A three-state prediction of single point mutations on protein stability changes.
ABSTRACT: BACKGROUND: A basic question of protein structural studies is to which extent mutations affect the stability. This question may be addressed starting from sequence and/or from structure. In proteomics and genomics studies prediction of protein stability free energy change (DeltaDeltaG) upon single point mutation may also help the annotation process. The experimental DeltaDeltaG values are affected by uncertainty as measured by standard deviations. Most of the DeltaDeltaG values are nearly zero (about 32% of the DeltaDeltaG data set ranges from -0.5 to 0.5 kcal/mole) and both the value and sign of DeltaDeltaG may be either positive or negative for the same mutation blurring the relationship among mutations and expected DeltaDeltaG value. In order to overcome this problem we describe a new predictor that discriminates between 3 mutation classes: destabilizing mutations (DeltaDeltaG<-1.0 kcal/mol), stabilizing mutations (DeltaDeltaG>1.0 kcal/mole) and neutral mutations (-1.0
Project description:Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (DeltaDeltaG), thus suggesting a tradeoff between the acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (DeltaDeltaG>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied DeltaDeltaG computations by FoldX to analyze the effects of 548 mutations that arose from the directed evolution of 22 different enzymes. The stability effects, location, and type of function-altering mutations were compared to DeltaDeltaG changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average DeltaDeltaG = +0.9 kcal/mol), and are almost as destabilizing as the "average" mutation in these enzymes (+1.3 kcal/mol). Although their stability effects are not as dramatic as in key catalytic residues, mutations that modify the substrate binding pockets, and thus mediate new enzymatic specificities, place a larger stability burden than surface mutations that underline neutral, non-adaptive evolutionary changes. How are the destabilizing effects of functional mutations balanced to enable adaptation? Our analysis also indicated that many mutations that appear in directed evolution variants with no obvious role in the new function exert stabilizing effects that may compensate for the destabilizing effects of the crucial function-altering mutations. Thus, the evolution of new enzymatic activities, both in nature and in the laboratory, is dependent on the compensatory, stabilizing effect of apparently "silent" mutations in regions of the protein that are irrelevant to its function.
Project description:Accurately predicting changes in protein stability upon amino acid substitution is a much sought after goal. Destabilizing mutations are often implicated in disease, whereas stabilizing mutations are of great value for industrial and therapeutic biotechnology. Increasing protein stability is an especially challenging task, with random substitution yielding stabilizing mutations in only ∼2% of cases. To overcome this bottleneck, computational tools that aim to predict the effect of mutations have been developed; however, achieving accuracy and consistency remains challenging. Here, we combined 11 freely available tools into a meta-predictor (meieringlab.uwaterloo.ca/stabilitypredict/). Validation against ∼600 experimental mutations indicated that our meta-predictor has improved performance over any of the individual tools. The meta-predictor was then used to recommend 10 mutations in a previously designed protein of moderate thermodynamic stability, ThreeFoil. Experimental characterization showed that four mutations increased protein stability and could be amplified through ThreeFoil's structural symmetry to yield several multiple mutants with >2-kcal/mol stabilization. By avoiding residues within functional ties, we could maintain ThreeFoil's glycan-binding capacity. Despite successfully achieving substantial stabilization, however, almost all mutations decreased protein solubility, the most common cause of protein design failure. Examination of the 600-mutation data set revealed that stabilizing mutations on the protein surface tend to increase hydrophobicity and that the individual tools favor this approach to gain stability. Thus, whereas currently available tools can increase protein stability and combining them into a meta-predictor yields enhanced reliability, improvements to the potentials/force fields underlying these tools are needed to avoid gaining protein stability at the cost of solubility.
Project description:A theoretical approach for estimating association free energies of alpha-helices in nonpolar media has been developed. The parameters of energy functions have been derived from DeltaDeltaG values of mutants in water-soluble proteins and partitioning of organic solutes between water and nonpolar solvents. The proposed approach was verified successfully against three sets of published data: (1) dissociation constants of alpha-helical oligomers formed by 27 hydrophobic peptides; (2) stabilities of 22 bacteriorhodopsin mutants, and (3) protein-ligand binding affinities in aqueous solution. It has been found that coalescence of helices is driven exclusively by van der Waals interactions and H-bonds, whereas the principal destabilizing contributions are represented by side-chain conformational entropy and transfer energy of atoms from a detergent or lipid to the protein interior. Electrostatic interactions of alpha-helices were relatively weak but important for reproducing the experimental data. Immobilization free energy, which originates from restricting rotational and translational rigid-body movements of molecules during their association, was found to be less than 1 kcal/mole. The energetics of amino acid substitutions in bacteriorhodopsin was complicated by specific binding of lipid and water molecules to cavities created in certain mutants.
Project description:The hen (chicken) egg-white lysozyme (HEWL) epitope for the monoclonal antibody HyHEL-10 Fab (Fab-10) was investigated by alanine scan mutagenesis. The association rate constants (k(on)) for the HEWL Fab-10 complexes were obtained from the homogenous solution method described in the preceding paper (Taylor et al., 1998). A new method for determining the dissociation rate constant (k(off)) for the complex, by trapping nascent free antibody with an inactive HEWL mutant is described. The values of k(on) fall within a factor of 2 of the wild-type (WT) HEWL value (1.43+/-0.13 X 10(6)M(-1)s(-1)), while the increases in k(off)more nearly reflect the total change in free energies of the complex (deltadeltaG(D)). The dissociation constants (K(D)) were measured directly in those cases where satisfactory kinetic data could not be obtained. The Y20A, K96A, and K97A HEWL.Fab-10 complexes are destabilized by more than 4 kcal/mol compared to the WT complex. The R21A, L75A, and D101A antibody complexes are moderately destabilized (0.7 < deltadeltaG(D)< or = 1.0 kcal/mol). Additional mutations of the "hotspot" residues (Tyr20, Lys96, Lys97) were constructed to probe, more precisely, the nature of their contributions to complex formation. The results show that the entire hydrocarbon side chains of Tyr20 and Lys97, and only the epsilon-amino group of Lys96, contribute to the stability of the complex. The value of deltadeltaG(D) for the R21A mutant complex is a distinct outlier in the Arg21 replacement series demonstrating the importance of supplementing alanine scan mutagenesis with additional mutations.
Project description:Reverse cholesterol transport in plasma involves variations in HDL cholesterol concentration. To understand physicochemical and functional implications of such variations, we analyzed stability of reconstituted HDL containing human apolipoproteins (apoA-I, apoA-II, or apoC-I), phosphatidylcholines varying in chain length (12-18 carbons) and unsaturation (0 or 1), and 0-35 mol% cholesterol. Lipoprotein heat denaturation was monitored by circular dichroism for protein unfolding/dissociation and by light scattering for particle fusion. We found that cholesterol stabilizes relatively unstable complexes; for example, incorporation of 10-30 mol% cholesterol in apoC-I:dimyristoyl phosphatidylcholine complexes increased their kinetic stability by deltaDeltaG* congruent with 1 kcal/mol. In more stable complexes containing larger proteins and/or longer-chain lipids, incorporation of 10% cholesterol did not significantly alter the disk stability; however, 15% or more cholesterol destabilized the apoA-I-containing complexes and led to vesicle formation. Thus, cholesterol tends to stabilize less stable lipoproteins, apparently by enhancing favorable packing interactions, but in more stable lipoproteins, where such interactions are already highly optimized, the stabilizing effect of cholesterol decreases and, eventually, becomes destabilizing. These results help uncouple the functional roles of particle stability and chain fluidity and suggest that structural disorder in HDL surface, rather than chain fluidity, is an important physicochemical determinant of HDL function.
Project description:We carried our Poisson-Boltzmann (PB) calculations for the effects of charge reversal at five exposed sites (K16E, R119E, K135E, K147E, and R154E) and charge neutralization and proton titration of the H31-D70 semi-buried salt bridge on the stability of T4 lysozyme. Instead of the widely used solvent-exclusion (SE) surface, we used the van der Waals (vdW) surface as the boundary between the protein and solvent dielectrics (a protocol established in our earlier study on charge mutations in barnase). By including residual charge-charge interactions in the unfolded state, the five charge reversal mutations were found to have DeltaDeltaG(unfold) from -1.6 to 1.3 kcal/mol. This indicates that the variable effects of charge reversal observed by Matthews and co-workers are not unexpected. The H31N, D70N, and H31N/D70N mutations were found to destabilize the protein by 2.9, 1.3, and 1.6 kcal/mol, and the pK(a) values of H31 and D70 were shifted to 9.4 and 0.6, respectively. These results are in good accord with experimental data of Dahlquist and co-workers. In contrast, if the SE surface were used, the H31N/D70N mutant would be more stable than the wild-type protein by 1.3 kcal/mol. From these and additional results for 27 charge mutations on five other proteins, we conclude that 1) the popular view that electrostatic interactions are generally destabilizing may have been based on overestimated desolvation cost as a result of using the SE surface as the dielectric boundary; and 2) while solvent-exposed charges may not reliably contribute to protein stability, semi-buried salt bridges can provide significant stabilization.
Project description:Alanine scanning mutagenesis of the HyHEL-10 paratope of the HyHEL-10/HEWL complex demonstrates that the energetically important side chains (hot spots) of both partners are in contact. A plot of deltadeltaG(HyHEL-10_mutant) vs. deltadeltaG(HEWL_mutant) for the five of six interacting side-chain hydrogen bonds is linear (Slope = 1). Only 3 of the 13 residues in the HEWL epitope contribute >4 kcal/mol to the free energy of formation of the complex when replaced by alanine, but 6 of the 12 HyHEL-10 paratope amino acids do. Double mutant cycle analysis of the single crystallographically identified salt bridge, D32H/K97, shows that there is a significant energetic penalty when either partner is replaced with a neutral side-chain amino acid, but the D32(H)N/K97M complex is as stable as the WT. The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing the deltadeltaG values of the Tyr --> Phe vs. the corresponding Tyr --> Ala mutations. The nonpolar contacts in the light chain contribute only about one-half of the total deltadeltaG observed for the Tyr --> Ala mutation, while they are significantly more important in the heavy chain. Replacement of the N31L/K96 hydrogen bond with a salt bridge, N31D(L)/K96, destabilizes the complex by 1.4 kcal/mol. The free energy of interaction, deltadeltaG(int), obtained from double mutant cycle analysis showed that deltadeltaG(int) for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL-10 single mutant. Error propagation analysis of double mutant cycles shows that data of atypically high precision are required to use this method meaningfully, except where large deltadeltaG values are analyzed.
Project description:The thermodynamics of base pairing is of fundamental importance. Fluorinated base analogs are valuable tools for investigating pairing interactions. To understand the influence of direct base-base interactions in relation to the role of water, pairing free energies between natural nucleobases and fluorinated analogs are estimated by potential of mean force calculations. Compared to pairing of AU and GC, pairing involving fluorinated analogs is unfavorable by 0.5-1.0 kcal mol(-1). Decomposing the pairing free energies into enthalpic and entropic contributions reveals fundamental differences for Watson-Crick pairs compared to pairs involving fluorinated analogs. These differences originate from direct base-base interactions and contributions of water. Pairing free energies of fluorinated base analogs with natural bases are less unfavorable by 0.5-1.0 kcal mol(-1) compared to non-fluorinated analogs. This is attributed to stabilizing C-F(...)H-N dipolar interactions and stronger N(...)H-C hydrogen bonds, demonstrating direct and indirect influences of fluorine. 7-methyl-7H-purine and its 9-deaza analog (Z) have been suggested as members of a new class of non-fluorinated base analogs. Z is found to be the least destabilizing universal base in the context of RNA known to date. This is the first experimental evidence for nitrogen-containing heterocylces as bioisosteres of aromatic rings bearing fluorine atoms.
Project description:There is a compelling and growing need to accurately predict the impact of amino acid mutations on protein stability for problems in personalized medicine and other applications. Here the ability of 10 computational tools to accurately predict mutation-induced perturbation of folding stability (??G) for membrane proteins of known structure was assessed. All methods for predicting ??G values performed significantly worse when applied to membrane proteins than when applied to soluble proteins, yielding estimated concordance, Pearson, and Spearman correlation coefficients of <0.4 for membrane proteins. Rosetta and PROVEAN showed a modest ability to classify mutations as destabilizing (??G < -0.5 kcal/mol), with a 7 in 10 chance of correctly discriminating a randomly chosen destabilizing variant from a randomly chosen stabilizing variant. However, even this performance is significantly worse than for soluble proteins. This study highlights the need for further development of reliable and reproducible methods for predicting thermodynamic folding stability in membrane proteins.
Project description:To investigate the cooperativity of insulin's structure, a cavity-forming substitution was introduced within the hydrophobic core of an engineered monomer. The substitution, Ile(A2)-->Ala in the A1-A8 alpha-helix, does not impair disulfide pairing between chains. In accord with past studies of cavity-forming mutations in globular proteins, a decrement was observed in thermodynamic stability (DeltaDeltaG(u) 0.4-1.2 kcal/mole). Unexpectedly, CD studies indicate an attenuated alpha-helix content, which is assigned by NMR spectroscopy to selective destabilization of the A1-A8 segment. The analog's solution structure is otherwise similar to that of native insulin, including the B chain's supersecondary structure and a major portion of the hydrophobic core. Our results show that (1) a cavity-forming mutation in a globular protein can lead to segmental unfolding, (2) tertiary packing of Ile(A2), a residue of low helical propensity, stabilizes the A1-A8 alpha-helix, and (3) folding of this segment is not required for native disulfide pairing or overall structure. We discuss these results in relation to a hierarchical pathway of protein folding and misfolding. The Ala(A2) analog's low biological activity (0.5% relative to the parent monomer) highlights the importance of the A1-A8 alpha-helix in receptor recognition.