3/Gab2pT391 site is a pre-formed pocket occupied by ISIR-005; binding of the Gab2pS210 motif to 14-3-3 does not create an interface pocket suitable for the molecule. Accordingly, ISIR-005 only stabilizes the binding of the Gab2pT391 but not the Gab2pS210 site. This study represents structural and biochemical proof of the druggability of the 14-3-3/Gab2 PPI interface with important implications for the development of PPI stabilizers. PMID:26644359

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

    PubMed Central

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

    2010-01-01

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

  15. 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. PMID:20810950

  16. Direct observation of multimer stabilization in the mechanical unfolding pathway of a protein undergoing oligomerization.

    PubMed

    Scholl, Zackary N; Yang, Weitao; Marszalek, Piotr E

    2015-02-24

    Understanding how protein oligomerization affects the stability of monomers in self-assembled structures is crucial to the development of new protein-based nanomaterials and protein cages for drug delivery. Here, we use single-molecule force spectroscopy (AFM-SMFS), protein engineering, and computer simulations to evaluate how dimerization and tetramerization affects the stability of the monomer of Streptavidin, a model homotetrameric protein. The unfolding force directly relates to the folding stability, and we find that monomer of Streptavidin is mechanically stabilized by 40% upon dimerization, and that it is stabilized an additional 24% upon tetramerization. We also find that biotin binding increases stability by another 50% as compared to the apo-tetrameric form. We used the distribution of unfolding forces to extract properties of the underlying energy landscape and found that the distance to the transition state is decreased and the barrier height is increased upon multimerization. Finally, we investigated the origin of the strengthening by ligand binding. We found that, rather than being strengthened through intramolecular contacts, it is strengthened due to the contacts provided by the biotin-binding loop that crosses the interface between the dimers. PMID:25639698

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed

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

    2014-06-10

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

  19. CYP3A4 Mediates Oxidative Metabolism of the Synthetic Cannabinoid AKB-48.

    PubMed

    Holm, Niels Bjerre; Nielsen, Line Marie; Linnet, Kristian

    2015-09-01

    Synthetic cannabinoid designer drugs have emerged as drugs of abuse during the last decade, and acute intoxication cases are documented in the scientific literature. Synthetic cannabinoids are extensively metabolized, but our knowledge of the involved enzymes is limited. Here, we investigated the metabolism of N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB-48), a compound identified in herbal blends from 2012 and onwards. We screened for metabolite formation using a panel of nine recombinant cytochrome P450 (CYP) enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) and compared the formed metabolites to human liver microsomal (HLM) incubations with specific inhibitors against CYP2D6, 2C19, and 3A4, respectively. The data reported here demonstrate CYP3A4 to be the major CYP enzyme responsible for the oxidative metabolism of AKB-48, preferentially performing the oxidation on the adamantyl moiety. Genetic polymorphisms are likely not important with regard to toxicity given the major involvement of CYP3A4. Adverse drug-drug interactions (DDIs) could potentially occur in cases with co-intake of strong CYP3A4 inhibitors, e.g., HIV antivirals and azole antifungal agents. PMID:26002511

  20. Metabolic behavior prediction of pazopanib by cytochrome P450 (CYP) 3A4 by molecular docking.

    PubMed

    Liu, Xing-Jie; Lu, Hua; Sun, Ju-Xiang; Wang, Su-Rong; Mo, Yan-Shuai; Yang, Xing-Sheng; Shi, Ben-Kang

    2016-08-01

    Metabolism-mediated drug adverse effects (e.g., drug-drug interaction, bioactivation, etc.) strongly limit the utilization of clinical drugs. The present study aims to predict the metabolic capability of cytochrome P450 (CYP) 3A4 toward pazopanib which is an excellent drug exhibiting therapeutic role toward various cancers especially for ovarian cancer. Pazopanib can be well docked into the activity cavity of CYP3A4, and the interaction structure in pazopanib was methyl group located besides nitrogen in the five-membered ring. The distance between the hydrogen atom in methyl group and active center is 3.64 Å. The interaction amino acid is Glu374. Furthermore, both pazopanib and ketoconazole were docked into the activity cavity of CYP3A4 to compare their binding potential. The distance between ketoconazole and activity center (2.10 Å) is closer than the distance between pazopanib and activity center of CYP3A4, indicating the easy influence of CYP3A4 inhibitor toward the metabolism of pazopanib. All these data were helpful for the clinical application of pazopanib, and R&D of other tinib drug candidates as new anti-tumor drugs. PMID:25737032

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

    PubMed

    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

  2. Effect of Glutathione on Homo- and Heterotropic Cooperativity in Cytochrome P450 3A4

    PubMed Central

    Davydov, Dmitri R.; Davydova, Nadezhda Y.; Tsalkova, Tamara N.; Halpert, James R.

    2008-01-01

    Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1–4 mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of α-helix A. Importantly, the KS value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo. PMID:18206979

  3. 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

  4. 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.

  5. Impact of Protein Stability, Cellular Localization, and Abundance on Proteomic Detection of Tumor-Derived Proteins in Plasma

    PubMed Central

    Faca, Vitor M.; Zhang, Wenxuan; Zhang, Qing; Jain, Anjali; Hanash, Sam; Agus, David B.; McIntosh, Martin W.; Mallick, Parag

    2011-01-01

    Tumor-derived, circulating proteins are potentially useful as biomarkers for detection of cancer, for monitoring of disease progression, regression and recurrence, and for assessment of therapeutic response. Here we interrogated how a protein's stability, cellular localization, and abundance affect its observability in blood by mass-spectrometry-based proteomics techniques. We performed proteomic profiling on tumors and plasma from two different xenograft mouse models. A statistical analysis of this data revealed protein properties indicative of the detection level in plasma. Though 20% of the proteins identified in plasma were tumor-derived, only 5% of the proteins observed in the tumor tissue were found in plasma. Both intracellular and extracellular tumor proteins were observed in plasma; however, after normalizing for tumor abundance, extracellular proteins were seven times more likely to be detected. Although proteins that were more abundant in the tumor were also more likely to be observed in plasma, the relationship was nonlinear: Doubling the spectral count increased detection rate by only 50%. Many secreted proteins, even those with relatively low spectral count, were observed in plasma, but few low abundance intracellular proteins were observed. Proteins predicted to be stable by dipeptide composition were significantly more likely to be identified in plasma than less stable proteins. The number of tryptic peptides in a protein was not significantly related to the chance of a protein being observed in plasma. Quantitative comparison of large versus small tumors revealed that the abundance of proteins in plasma as measured by spectral count was associated with the tumor size, but the relationship was not one-to-one; a 3-fold decrease in tumor size resulted in a 16-fold decrease in protein abundance in plasma. This study provides quantitative support for a tumor-derived marker prioritization strategy that favors secreted and stable proteins over all but the

  6. Impact of protein stability, cellular localization, and abundance on proteomic detection of tumor-derived proteins in plasma.

    PubMed

    Fang, Qiaojun; Kani, Kian; Faca, Vitor M; Zhang, Wenxuan; Zhang, Qing; Jain, Anjali; Hanash, Sam; Agus, David B; McIntosh, Martin W; Mallick, Parag

    2011-01-01

    Tumor-derived, circulating proteins are potentially useful as biomarkers for detection of cancer, for monitoring of disease progression, regression and recurrence, and for assessment of therapeutic response. Here we interrogated how a protein's stability, cellular localization, and abundance affect its observability in blood by mass-spectrometry-based proteomics techniques. We performed proteomic profiling on tumors and plasma from two different xenograft mouse models. A statistical analysis of this data revealed protein properties indicative of the detection level in plasma. Though 20% of the proteins identified in plasma were tumor-derived, only 5% of the proteins observed in the tumor tissue were found in plasma. Both intracellular and extracellular tumor proteins were observed in plasma; however, after normalizing for tumor abundance, extracellular proteins were seven times more likely to be detected. Although proteins that were more abundant in the tumor were also more likely to be observed in plasma, the relationship was nonlinear: Doubling the spectral count increased detection rate by only 50%. Many secreted proteins, even those with relatively low spectral count, were observed in plasma, but few low abundance intracellular proteins were observed. Proteins predicted to be stable by dipeptide composition were significantly more likely to be identified in plasma than less stable proteins. The number of tryptic peptides in a protein was not significantly related to the chance of a protein being observed in plasma. Quantitative comparison of large versus small tumors revealed that the abundance of proteins in plasma as measured by spectral count was associated with the tumor size, but the relationship was not one-to-one; a 3-fold decrease in tumor size resulted in a 16-fold decrease in protein abundance in plasma. This study provides quantitative support for a tumor-derived marker prioritization strategy that favors secreted and stable proteins over all but the

  7. 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.

  8. 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. PMID:26830565

  9. Identifying Protein Stabilizing Ligands Using GroEL

    PubMed Central

    Naik, Subhashchandra; Haque, Inamul; Degner, Nick; Kornilayev, Boris; Bomhoff, Gregory; Hodges, Jacob; Khorassani, Ara-Azad; Katayama, Hiroo; Morris, Jill; Kelly, Jeffery; Seed, John; Fisher, Mark T.

    2010-01-01

    Over the past five years, it has become increasingly apparent to researchers that the initial promise and excitement of using gene replacement therapies to ameliorate folding diseases are still far from being broadly or easily applicable. Because a large number of human diseases are protein folding diseases (~30 to 50%), many researchers now realize that more directed approaches to target and reverse the fundamental misfolding reactions preceding disease are highly feasible and offer the potential of developing more targeted drug therapies. This is also true with a large number of so called “orphan protein folding diseases”. The development of a broad-based general screening array method using the chaperonin as a detection platform will enable us to screen large chemical combinatorial libraries for specific ligands against the elusive transient, primary reactions that often lead to protein misfolding. This development will provide a highly desirable tool for the pharmaceutical, academic and medical professions. PMID:19802819

  10. Nanocomplexation between curcumin and soy protein isolate: influence on curcumin stability/bioaccessibility and in vitro protein digestibility.

    PubMed

    Chen, Fei-Ping; Li, Bian-Sheng; Tang, Chuan-He

    2015-04-01

    The complexation of nanoparticles in unheated and heated (at 75-95°) soy protein isolate (SPI) with curcumin and the effects on curcumin stability/bioaccessibility and in vitro protein digestibility were investigated. The nanoparticles did not display noticeable changes in size and morphology upon nanocomplexation with curcumin, except their surface hydrophobicity. The encapsulation efficiency of curcumin progressively decreased with increasing initial curcumin concentration in the dispersion, while the load amount linearly increased. The solubility of curcumin in water was enhanced by the complexation above 98000-fold (vs free curcumin in water). The formation of the nanocomplexes considerably improved the storage stability of curcumin. In vitro simulated digestion experiments indicated that the complexation also improved the bioaccessibility of curcumin; the bioaccessibility was greatly impaired by hydrolysis-induced protein aggregation. Addtionally, the nanocomplexation significantly improved the in vitro protein digestibility of both unheated and heated SPI. PMID:25779681

  11. 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

  12. Concentration-dependent changes in apparent diffusion coefficients as indicator for colloidal stability of protein solutions.

    PubMed

    Bauer, Katharina Christin; Göbel, Mathias; Schwab, Marie-Luise; Schermeyer, Marie-Therese; Hubbuch, Jürgen

    2016-09-10

    The colloidal stability of a protein solution during downstream processing, formulation, and storage is a key issue for the biopharmaceutical production process. Thus, knowledge about colloidal solution characteristics, such as the tendency to form aggregates or high viscosity, at various processing conditions is of interest. This work correlates changes in the apparent diffusion coefficient as a parameter of protein interactions with observed protein aggregation and dynamic viscosity of the respective protein samples. For this purpose, the diffusion coefficient, the protein phase behavior, and the dynamic viscosity in various systems containing the model proteins α-lactalbumin, lysozyme, and glucose oxidase were studied. Each of these experiments revealed a wide range of variations in protein interactions depending on protein type, protein concentration, pH, and the NaCl concentration. All these variations showed to be mirrored by changes in the apparent diffusion coefficient in the respective samples. Whereas stable samples with relatively low viscosity showed an almost linear dependence, the deviation from the concentration-dependent linearity indicated both an increase in the sample viscosity and probability of protein aggregation. This deviation of the apparent diffusion coefficient from concentration-dependent linearity was independent of protein type and solution properties for this study. Thus, this single parameter shows the potential to act as a prognostic tool for colloidal stability of protein solutions. PMID:27421911

  13. Evolutionary Dynamics on Protein Bi-stability Landscapes can Potentially Resolve Adaptive Conflicts

    PubMed Central

    Sikosek, Tobias; Bornberg-Bauer, Erich; Chan, Hue Sun

    2012-01-01

    Experimental studies have shown that some proteins exist in two alternative native-state conformations. It has been proposed that such bi-stable proteins can potentially function as evolutionary bridges at the interface between two neutral networks of protein sequences that fold uniquely into the two different native conformations. Under adaptive conflict scenarios, bi-stable proteins may be of particular advantage if they simultaneously provide two beneficial biological functions. However, computational models that simulate protein structure evolution do not yet recognize the importance of bi-stability. Here we use a biophysical model to analyze sequence space to identify bi-stable or multi-stable proteins with two or more equally stable native-state structures. The inclusion of such proteins enhances phenotype connectivity between neutral networks in sequence space. Consideration of the sequence space neighborhood of bridge proteins revealed that bi-stability decreases gradually with each mutation that takes the sequence further away from an exactly bi-stable protein. With relaxed selection pressures, we found that bi-stable proteins in our model are highly successful under simulated adaptive conflict. Inspired by these model predictions, we developed a method to identify real proteins in the PDB with bridge-like properties, and have verified a clear bi-stability gradient for a series of mutants studied by Alexander et al. (Proc Nat Acad Sci USA 2009, 106:21149–21154) that connect two sequences that fold uniquely into two different native structures via a bridge-like intermediate mutant sequence. Based on these findings, new testable predictions for future studies on protein bi-stability and evolution are discussed. PMID:23028272

  14. 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

  15. The ubiquitin–protein ligase Itch regulates p73 stability

    PubMed Central

    Rossi, Mario; De Laurenzi, Vincenzo; Munarriz, Eliana; Green, Douglas R; Liu, Yun-Cai; Vousden, Karen H; Cesareni, Gianni; Melino, Gerry

    2005-01-01

    p73, a member of the p53 family of transcription factors, is upregulated in response to DNA damage, inducing cell cycle arrest and apoptosis. Besides indications that this p73 response is post-transcriptional, little is known about the underlying molecular mechanisms of p73 protein degradation. Ubiquitination and proteasomal-dependent degradation of p53 are regulated by its transcriptional target MDM2. However, unlike p53, p73 binds to, but is not degraded by, MDM2. Here we describe the binding of p73 to Itch, a Hect ubiquitin–protein ligase. Itch selectively binds and ubiquitinates p73 but not p53; this results in the rapid proteasome-dependent degradation of p73. Upon DNA damage Itch itself is downregulated, allowing p73 protein levels to rise and thus interfere with p73 function. In conclusion, we have identified a key mechanism in the control of p73 protein levels both in normal as well as in stress conditions. PMID:15678106

  16. Mechanical stability of multidomain proteins and novel mechanical clamps.

    PubMed

    Sikora, Mateusz; Cieplak, Marek

    2011-06-01

    We estimate the size of mechanostability for 318 multidomain proteins which are single-chain and contain up to 1021 amino acids. We predict existence of novel types of mechanical clamps in which interdomain contacts play an essential role. Mechanical clamps are structural regions which are the primary source of a protein's resistance to pulling. Among these clamps there is one that opposes tensile stress due to two domains swinging apart. This movement strains and then ruptures the contacts that hold the two domains together. Another clamp also involves tensile stress but it originates from an immobilization of a structural region by a surrounding knot-loop (without involving any disulfide bonds). Still another mechanism involves shear between helical regions belonging to two domains. We also consider the amyloid-prone cystatin C which provides an example of a two-chain 3D domain-swapped protein. We predict that this protein should withstand remarkably large stress, perhaps of order 800 pN, when inducing a shearing strain. The survey is generated through molecular dynamics simulations performed within a structure-based coarse grained model. PMID:21465555

  17. 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

  18. Regulation of Paramyxovirus Fusion Activation: the Hemagglutinin-Neuraminidase Protein Stabilizes the Fusion Protein in a Pretriggered State

    PubMed Central

    Salah, Zuhair W.; Gui, Long; DeVito, Ilaria; Jurgens, Eric M.; Lu, Hong; Yokoyama, Christine C.; Palermo, Laura M.; Lee, Kelly K.

    2012-01-01

    The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. However, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect. PMID:22993149

  19. 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.

  20. 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.

    PubMed

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

    2016-06-15

    A novel organotin DFDPT was synthesized and characterized by elemental analysis, IR, (1)H, (13)C, (119)Sn, 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 310K were 2.51×10(7) and 3.09×10(5), 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. PMID:27049867

  1. Influence of Miscibility of Protein-Sugar Lyophilizates on Their Storage Stability.

    PubMed

    Mensink, Maarten A; Nethercott, Matthew J; Hinrichs, Wouter L J; van der Voort Maarschalk, Kees; Frijlink, Henderik W; Munson, Eric J; Pikal, Michael J

    2016-09-01

    For sugars to act as successful stabilizers of proteins during lyophilization and subsequent storage, they need to have several characteristics. One of them is that they need to be able to form interactions with the protein and for that miscibility is essential. To evaluate the influence of protein-sugar miscibility on protein storage stability, model protein IgG was lyophilized in the presence of various sugars of different molecular weight. By comparing solid-state nuclear magnetic resonance spectroscopy relaxation times of both protein and sugar on two different timescales, i.e., (1)H T1 and (1)H T1ρ, miscibility of the two components was established on a 2-5- and a 20-50-nm length scale, respectively, and related to protein storage stability. Smaller sugars showed better miscibility with IgG, and the tendency of IgG to aggregate during storage was lower for smaller sugars. The largest sugar performed worst and was phase separated on both length scales. Additionally, shorter protein (1)H T1 relaxation times correlated with higher aggregation rates during storage. The enzyme-linked immunosorbent assay (ELISA) assay showed overlapping effects of aggregation and chemical degradation and did not correspond as well with the miscibility. Because of the small scale at which miscibility was determined (2-5 nm) and the size of the protein domains (∼2.5 × 2.5 × 5 nm), the miscibility data give an indirect measure of interaction between protein and sugar. This reduced interaction could be the result of steric hindrance, providing a possible explanation as to why smaller sugars show better miscibility and storage stability with the protein. PMID:27301753

  2. In vivo protein stabilization based on fragment complementation and a split GFP system

    PubMed Central

    Lindman, Stina; Hernandez-Garcia, Armando; Szczepankiewicz, Olga; Frohm, Birgitta; Linse, Sara

    2010-01-01

    Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state. PMID:21041669

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Using tryptophan fluorescence to measure the stability of membrane proteins folded in liposomes

    PubMed Central

    Moon, C. Preston; Fleming, Karen G.

    2013-01-01

    Accurate measurements of the thermodynamic stability of folded membrane proteins require methods for monitoring their conformation that are free of experimental artifacts. For tryptophan fluorescence emission experiments with membrane proteins folded into liposomes, there are two significant sources of artifacts: the first is light scattering by the liposomes; the second is the nonlinear relationship of some tryptophan spectral parameters with changes in protein conformation. Both of these sources of error can interfere with the method of determining the reversible equilibrium thermodynamic stability of proteins using titrations of chemical denaturants. Here, we present methods to manage light scattering by liposomes for tryptophan emission experiments and to properly monitor tryptophan spectra as a function of protein conformation. Our methods are tailored to the titrations of membrane proteins using common chemical denaturants. One of our recommendations is to collect and analyze the right-angle light scattering peak that occurs around the excitation wave- length in a fluorescence experiment. Another recommendation is to use only those tryptophan spectral parameters that are linearly proportional to the protein conformational population. We show that other commonly used spectral commonly used parameters lead to errors in protein stability measurements. PMID:21333792