Conservative mutation Met8 --> Leu affects the folding process and structural stability of squash trypsin inhibitor CMTI-I

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process.     

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

Highly fluorinated analogs of hydrophobic amino acids are well known to increase the stability of proteins toward thermal unfolding and chemical denaturation, but there is very little data on the structural consequences of fluorination. We have determined the structures and folding energies of three variants of a de novo designed 4‐helix bundle protein whose hydrophobic cores contain either hexafluoroleucine (hFLeu) or t‐butylalanine (tBAla). Although the buried hydrophobic surface area is the same for all three proteins, the incorporation of tBAla causes a rearrangement of the core packing, resulting in the formation of a destabilizing hydrophobic cavity at the center of the protein. In contrast, incorporation of hFLeu, causes no changes in core packing with respect to the structure of the nonfluorinated parent protein which contains only leucine in the core. These results support the idea that fluorinated residues are especially effective at stabilizing proteins because they closely mimic the shape of the natural residues they replace while increasing buried hydrophobic surface area.     

Polar residues in the protein core of Escherichia coli thioredoxin are important for fold specificity

Most globular proteins contain a core of hydrophobic residues that are inaccessible to solvent in the folded state. In general, polar residues in the core are thermodynamically unfavorable except when they are able to form intramolecular hydrogen bonds. Compared to hydrophobic interactions, polar interactions are more directional in character and may aid in fold specificity. In a survey of 263 globular protein structures, we found a strong positive correlation between the number of polar residues at core positions and protein size. To probe the importance of buried polar residues, we experimentally tested the effects of hydrophobic mutations at the five polar core residues in Escherichia coli thioredoxin. Proteins with single hydrophobic mutations (D26I, C32A, C35A, T66L, and T77V) all have cooperative unfolding transitions like the wild type (wt), as determined by chemical denaturation. Relative to wt, D26I is more stable while the other point mutants are less stable. The combined 5-fold mutant protein (IAALV) is less stable than wt and has an unfolding transition that is substantially less cooperative than that of wt. NMR spectra as well as amide deuterium exchange indicate that IAALV is likely sampling a number of low-energy structures in the folded state, suggesting that polar residues in the core are important for specifying a well-folded native structure.     

Structural insights into the evolution of a non-biological protein: importance of surface residues in protein fold optimization

Phylogenetic profiling of amino acid substitution patterns in proteins has led many to conclude that most structural information is carried by interior core residues that are solvent inaccessible. This conclusion is based on the observation that buried residues generally tolerate only conserved sequence changes, while surface residues allow more diverse chemical substitutions. This notion is now changing as it has become apparent that both core and surface residues play important roles in protein folding and stability. Unfortunately, the ability to identify specific mutations that will lead to enhanced stability remains a challenging problem. Here we discuss two mutations that emerged from an in vitro selection experiment designed to improve the folding stability of a non-biological ATP binding protein. These mutations alter two solvent accessible residues, and dramatically enhance the expression, solubility, thermal stability, and ligand binding affinity of the protein. The significance of both mutations was investigated individually and together, and the X-ray crystal structures of the parent sequence and double mutant protein were solved to a resolution limit of 2.8 and 1.65 A, respectively. Comparative structural analysis of the evolved protein to proteins found in nature reveals that our non-biological protein evolved certain structural features shared by many thermophilic proteins. This experimental result suggests that protein fold optimization by in vitro selection offers a viable approach to generating stable variants of many naturally occurring proteins whose structures and functions are otherwise difficult to study.     

Using physical features of protein core packing to distinguish real proteins from decoys

The ability to consistently distinguish real protein structures from computationally generated model decoys is not yet a solved problem. One route to distinguish real protein structures from decoys is to delineate the important physical features that specify a real protein. For example, it has long been appreciated that the hydrophobic cores of proteins contribute significantly to their stability. We used two sources to obtain datasets of decoys to compare with real protein structures: submissions to the biennial Critical Assessment of protein Structure Prediction competition, in which researchers attempt to predict the structure of a protein only knowing its amino acid sequence, and also decoys generated by 3DRobot, which have user‐specified global root‐mean‐squared deviations from experimentally determined structures. Our analysis revealed that both sets of decoys possess cores that do not recapitulate the key features that define real protein cores. In particular, the model structures appear more densely packed (because of energetically unfavorable atomic overlaps), contain too few residues in the core, and have improper distributions of hydrophobic residues throughout the structure. Based on these observations, we developed a feed‐forward neural network, which incorporates key physical features of protein cores, to predict how well a computational model recapitulates the real protein structure without knowledge of the structure of the target sequence. By identifying the important features of protein structure, our method is able to rank decoy structures with similar accuracy to that obtained by state‐of‐the‐art methods that incorporate many additional features. The small number of physical features makes our model interpretable, emphasizing the importance of protein packing and hydrophobicity in protein structure prediction.     

Increasing the thermostability of staphylococcal nuclease: implications for the origin of protein thermostability

Seven hyper-stable multiple mutants have been constructed in staphylococcal nuclease by various combinations of eight different stabilizing single mutants. The stabilities of these multiple mutants determined by guanidine hydrochloride denaturation were 3.4 to 5.6 kcal/mol higher than that of the wild-type. Their thermal denaturation midpoint temperatures were 12.6 to 22.9 deg. C higher than that of the wild-type. These are among the greatest increases in protein stability and thermal denaturation midpoint temperature relative to the wild-type yet attained. There has been great interest in understanding how proteins found in thermophilic organisms are stabilized. One frequently cited theory is that the packing of hydrophobic side-chains is improved in the cores of proteins isolated from thermophiles when compared to proteins from mesophiles. The crystal structures of four single and five multiple stabilizing mutants of staphylococcal nuclease were solved to high resolution. No large overall structural change was found, with most changes localized around the sites of mutation. Rearrangements were observed in the packing of side-chains in the major hydrophobic core, although none of the mutations was in the core. It is surprising that detailed structural analysis showed that packing had improved, with the volume of the mutant protein's hydrophobic cores decreasing as protein stability increased. Further, the number of van der Waals interactions in the entire protein showed an experimentally significant increase correlated with increasing stability. These results indicate that optimization of packing follows as a natural consequence of increased protein thermostability and that good packing is not necessarily the proximate cause of high stability. Another popular theory is that thermostable proteins have more electrostatic and hydrogen bonding interactions and these are responsible for the high stabilities. The mutants here show that increased numbers of electrostatic and hydrogen bonding interactions are not obligatory for large increases in protein stability.     

Stabilization of proteins by enhancement of inter-residue hydrophobic contacts: lessons of T4 lysozyme and barnase

Although the hydrophobic interactions are considered as the main contributors to the protein stability, not much examples of protein stabilization by rational increasing of this type of interactions still can be found in literature. This is partly due to the lack of proper theoretical "measure" of hydrophobic interactions and their changes upon mutations. In the present paper the molecular hydrophobicity potential approach is used to assess how the changes in type and the strength of inter-residue contacts upon single amino acid mutations are correlated with the changes in thermodynamic stability of T4 lysozyme and barnase mutants, and which factors affect these correlations. Mutations changing unfavorable hydrophilic-to-hydrophobic contacts into favorable hydrophobic were found to enhance the thermodynamic stability in more than 81 % of cases, if these mutations do not create steric bumps and do not involve proline residues and hydrogen-bonded side-chains. Mutations increasing hydrophobic contributions (according to molecular hydrophobicity potential formalism) lead to increase of thermodynamic stability in more than 94% of cases for certain type of mutations (i.e., mutations not involving charged residues, Pro and residues with side-chain hydrogen bonds, when these mutations do not introduce steric bumps and do not involve strongly exposed residues and residues situated at helix N- and C-cap positions). For this type of mutations the correlation was found between the change in hydrophobic contributions of mutated residues deltaCphob and thermodynamic parameters deltaTm (change in melting temperature) and deltadeltaG (change in free energy of unfolding). Although the correlation coefficients were larger if the experimental structures of mutants were used for the calculations (correlation coefficients r(exp) deltaC,deltaT = .85 and r(exp) deltaC,deltadeltaG = 0.87) than if the modeled structures were used instead (r(mod) deltaC,deltaT = 0.74 and r(mod)deltaC,deltadeltaG = 0.76), the modelled structures of mutants in the vast majority of cases can be used for qualitative predictition of the protein stabilization. Basing on the analysis of mutations increasing hydrophobic contributions in T4 lysozyme the substitution matrix was derived, which can be used to decide which new residue should be put instead the old one to increase the stability of protein. The estimation shows that the number of potential mutation sites for enhancement of hydrophobic interactions in T4 lysozyme is quite large, and only approximately 10 per cent of them were studied thus far. Basing on the current analysis of T4 lysozyme and barnase mutations the algorithm for increasing of protein stability via increasing of hydrophobic interactions for the proteins with known spatial structure is proposed.     

Effect of site-directed point mutations on protein misfolding: A simulation study

A Monte Carlo simulation based sequence design method is proposed to investigate the role of site-directed point mutations in protein misfolding. Site-directed point mutations are incorporated in the designed sequences of selected proteins. While most mutated sequences correctly fold to their native conformation, some of them stabilize in other nonnative conformations and thus misfold/unfold. The results suggest that a critical number of hydrophobic amino acid residues must be present in the core of the correctly folded proteins, whereas proteins misfold/unfold if this number of hydrophobic residues falls below the critical limit. A protein can accommodate only a particular number of hydrophobic residues at the surface, provided a large number of hydrophilic residues are present at the surface and critical hydrophobicity of the core is preserved. Some surface sites are observed to be equally sensitive toward site-directed point mutations as the core sites. Point mutations with highly polar and charged amino acids increases the misfold/unfold propensity of proteins. Substitution of natural amino acids at sites with different number of nonbonded contacts suggests that both amino acid identity and its respective site-specificity determine the stability of a protein. A clash-match method is developed to calculate the number of matching and clashing interactions in the mutated protein sequences. While misfolded/unfolded sequences have a higher number of clashing and a lower number of matching interactions, the correctly folded sequences have a lower number of clashing and a higher number of matching interactions. These results are valid for different SCOP classes of proteins.     

Charge centers and formation of the protein folding core

Electrostatic interactions are important for protein folding. At low resolution, the electrostatic field of the whole molecule can be described in terms of charge center(s). To study electrostatic effects, the centers of positive and negative charge were calculated for 20 small proteins of known structure, for which hydrogen exchange cores had been determined experimentally. Two observations seem to be important. First, in all 20 proteins studied 30-100% of the residues forming hydrogen exchange core(s) were clustered around the charge centers. Moreover, in each protein more than half of the core sequences are located near the centers of charge. Second, the general architecture of all-alpha proteins from the set seems to be stabilized by interactions of residues surrounding the charge centers. In most of the alpha-beta proteins, either or both of the centers are located near a pair of consecutive strands, and this is even more characteristic for alpha/Beta and all-beta structures. Consecutive strands are very probable sites of early folding events. These two points lead to the conclusion that charge centers, defined solely from the structure of the folded protein may indicate the location of a protein's hydrogen exchange/folding core. In addition, almost all the proteins contain well-conserved continuous hydrophobic sequences of three or more residues located in the vicinity of the charge centers. These hydrophobic sequences may be primary nucleation sites for protein folding. The results suggest the following scheme for the order of events in folding: local hydrophobic nucleation, electrostatic collapse of the core, global hydrophobic collapse, and slow annealing to the native state. This analysis emphasizes the importance of treating electrostatics during protein-folding simulations.     

X-ray structures of three interface mutants of gammaB-crystallin from bovine eye lens.

GammaB-crystallin consists of two domains each comprising two "Greek key" motifs. Both domains fold independently, and domain interactions contribute significantly to the stability of the C-terminal domain. In a previous study (Palme S et al., 1996, Protein Sci 6:1529-1636) it was shown that Phe56 from the N-terminal domain, a residue involved in forming a hydrophobic core at the domain interface, effects the interaction of the two domains, and therefore, the stability of the C-terminal domain. Ala or Asp at position 56 drastically decreased the stability of the C-terminal domain, whereas Trp had a more moderate effect. In this article we present the X-ray structures of these interface mutants and correlate them with the stability data. The mutations do not effect the overall structure of the molecule. No structural changes are observed in the vicinity of the replaced residue, suggesting that the local structure is too rigid to allow compensations for the amino acid replacements. In the mutants gammaB-F56A and -F56D, a solvent-filled groove accessible to the bulk solvent is created by the replacement of the bulky Phe side chain. In gammaB-F56W, the pyrrole moiety of the indole ring replaces the phenyl side chain of the wild type. With the exception of gammaB-F56W, there is a good correlation between the hydrophobicity of the amino acid at position 56 according to the octanol scale and the stability of the C-terminal domain. In gammaB-F56W, the C-terminal domain is less stable than estimated from the hydrophobicity, presumably because the ring nitrogen (Nepsilon1) has no partner to form hydrogen bonds. The data suggest that the packing of hydrophobic residues in the interface core is important for domain interactions and the stability of gammaB-crystallin. Apparently, for protein stability, the same principles apply for hydrophobic cores within domains and at domain interfaces.     

Surface hydrophobic groups, stability, and flip-flopping in lattice proteins

Two-dimensional lattice protein models were studied in two approximations of the conformational equilibrium to elucidate the role of surface hydrophobic groups in their stabilities. We demonstrate that stability of any compactly folded sequence is determined by its ability to "flip-flop" (refold) into alternative compact structures. The degree of stability required for folded sequences determines the average numbers of surface hydrophobic groups in stable lattice structures which are in good agreement with ratios of core to surface hydrophobic groups in real proteins. However, the average destabilization of the native structure per surface hydrophobic group is small (0-0.25 kcal/mol), often disagrees with the free energies derived from the ratios of core to surface hydrophobic groups in the same structures, and has a combinatorial entropic nature independent of the strength of structure stabilizing interactions. This suggests that the free energies derived from the core to surface ratios of hydrophobic groups in real proteins have little to do with folding thermodynamics. On average, sequences with highly stable native structures are the least hydrophobic. The results suggest that in designing novel stable proteins hydrophobic groups on the surface should be avoided to reduce the possibility of flip-flopping. The average stability of highly designable structures is never higher than that of some low designability structures, contrary to the accepted view. In the equilibrium approximation with alternative compact and partially unfolded structures, the requirement of high stability selects a unique 5 x 5 structure formed by only a few sequences, suggesting much stronger sequence selectivity than commonly thought.     

Differential stabilization of two hydrophobic cores in the transition state of the villin 14T folding reaction

We report the distribution of hydrophobic core contacts during the folding reaction transition state for villin 14T, a small 126-residue protein domain. The solution structure of villin 14T contains a central beta-sheet with two flanking hydrophobic cores; transition states for this protein topology have not been previously studied. Villin 14T has no disulfide bonds or cis-proline residues in its native state; it folds reversibly, and in an apparently two-state manner under some conditions. To map the hydrophobic core contacts in the transition state, 27 point mutations were generated at positions spread throughout the two hydrophobic cores. After each point mutation, comparison of the change in folding kinetics with the equilibrium destabilization indicates whether the site of mutation is stabilized in the transition state. The results show that the folding nucleus, or the sub-region with the strongest transition state contacts, is located in one of the two hydrophobic cores (the predominantly aliphatic core). The other hydrophobic core, which is mostly aromatic, makes much weaker contacts in the transition state. This work is the first transition state mapping for a protein with multiple major hydrophobic cores in a single folding unit; the hydrophobic cores cannot be separated into individual folding subdomains. The stabilization of only one hydrophobic core in the transition state illustrates that hydrophobic core formation is not intrinsically capable of nucleating folding, but must also involve the right specific interactions or topological factors in order to be kinetically important.     

Prediction of solvent accessibility and sites of deleterious mutations from protein sequence

Residues that form the hydrophobic core of a protein are critical for its stability. A number of approaches have been developed to classify residues as buried or exposed. In order to optimize the classification, we have refined a suite of five methods over a large dataset and proposed a metamethod based on an ensemble average of the individual methods, leading to a two-state classification accuracy of 80%. Many studies have suggested that hydrophobic core residues are likely sites of deleterious mutations, so we wanted to see to what extent these sites can be predicted from the putative buried residues. Residues that were most confidently classified as buried were proposed as sites of deleterious mutations. This proposition was tested on six proteins for which sites of deleterious mutations have previously been identified by stability measurement or functional assay. Of the total of 130 residues predicted as sites of deleterious mutations, 104 (or 80%) were correct.     

Correlation between sequence hydrophobicity and surface-exposure pattern of database proteins

Hydrophobicity is thought to be one of the primary forces driving the folding of proteins. On average, hydrophobic residues occur preferentially in the core, whereas polar residues tend to occur at the surface of a folded protein. By analyzing the known protein structures, we quantify the degree to which the hydrophobicity sequence of a protein correlates with its pattern of surface exposure. We have assessed the statistical significance of this correlation for several hydrophobicity scales in the literature, and find that the computed correlations are significant but far from optimal. We show that this less than optimal correlation arises primarily from the large degree of mutations that naturally occurring proteins can tolerate. Lesser effects are due in part to forces other than hydrophobicity, and we quantify this by analyzing the surface-exposure distributions of all amino acids. Lastly, we show that our database findings are consistent with those found from an off-lattice hydrophobic-polar model of protein folding.     

Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein's physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein's global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.     

Insights from the structural analysis of protein heterodimer interfaces

Protein heterodimer complexes are often involved in catalysis, regulation, assembly, immunity and inhibition. This involves the formation of stable interfaces between the interacting partners. Hence, it is of interest to describe heterodimer interfaces using known structural complexes. We use a non-redundant dataset of 192 heterodimer complex structures from the protein databank (PDB) to identify interface residues and describe their interfaces using amino-acids residue property preference. Analysis of the dataset shows that the heterodimer interfaces are often abundant in polar residues. The analysis also shows the presence of two classes of interfaces in heterodimer complexes. The first class of interfaces (class A) with more polar residues than core but less than surface is known. These interfaces are more hydrophobic than surfaces, where protein-protein binding is largely hydrophobic. The second class of interfaces (class B) with more polar residues than core and surface is shown. These interfaces are more polar than surfaces, where binding is mainly polar. Thus, these findings provide insights to the understanding of protein-protein interactions.     

Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.     

Increasing protein stability using a rational approach combining sequence homology and structural alignment: Stabilizing the WW domain

This study shows that a combination of sequence homology and structural information can be used to increase the stability of the WW domain by 2.5 kcal mol(-1) and increase the T(m) by 28 degrees C. Previous homology-based protein design efforts typically investigate positions with low sequence identity, whereas this study focuses on semi-conserved core residues and proximal residues, exploring their role(s) in mediating stabilizing interactions on the basis of structural considerations. The A20R and L30Y mutations allow increased hydrophobic interactions because of complimentary surfaces and an electrostatic interaction with a third residue adjacent to the ligand-binding hydrophobic cluster, increasing stability significantly beyond what additivity would predict for the single mutations. The D34T mutation situated in a pi-turn possibly disengages Asn31, allowing it to make up to three hydrogen bonds with the backbone in strand 1 and loop 2. The synergistic mutations A20R/L30Y in combination with the remotely located mutation D34T add together to create a hYap WW domain that is significantly more stable than any of the protein structures on which the design was based (Pin and FBP28 WW domains).     

Fluorous effect in proteins: de novo design and characterization of a four-alpha-helix bundle protein containing hexafluoroleucine

Several studies have demonstrated that proteins incorporating fluorinated analogues of hydrophobic amino acids such as leucine and valine into their hydrophobic cores exhibit increased stability toward thermal denaturation and unfolding by guanidinium chloride. However, estimates for the increase in the thermodynamic stability of a protein (DeltaDeltaG(unfold)) afforded by the substitution of a hydrophobic amino acid with its fluorinated analogue vary quite significantly. To address this, we have designed a peptide that adopts an antiparallel four-helix bundle structure in which the hydrophobic core is packed with leucine, and investigated the effects of substituting the central two layers of the core with L-5,5,5,5',5',5'-hexafluoroleucine (hFLeu). We find that DeltaDeltaG(unfold) is increased by 0.3 kcal/mol per hFLeu residue. This is in good agreement with the predicted increase in DeltaDeltaG(unfold) of 0.4 kcal/mol per residue arising from the increased hydrophobicity of the hFLeu side chain, which we determined experimentally from partitioning measurements on hFLeu and leucine. The increased stability of this fluorinated protein may therefore be ascribed to simple hydrophobic effects, rather than specific "fluorous" interactions between the hFLeu residues.