Interactions In Protein Stability Research Articles

A computational study on heat flow and thermostability of thermophilic and mesophilic proteins

Thermophilic and mesophilic proteins exhibit distinct responses to temperature variations, a critical aspect of their biological functionality. Understanding the microscopic mechanisms governing protein stability and flexibility at different temperatures is essential. Vibrational energy transfer within proteins plays a pivotal role in their diverse biological processes. We employed vibrational energy diffusivity methodology to investigate this phenomenon. Molecular dynamics simulations were conducted at multiple temperatures such as 308, 325, and 350 K, revealing that fluctuations persist in both thermophilic and mesophilic proteins. To delve deeper into the system's stability, we constructed frequency-resolved communication maps, highlighting key residues involved in energy transfer pathways. Our results identify highly active residues that influence protein stabilization or destabilization. Furthermore, the role of salt bridges and non-covalent interactions in protein stability is explored. Our findings offer insights into the relationship between protein activity and thermal resistance, contributing to a comprehensive understanding of thermophilic protein's behavior at different temperatures. This study provides valuable insights into the protein's flexibility and stability, shedding light on the mechanisms that underpin their biological functions.

A pH-dependent protein stability switch coupled to the perturbed pKa of a single ionizable residue

The contribution of electrostatic interactions in protein stability has not been fully understood. Burial of an ionizable amino acid inside the hydrophobic protein core can affect its ionization equilibrium and shift its pKa differentially in the native (N) and unfolded (U) states of a protein and this coupling between the folding/unfolding cycle and the ionization equilibria of the ionizable residue can substantially influence the protein stability. Here, we studied the coupling of the folding/unfolding cycle with the ionization of a buried ionizable residue in a multi-domain protein, Human Serum Albumin (HSA) using fluorescence spectroscopy. A pH-dependent change in the stability of HSA was observed in the near native pH range (pH 6.0–9.0). The protonation-deprotonation equilibrium of a single thiol residue that is buried in the protein structure was identified to give rise to the pH-dependent protein stability. We quantified the pKa of the thiol residue in the N and the U states. The mean pKa of the thiol in the N state was upshifted by 0.5 units to 8.7 due to the burial of the thiol in the protein structure. Surprisingly, the mean pKa of the thiol in the U state was observed to be downshifted by 1.3 units to 6.9. These results indicate that some charged residues are spatially proximal to the thiol group in the U state. Our results suggest that, in addition to the N state, electrostatic interactions in the U state are important determinants of protein stability.

Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability

A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge–charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme.

Characterization and evolution of a metagenome-derived lipase towards enhanced enzyme activity and thermostability

In the present investigation, we used directed evolution approach to engineer a lipase from metagenomic origin. A variant S311C, was generated, characterized in detail and compared with wild type. Wild type and variant lipases were overexpressed and purified to homogeneity. The temperature optima of the purified lipases (Variant and wild type) were almost same, and found to be 45 and 50 °C, respectively. The variant protein was highly thermostable (54 times) as compared with the wild type at 60 °C. The variant displayed very high kinetic efficiency over the wild type protein. Analysis of the homology models of wild type and variant lipase showed that the substitution is on the surface of the protein. This substitution, along with hydrophobic residues in near vicinity may be involved in formation of strong hydrophobic channel leading to active site. This study identifies the role of hydrophobic interactions in protein stability along with enhancement of enzyme activity.

The Role of Electrostatic Interactions on Klentaq1 Insight for Domain Separation

We investigated the relationship between the thermostability of Klentaq1 and factors stabilizing interdomain interactions. When thermal adaptation of Klentaq1 was analyzed at the atomic level, the protein was stable at 300 and 350 K. It gradually unfolded at 373 K and almost spontaneously unfolded at 400 K. Domain separation was induced by disrupting electrostatic interactions in two salt bridges formed by Lys354-Glu445 and Asp371-Arg435 on the interface domain. The role of these interactions in protein stability was evaluated by comparing free energy solvation (ΔΔGsolv) between wild type and mutants. Substitution of Asp371 by Glu or Asn, and also Glu445 by Asn resulted in a positive value of ΔΔGsolv, suggesting that mutations destabilized the protein structure. Nevertheless, substitution of Glu445 by Asp gave a negative value to ΔΔGsolv reflecting increasing protein stability. Our results demonstrate that interactions at the interface domains of Klentaq1 are essential factors correlated with the Klentaq1 thermostability.

Role of charged residues in stabilization of Pyrococcus horikoshii CutA1, which has a denaturation temperature of nearly 150 °C

The CutA1 protein from Pyrococcus horikoshii (PhCutA1), a hyperthermophile, has an unusually high content of charged residues and an unusually high denaturation temperature. To elucidate the role of ion-ion interactions in protein stability, mutant proteins of PhCutA1 in which charged residues were substituted by noncharged residues were comprehensively examined. The denaturation temperatures (T(d)) for 13 of 53 examined mutant proteins were higher than that of the wild-type (148.5 °C at pH 7.0), among which E99Q had the highest T(d) at 154.9 °C. R25A had the largest decrease in T(d) among single mutants at ΔT(d) = -12.4 °C. The average decrease in T(d) of Lys or Arg mutants was greater than that of Glu or Asp mutants, and the average change in T(d) (ΔT(d)) of 21 Glu mutants was negligible, at 0.03 ± 2.05 °C. However, the electrostatic energy (-159.3 kJ·mol(-1)) of PhCutA1 was quite high, compared with that of CutA1 from Escherichia coli (-9.7 kJ·mol(-1)), a mesophile. These results indicate that: (a) many Glu and Asp residues of PhCutA1 should be essential for highly efficient interactions with positively charged residues and for generating high electrostatic energy, although they were forced to be partially repulsive to each other; (b) the changes in stability of mutant proteins with a T(d) value of ~140-150 °C were able to be explained by considering factors important for protein stability and the structural features of mutant sites; and (c) these findings are useful for the design of proteins that are stable at temperatures > 100 °C.

Distinct roles of conventional non-covalent and cation–π interactions in protein stability

The different roles played by conventional non-covalent and cation–π interactions in stabilizing protein structures have been investigated using a dataset of 62 non-redundant DNA binding proteins. The stabilizing residues have been identified by a consensus approach using the concepts of hydrophobicity, long-range interactions and conservation of amino acid residues. The cation–π interactions have been delineated based on a geometric approach, such as, distance and energy criteria. I have identified 138 and 196 stabilizing residues, based on non-covalent and cation–π interactions, respectively. Interestingly, the stabilizing residues identified by consensus approach are not contributing to cation–π interactions. Further, 99% of the cation–π interactions forming residues are not identified as stabilizing ones. These results demonstrate that the roles of cation–π and non-covalent interactions are different from each other to the stability of protein structures. I have further evaluated the range of surrounding hydrophobicity, long-range order, stabilization center and conservation score for the residues that are contributing to cation–π interactions. The average surrounding hydrophobicity and long-range order of cation–π interaction forming residues are in the range of 7–15 kcal/mol and 0–0.03, respectively. I suggest that the inclusion of cation–π interaction along with conventional non-covalent interactions would provide deep insights for understanding the stability of protein structures.

Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments.

Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9-kD and 5-kD peptide fragments formed by limited proteolytic digestion of the N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3'-kinase fold into an active native-like structure on interaction with one another. The corresponding 5-kD fragment of the homologous Src protein, however, was not capable of structurally complementing the p85 9-kD fragment, indicating that fragment complementation among these SH2 domains is sensitive to the sequence differences between the Src and p85 domains. Partial complementation and folding activity could be recovered with hybrid sequences of these SH2 domains. Complete alanine scanning of the 5-kD p85 fragment was used to identify the sequence recognition elements required for complex formation. The alanine substitutions in the p85 5-kD fragment that abolished binding affinity with the cognate 9-kD fragment correlate well with highly conserved residues among SH2 domains that are either integrally involved in core packing or found at the interface between fragments. Surprisingly, however, mutation of a nonconserved surface-exposed aspartic acid to alanine was found to have a significant effect on complementation. A single additional mutation of arginine to aspartic acid allowed for recovery of native structure and increased the thermal stability of the designed Src-p85 chimera by 18 degrees C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability.

Teaching Noncovalent Interactions in the Biochemistry Curriculum through Molecular Visualization: The Search for pi Interactions

Teaching students how proteins adopt a stable three-dimensional structure is a major goal in the biochemistry curriculum. Inherent in this task is the introduction of the structure of the amino acids and the various types of noncovalent interactions. When these two topics are taught together, the students gain a better appreciation for the importance of noncovalent interactions in stabilizing the structure of proteins and protein complexes. Biochemistry textbooks have not recognized the ability of aromatic amino acids to participate in π interactions with other amino acids and the role of these interactions in protein stability. To introduce these ideas, RasMol, a molecular visualization program, was used by students to search for specific examples of noncovalent interactions in a variety of protein structures. The students found examples of the traditional weak interactions as well as examples of stabilizing interactions involving aromatic amino acids. These interactions are described in this report and can be viewed at an accompanying Web site. This work clearly demonstrates that π-type interactions help stabilize the structure and complexes of many proteins and that such interactions should be integrated into the biochemistry curriculum.

A tale of two secondary structure elements: when a β-hairpin becomes an α-helix

In this work, we have analyzed the relative importance of secondary versus tertiary interactions in stabilizing and guiding protein folding. For this purpose, we have designed four different mutants to replace the α-helix of the GB1 domain by a sequence with strong β-hairpin propensity in isolation. In particular, we have chosen the sequence of the second β-hairpin of the GB1 domain, which populates the native conformation in aqueous solution to a significant extent. The resulting protein has roughly 30 % of its sequence duplicated and maintains the 3D-structure of the wild-type protein, but with lower stability (up to −5 kcal/mol). The loss of intrinsic helix stability accounts for about 80 % of the decrease in free energy, illustrating the importance of local interactions in protein stability. Interestingly enough, all the mutant proteins, included the one with the duplicated β-hairpin sequence, fold with similar rates as the GB1 domain. Essentially, it is the nature of the rate-limiting step in the folding reaction that determines whether a particular interaction will speed up, or not, the folding rates. While local contacts are important in determining protein stability, residues involved in tertiary contacts in combination with the topology of the native fold, seem to be responsible for the specificity of protein structures. Proteins with non-native secondary structure tendencies can adopt stable folds and be as efficient in folding as those proteins with native-like propensities.

Cation selective promotion of tubulin polymerization by alkali metal chlorides

A role for charge-based interactions in protein stability at the monomer or dimer level is well known. We show here that such interactions can also be important for the higher-order structures of microtubule assembly. Alkali metal chlorides increase the rate of polymerization of pure tubulin driven by either taxol or dimethyl sulfoxide. The effect is cation selective, exhibiting a sequence Na+ > K+ > Li+ > Cs+, with optimal concentrations for Na+ at approximately 160 mM. Hofmeister anion effects are additive with these rate stimulations. Sodium is less potent than guanidinium ion stimulation reported previously, but produces a larger fraction of normal microtubules. Alkali metal cations lower the critical concentration by a factor of approximately 2, produce cold reversible polymers whose formation is sensitive to podophyllotoxin inhibition, increase the fraction of polymers present as microtubules from approximately 0.9 to 0.99, and reverse or prevent urea-induced depolymerization of microtubules. In the presence of microtubule-associated proteins, the promotion of polymerization is no longer cation selective. In the polymerization of tubulin S, in which the acidic C termini of both monomers have been cleaved, the cation enhancement is markedly decreased, although selective persists. Because the selectivity sequence is similar to that of the coil/helix transition of polyglutamic acid, we suggest that a major part, although not all, of the cation selective enhancement of polymerization results from shielding of the glutamate-rich C termini of the tubulin monomers.

Dissecting the roles of individual interactions in protein stability: lessons from a circularized protein.

A circular form of bovine pancreatic trypsin inhibitor (BPTI) has been prepared by introducing a peptide bond between the N- and C-termini, which are in close proximity in the native conformation. The pathway and energetics of the disulphide-coupled folding transition of the circular protein have been studied using methods applied previously to the unmodified protein. The cross-link between the termini was found not to significantly stabilize the native state in spite of the expected reduction in entropy of the unfolded protein. This unexpected result has led to a reexamination of the stabilization expected from a cross-link, considering effects on the native, as well as unfolded, states of the protein. The greatest stabilization is expected when the cross-linked groups are held rigidly in the native protein in the optimum orientation for forming the cross-link. Similar analyses, utilizing thermodynamic cycles, can be applied to other interactions that stabilize native proteins, including disulphide bonds, salt bridges, and hydrogen bonds and to modifications to the protein that remove them. In general, the contribution of an individual interaction to the stability of the native state depends on the extent to which the interaction is favored in the native conformation, which can vary greatly depending on the local environment of the interacting groups.