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Alexey V Gribenko

Publications and source records attributed to Alexey V Gribenko.

7 recordsLinked to original sources
PubMed2006

Role of the charge-charge interactions in defining stability and halophilicity of the CspB proteins.

Charge-charge interactions on the surface of native proteins are important for protein stability and can be computationally redesigned in a rational way to modulate protein stability. Such computational effort led to an engineered protein, CspB-TB that has the same core as the mesophilic cold shock protein CspB-Bs from Bacillus subtilis, but optimized distribution of charge-charge interactions on the surface. The CspB-TB protein shows an increase in the transition temperature by 20 degrees C relative to the unfolding temperature of CspB-Bs. The CspB-TB and CspB-Bs protein pair offers a unique opportunity to further explore the energetics of charge-charge interactions as the substitutions at the same sequence positions are done in largely similar structural but different electrostatic environments. In particular we addressed two questions. What is the contribution of charge-charge interactions in the unfolded state to the protein stability and how amino acid substitutions modulate the effect of increase in ionic strength on protein stability (i.e. protein halophilicity). To this end, we experimentally measured the stabilities of over 100 variants of CspB-TB and CspB-Bs proteins with substitutions at charged residues. We also performed computational modeling of these protein variants. Analysis of the experimental and computational data allowed us to conclude that the charge-charge interactions in the unfolded state of two model proteins CspB-Bs and CspB-TB are not very significant and computational models that are based only on the native state structure can adequately, i.e. qualitatively (stabilizing versus destabilizing) and semi-quantitatively (relative rank order), predict the effects of surface charge neutralization or reversal on protein stability. We also show that the effect of ionic strength on protein stability (protein halophilicity) appears to be mainly due to the screening of the long-range charge-charge interactions.

Amino Acid Sequence↗
PubMed2006

Amino acid substitutions affecting protein dynamics in eglin C do not affect heat capacity change upon unfolding.

The heat capacity change upon unfolding (deltaC(p)) is a thermodynamic parameter that defines the temperature dependence of the thermodynamic stability of proteins; however, physical basis of the heat capacity change is not completely understood. Although empirical surface area-based calculations can predict heat capacity changes reasonably well, accumulating evidence suggests that changes in hydration of those surfaces is not the only parameter contributing to the observed heat capacity changes upon unfolding. Because packing density in the protein interior is similar to that observed in organic crystals, we hypothesized that changes in protein dynamics resulting in increased rigidity of the protein structure might contribute to the observed heat capacity change upon unfolding. Using differential scanning calorimetry we characterized the thermodynamic behavior of a serine protease inhibitor eglin C and two eglin C variants with altered native state dynamics, as determined by NMR. We found no evidence of changes in deltaC(p) in either of the variants, suggesting that changes in rigidity do not contribute to the heat capacity change upon unfolding in this model system.

Amino Acid Sequence↗
PubMed2006

Protein stability and surface electrostatics: a charged relationship.

Engineering proteins to withstand a broad range of conditions continues to be a coveted objective, holding the potential to advance biomedicine, industry, and our understanding of disease. One way of achieving this goal lies in elucidating the underlying interactions that define protein stability. It has been shown that the hydrophobic effect, hydrogen bonding, and packing interactions between residues in the protein interior are dominant factors that define protein stability. The role of surface residues in protein stability has received much less attention. It has been believed that surface residues are not important for protein stability particularly because their interactions with the solvent should be similar in the native and unfolded states. In the case of surface charged residues, it was sometimes argued that solvent exposure meant that the high dielectric of the solvent will further decrease the strength of the charge-charge interactions. In this paper, we challenge the notion that the surface charged residues are not important for protein stability. We computationally redesigned sequences of five different proteins to optimize the surface charge-charge interactions. All redesigned proteins exhibited a significant increase in stability relative to their parent proteins, as experimentally determined by circular dichroism spectroscopy and differential scanning calorimetry. These results suggest that surface charge-charge interactions are important for protein stability and that rational optimization of charge-charge interactions on the protein surface can be a viable strategy for enhancing protein stability.

Acid Anhydride Hydrolases↗
PubMed2006

Dissecting the energetics of protein alpha-helix C-cap termination through chemical protein synthesis.

The alpha-helix is a fundamental protein structural motif and is frequently terminated by a glycine residue. Explanations for the predominance of glycine at the C-cap terminal portions of alpha-helices have invoked uniquely favorable energetics of this residue in a left-handed conformation or enhanced solvation of the peptide backbone because of the absence of a side chain. Attempts to quantify the contributions of these two effects have been made previously, but the issue remains unresolved. Here we have used chemical protein synthesis to dissect the energetic basis of alpha-helix termination by comparing a series of ubiquitin variants containing an L-amino acid or the corresponding D-amino acid at the C-cap Gly35 position. D-Amino acids can adopt a left-handed conformation without energetic penalty, so the contributions of conformational strain and backbone solvation can thus be separated. Analysis of the thermodynamic data revealed that the preference for glycine at the C' position of a helix is predominantly a conformational effect.

Calorimetry, Differential Scanning↗
PubMed2004

Mechanism of thermostabilization in a designed cold shock protein with optimized surface electrostatic interactions.

Using computational and sequence analysis of bacterial cold shock proteins, we designed a protein (CspB-TB) that has the core residues of mesophilic protein from Bacillus subtilis(CspB-Bs) and altered distribution of surface charged residues. This designed protein was characterized by circular dichroism spectroscopy, and found to have secondary and tertiary structure similar to that of CspB-Bs. The activity of the CspB-TB protein as measured by the affinity to a single-stranded DNA (ssDNA) template at 25 degrees C is somewhat higher than that of CspB-Bs. Furthermore, the decrease in the apparent binding constant to ssDNA upon increase in temperature is much more pronounced for CspB-Bs than for CspB-TB. Temperature-induced unfolding (as monitored by differential scanning calorimetry and circular dichroism spectroscopy) and urea-induced unfolding experiments were used to compare the stabilities of CspB-Bs and CspB-TB. It was found that CspB-TB is approximately 20 degrees C more thermostable than CspB-Bs. The thermostabilization of CspB-TB relative to CspB-Bs is achieved by decrease in the enthalpy and entropy of unfolding without affecting their temperature dependencies, i.e. these proteins have similar heat capacity changes upon unfolding. These changes in the thermodynamic parameters result in the global stability function, i.e. Gibbs energy, deltaG(T), that is shifted to higher temperatures with only small changes in the maximum stability. Such a mechanism of thermostabilization, although predicted from the basic thermodynamic considerations, has never been identified experimentally.

Amino Acid Sequence↗
PubMed2002

Conformational and thermodynamic properties of peptide binding to the human S100P protein.

S100P is a member of the S100 subfamily of calcium-binding proteins that are believed to be associated with various diseases, and in particular deregulation of S100P expression has been documented for prostate and breast cancer. Previously, we characterized the effects of metal binding on the conformational properties of S100P and proposed that S100P could function as a Ca2+ conformational switch. In this study we used fluorescence and CD spectroscopies and isothermal titration calorimetry to characterize the target-recognition properties of S100P using a model peptide, melittin. Based on these experimental data we show that S100P and melittin can interact in a Ca2+-dependent and -independent manner. Ca2+-independent binding occurs with low affinity (Kd approximately 0.2 mM), has a stoichiometry of four melittin molecules per S100P dimer and is presumably driven by favorable electrostatic interactions between the acidic protein and the basic peptide. In contrast, Ca2+-dependent binding of melittin to S100P occurs with high affinity (Kd approximately 5 microM) has a stoichiometry of two molecules of melittin per S100P dimer, appears to have positive cooperativity, and is driven by hydrophobic interactions. Furthermore, Ca2+-dependent S100P-melittin complex formation is accompanied by significant conformational changes: Melittin, otherwise unstructured in solution, adopts a helical conformation upon interaction with Ca2+-S100P. These results support a model for the Ca2+-dependent conformational switch in S100P for functional target recognition.

Calcium↗