Unexpectedly strong energy stabilization inside the hydrophobic core of small protein rubredoxin mediated by aromatic residues: correlated ab initio quantum chemical calculations

The formation of a hydrophobic core of globular proteins is believed to be the consequence of exterior hydrophobic forces of entropic nature. This, together with the low occurrence of hydrogen bonds in the protein core, leads to the opinion that the energy contribution of core formation to protein folding and stability is negligible. We show that stabilization inside the hydrophobic core of a small protein, rubredoxin, determined by means of high-level correlated ab initio calculations (complete basis set limit of MP2 stabilization energy + CCSD(T) correction term), amounted to approximately 50 kcal/mol. These results clearly demonstrate strong attraction inside a hydrophobic core. This finding may lead to substantial changes in the current view of protein folding. We also point out the inability of the DFT/B3LYP method to describe a strong attraction between studied amino acids.     

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

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

Critical residue that promotes protein dimerization: a story of partially exposed Phe25 in 14-3-3σ

Many proteins exist and function as oligomers. While hydrophobic interactions have been recognized as the major driving force for oligomerization, detailed molecular mechanisms for the assembly are unknown. Here, we used 14-3-3σ as a model protein and investigated the role of hydrophobic residues at the dimeric interface using MD simulations and coimmunoprecipitations. We found that a half-exposed and half-buried residue in the interface, Phe(25), plays a more important role in promoting homodimerization than the hydrophobic core residues by organizing both favorable hydrophobic and hydrophilic interactions. Phe(25) is critical in packing and stabilizing hydrophobic core residues. We conclude that the structural stability of hydrophobic cores is critical for a stable homodimer complex and this stable property can be bestowed by residues outside of hydrophobic core. The important organizing activity of Phe(25) for homodimerization of 14-3-3σ originates from its unique physical location, rigidity, size, and hydrophobicity. Thus, hydrophobic residues that are not deeply buried at the oligomeric interface may play important but different roles from the buried core residues and they may promote oligomerization by organizing co-operativity of core and other residues for favorable hydrophobic and electrostatic interactions.     

Importance of individual side chains for the stability of a protein fold: computational alanine scanning of the insulin monomer

A new computational approach is proposed to probe the importance of residue side chains for the stability of a protein fold. Computational mutations to estimate protein stability (CMEPS) is based on the notion that the binding free energy corresponding to the complexation of a given side chain, considered as a "pseudo-ligand" of the wild type protein, reflects the importance of this side chain to the thermodynamic stability of the protein. The contribution of a particular side chain to the folding energy is estimated according to the molecular mechanics-generalized born surface area MM-GBSA approach, using a single molecular dynamics simulation trajectory of the wild type protein. CMEPS is a first principles method which does not contain any adjustable parameter that could be fitted to experimental data. The approach is first validated for Barnase and the B1 domain of protein L, for which a correlation coefficient R = 0.73, between experimental and CMEPS calculated DeltaDeltaG values, is found and then applied to the insulin monomer. In the present application, CMEPS replaces each amino acid by an alanine residue. Therefore, most mutations lead to cavities in the protein. From this the change in stability can be correlated with increased cavity volume. For insulin, this correlation is very similar compared with data previously analyzed for T4 lysozyme from an experiment for buried apolar side chains. There, the increased cavity volume has been related to the hydrophobic effect. However, since CMEPS uses the energetics in terms of electrostatic and van der Waals interactions (and not the hydrophobic effect which is difficult to relate to physical interactions), it is possible to study the effect of mutations of polar and solvent accessible side chains. According to CMEPS, residues Leu A16, Tyr A19, Leu B11, Leu B15, and Arg B22 are most important for the stability of the monomeric insulin fold. This is in agreement with experimental data. As a consequence, mutation of these residues may lead to misfolded and inactive insulin analogues.     

Enhanced hairpin stability through loop design: the case of the protein G B1 domain hairpin

A mutational study of the peptide corresponding to the second hairpin of the protein G B1 domain (GB1p) provided a series of mutants with significantly increased fold stability. Mutations focused on improvement of the direction-reversing loop and the addition of favorable Coulombic interactions at the sequence termini. The loop optimization was based on a database search for residues that occur with the greatest probability in similar hairpin loops in proteins. This search suggested replacing the native DDATKT sequence with NPATGK, which resulted in a 4.5 kJ/mol stabilization of the hairpin fold. The introduction of positively charged lysines at the N-terminus provided an additional 2.4 kJ/mol of stabilization, affording a GB1p mutant that is 86 +/- 3% folded at 25 degrees C with a melting temperature of 60 +/- 2 degrees C. The trpzip version of this peptide, in which three of the hydrophobic core residues were mutated to tryptophan, yielded a sequence that melted at 85 degrees C. Throughout, fold populations and melting temperatures were derived from the mutation and temperature dependence of proton chemical shifts and were corroborated by circular dichroism (CD) melts. The study also suggests that the wild-type GB1p sequence is significantly less stable than reported in some other studies: only 30% folded in water at 25 degrees C.     

How ion properties determine the stability of a lipase enzyme in ionic liquids: a molecular dynamics study

The influence of eight different ionic liquid (IL) solvents on the stability of the lipase Candida antarctica lipase B (CAL-B) is investigated with molecular dynamics (MD) simulations. Considered ILs contain cations that are based either on imidazolium or guanidinium as well as nitrate, tetrafluoroborate or hexafluorophosphate anions. Partial unfolding of CAL-B is observed during high-temperature MD simulations and related changes of CAL-B regarding its radius of gyration, surface area, secondary structure, amount of solvent close to the backbone and interaction strength with the ILs are evaluated. CAL-B stability is influenced primarily by anions in the order NO(3)(-)? BF(4)(-) < PF(6)(-) of increasing stability, which agrees with experiments. Cations influence protein stability less than anions but still substantially. Long decyl side chains, polar methoxy groups and guanidinium-based cations destabilize CAL-B more than short methyl groups, other non-polar groups and imidazolium-based cations, respectively. Two distinct causes for CAL-B destabilization are identified: a destabilization of the protein surface is facilitated mostly by strong Coulomb interactions of CAL-B with anions that exhibit a localized charge and strong polarization as well as with polar cation groups. Surface instability is characterized by an unraveling of α-helices and an increase of surface area, radius of gyration and protein-IL total interaction strength of CAL-B, all of which describe a destabilization of the folded protein state. On the other hand, a destabilization of the protein core is facilitated when direct core-IL interactions are feasible. This is the case when long alkyl chains are involved or when particularly hydrophobic ILs induce major conformational changes that enable ILs direct access to the protein core. This core instability is characterized by a disintegration of β-sheets, diffusion of ions into CAL-B and increasing protein-IL van der Waals interactions. This process describes a stabilization of the unfolded protein state. Both of these processes reduce the folding free energy and thus destabilize CAL-B. The results of this work clarify the impact of ions on CAL-B stabilization. An extrapolation of the observed trends enables proposing novel ILs in which protein stability could be enhanced further.     

Mechanism of simultaneously refolding and purification of proteins by hydrophobic interaction chromatographic unit and applications

The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent the denatured protein molecules from aggregating with each other. The STHIC can provide high enough energy to a denatured protein molecule to make it dehydration and to refold it into its native or various intermediate states. The outcome not only depends on the specific interactions between amino acids, the structure of STHIC, but also depends on the association between the STHIC and mobile phase. The mechanism of protein refolding and the principle of its quality control by HPHIC were also presented. By appropriate selection of the chromatographic condition, several denatured proteins can be refolded and separated simultaneously in a single chromatographic run. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative     

Cold Denaturation Unveiled: Molecular Mechanism of the Asymmetric Unfolding of Yeast Frataxin

What is the mechanism that determines the denaturation of proteins at low temperatures, which is, by now, recognized as a fundamental property of all proteins? We present experimental evidence that clarifies the role of specific interactions that favor the entrance of water into the hydrophobic core, a mechanism originally proposed by Privalov but never proved experimentally. By using a combination of molecular dynamics simulation, molecular biology, and biophysics, we identified a cluster of negatively charged residues that represents a preferential gate for the entrance of water molecules into the core. Even single‐residue mutations in this cluster, from acidic to neutral residues, affect cold denaturation much more than heat denaturation, suppressing cold denaturation at temperatures above zero degrees. The molecular mechanism of the cold denaturation of yeast frataxin is intrinsically different from that of heat denaturation.     

Enhanced stability of a protein with increasing temperature

The unusual stability of a structured but locally flexible protein, human growth hormone (hGH) at pH 2.7, was investigated using the temperature dependence of the nanosecond-picosecond dynamics of the backbone amide groups obtained from (15)N NMR relaxation data. It is found that the flexibility of the backbone of the helices decreases with temperature in the range from 24 °C to ～40 °C, corresponding to an increasing stability. A concomitant increase with temperature of the electrostatic interactions between charged residues forming an interhelical network of salt bridges at the center of the four-helix core suggests that these interactions give rise to the decreasing flexibility and increasing stability of the protein. However, numerous hydrophobic interactions in the interior of the four-helix core may also contribute. Above ～40 °C, where the thermal energy overcomes the electrostatic and hydrophobic interactions, a substantial increase in the flexibility of the helix backbones results in a highly positive contribution from the local conformational heat capacity, C(p,?conf), of the helix backbones to the total heat capacity, C(p), of the protein. This reduces the change in heat capacity upon unfolding, ΔC(p), increases the change in the Gibbs free energy, ΔG(unfold), and stabilizes the protein at high temperatures. A similar decrease in flexibility is found near other salt bridges in hGH and in Calmodulin and may be of general importance for the thermostability of proteins and, in particular, of the salt bridge intensive thermophilic proteins.     

Combining a polarizable force-field and a coarse-grained polarizable solvent model. II. Accounting for hydrophobic effects

A revised and improved version of our efficient polarizable force-field/coarse grained solvent combined approach (Masella, Borgis, and Cuniasse, J. Comput. Chem. 2008, 29, 1707) is described. The polarizable pseudo-particle solvent model represents the macroscopic solvent polarization by induced dipoles placed on mobile pseudo-particles. In this study, we propose a new formulation of the energy term handling the nonelectrostatic interactions among the pseudo-particles. This term is now able to reproduce the energetic and structural response of liquid water due to the presence of a hydrophobic spherical cavity. Accordingly, the parameters of the energy term handling the nonpolar solute/solvent interactions have been refined to reproduce the free-solvation energy of small solutes, based on a standard thermodynamic integration scheme. The reliability of this new approach has been checked for the properties of solvated methane and of the solvated methane dimer, as well as by performing 10 × 20 ns molecular dynamics (MD) trajectories for three solvated proteins. A long-time stability of the protein structures along the trajectories is observed. Moreover, our method still provides a measure of the protein solvation thermodynamic at the same accuracy as standard Poisson-Boltzman continuum methods. These results show the relevance of our approach and its applicability to massively coupled MD schemes to accurately and intensively explore solvated macromolecule potential energy surfaces.     

The role of plastic beta-hairpin and weak hydrophobic core in the stability and unfolding of a full sequence design protein

In this study, the thermal stability of a designed alpha/beta protein FSD (full sequence design) was studied by explicit solvent simulations at three moderate temperatures, 273 K, 300 K, and 330 K. The average properties of the ten trajectories at each temperature were analyzed. The thermal unfolding, as judged by backbone root-mean-square deviation and percentage of native contacts, was displayed with increased sampling outside of the native basin as the temperature was raised. The positional fluctuation of the hairpin residues was significantly higher than that of the helix residues at all three temperatures. The hairpin segment displayed certain plasticity even at 273 K. Apart from the terminal residues, the highest fluctuation was shown in the turn residues 7-9. Secondary structure analysis manifested the structural heterogeneity of the hairpin segment. It was also revealed by the simulation that the hydrophobic core was vulnerable to thermal denaturation. Consistent with the experiment, the I7Y mutation in the double mutant FSD-EY (FSD with mutations Q1E and I7Y) dramatically increased the protein stability in the simulation, suggesting that the plasticity of the hairpin can be partially compensated by a stronger hydrophobic core. As for the unfolding pathway, the breathing of the hydrophobic core and the separation of the two secondary structure elements (alpha helix and beta hairpin) was the initiation step of the unfolding. The loss of global contacts from the separation further destabilized the hairpin structure and also led to the unwinding of the helix.     

Protein stability prediction: a Poisson-Boltzmann approach

Most proteins are only marginally stable at physiological temperatures. Thus a common defect due to mutation is the loss of protein stability, resulting in loss of their well-defined structures and functions at their functioning temperatures. Quantification of protein stability change upon mutation has attracted a large number of experimental and theoretical studies. In this work, we have extended the Poisson-Boltzmann theory that is originally used for predicting stability changes of charged mutations to predicting stability changes of all mutations. To achieve this, we have proposed a free energy model covering both electrostatic and hydrophobic interactions. A G?-like model for the denatured state that incorporates both nativeness and randomness of the denatured state has been used to calculate the hydrophobic contribution to protein stability. The new model is computationally simple and fast, and performs well for charged and hydrophobic mutations for all four tested proteins. Future directions for extending the method into pH-dependent effect and more accurate prediction for polar mutations are discussed.     

A structural study of model peptides derived from HIV-1 integrase central domain

The HIV-1 integrase (IN) catalyzes the integration of viral DNA in the human genome. In vitro the enzyme displays an equilibrium of monomers, dimers, tetramers and larger oligomers. However, its functional oligomeric form in vivo is not known. We report a study of the auto-associative properties of three peptides denoted K156, E156 and E159. These derive from the alpha4 helix of the IN catalytic core. The alpha4 helix is an amphipatic helix exposed at the surface of the protein and could be involved in the oligomerization process through its hydrophobic face. The peptides were obtained from the replacement of several amino acid residues by more helicogenic ones in the alpha4 helix peptide. K156 carries the basic residues Lys156 and Lys159, which have been shown important for the binding of IN to viral DNA. In E156 and E159 they are replaced with the acidic residue Glu. A fourth peptide K(E)156 obtained from the replacement of hydrophobic residues with Glu in K156 in order to abolish the auto-associative properties is used as a negative control. The capacity shown by peptides for alpha-helical formation is demonstrated by circular dichroism (CD) analysis performed in aqueous solution and in aqueous trifluoroethanol (TFE) mixtures. Both electrospray ionization mass spectrometry (ESI-MS) and glutaraldehyde chemical cross-linking show that peptides adopt different solvent-dependent equilibriums of monomers, dimers, trimers and tetramers. Oligomerization of peptides in aqueous solution is related to their ability to form helical structures. Addition of a small amount of TFE (<10%) stimulates helix stabilization and the interhelical hydrophobic contacts. Higher amounts of TFE alter the hydrophobic contacts and disrupt the oligomeric species. In addition to hydrophobic interactions, the patterns indicate that the biologically important Lys156 and Lys159 residues also participate in helix association. K(E)156 despite its ability to adopt a helical structure is unable to associate into oligomers, demonstrating the importance of hydrophobic contacts for oligomerization. Thus, the designed peptides provide us information on the functional properties of the alpha4 IN that seems to hold a dual role in DNA recognition and protein oligomerization.     

Self-organization in protein folding and the hydrophobic interaction

Self-organization is a critical aspect of living systems. During the folding of protein molecules, the hydrophobic interaction plays an important role in the collapse of the peptide chain to a compact shape. As the hydrophobic core tightens and excludes water, not only does the number of hydrophobic side chain contacts increase, but stabilization is further enhanced by an increase in strength of each hydrophobic interaction between side chains in the core. Thus, the self-organization of the protein folding process augments itself by enhancing the stability of the core against large-scale motions that would unfold the protein. Through calculations and computer simulations on a model four-helix bundle protein, we show how the strengthening of the hydrophobic interaction is crucial for stabilizing the core long enough for completion of the folding process and quantitatively manifests self-organizing dynamical behavior.     

Hydrophobic core repacking in a coiled-coil dimer via phage display: insights into plasticity and specificity at a protein-protein interface

The coiled-coil, which consists of two or more interwoven amphiphilic alpha-helices, is formed by sequences that have a characteristic heptad repeat (abcdefg) where a and d are hydrophobic residues. Most efforts to elucidate the origins of coiled-coil pairing selectivity have focused on electrostatic interactions among side chains that flank the core (positions e and g) and on polar side chains that occur occasionally at core positions. We have used phage display to explore another source of coiled-coil specificity: steric matching among nonpolar side chains in the core. We introduced a destabilizing Leu-->Ala mutation into the core of one helix in a known heterodimer and then screened a phage-based library of potential partner helices in search of compensating mutations. We identified a new heterodimer pair (30 residues/helix) that is comparable in stability to the GCN4-p1 homodimer (33 residues/helix). Furthermore, the Leu-->Ala mutant shows specificity for its phage-derived partner over the original partner despite their similar sequences. These results show that a phage-based approach can provide unique insights on coiled-coil pairing preferences that should facilitate both the analysis of natural sequences and the development of specific dimerization motifs that are orthogonal to one another.     

Single Molecule Force Spectroscopy Reveals Critical Roles of Hydrophobic Core Packing in Determining the Mechanical Stability of Protein GB1

Understanding molecular determinants of protein mechanical stability is important not only for elucidating how elastomeric proteins are designed and functioning in biological systems but also for designing protein building blocks with defined nanomechanical properties for constructing novel biomaterials. GB1 is a small α/β protein and exhibits significant mechanical stability. It is thought that the shear topology of GB1 plays an important role in determining its mechanical stability. Here, we combine single molecule atomic force microscopy and protein engineering techniques to investigate the effect of side chain reduction and hydrophobic core packing on the mechanical stability of GB1. We engineered seven point mutants and carried out mechanical ?-value analysis of the mechanical unfolding of GB1. We found that three mutations, which are across the surfaces of two subdomains that are to be sheared by the applied stretching force, in the hydrophobic core (F30L, Y45L, and F52L) result in significant decrease in mechanical unfolding force of GB1. The mechanical unfolding force of these mutants drop by 50-90 pN compared with wild-type GB1, which unfolds at around 180 pN at a pulling speed of 400 nm/s. These results indicate that hydrophobic core packing plays an important role in determining the mechanical stability of GB1 and suggest that optimizing hydrophobic interactions across the surfaces that are to be sheared will likely be an efficient method to enhance the mechanical stability of GB1 and GB1 homologues.     

Computational probe of cavitation events in protein systems

Previous all-atom simulations have identified several classes of proteins where hydrophobic de-wetting (cavitation) is at play. Here we develop and validate a computationally fast method that predicts in which protein systems water spontaneously cavitates. We implement a cubic lattice model, which incorporates the protein shape from crystallographic data and the protein-water interactions from thermodynamic data. Combining it with the previously developed coarse-grained model for water, we determine the extent of occupancy of water at protein-protein interfaces and in protein-ligand cavities. The model captures essential findings from all-atom molecular dynamics studies on the same systems by distinguishing protein cavities that dry from those that remain wet. We also interpret the origin of the cavitation inside the melittin tetramer on simple thermodynamic grounds, and show that part of the mellitin surface is sufficiently hydrophobic to trigger cavitation. Using Glauber/Kawasaki dynamics we obtain the time-scales for de-wetting events that are in agreement with those from all-atom simulations. The method can serve as an intermediate step between the necessary initial screening that identifies proteins with abundance of hydrophobic patches using bioinformatics tools (L. Hua, X. H. Huang, P. Liu, R. H. Zhou and B. J. Berne, J. Phys. Chem. B, 2007, 111, 9069), and computationally extensive studies that need to incorporate molecular details (e.g. single mutation studies of amino acid residues).