Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-α-helical protein ROP: Thermal unfolding studies

The structural and energetic perturbations caused by cavity-creating mutations (Leu-41 → Val and Leu-41 → Ala) in the dimeric 4-α-helical-bundle protein ROP have been characterized by CD spectroscopy and differential scanning calorimetry (DSC). Deconvolution of the CD spectra showed a decrease in α -helicity as a result of the amino acid exchanges that follows qualitatively the overall decrease in conformational stability. Transition enthalpies are sensitive probes of the energetic change associated with point mutations. ΔH⁰ values at the respective transition temperatures, T_1/2 (71.0, 65.3, and 52.9°C at 0.5 mg/ml) decrease from 580 ± 20 to 461 ± 20 kJ/(mol of dimmer) and 335 ± 20 kJ/(mol of dimmer) for wildtype ROP (Steif, C., Weber, P., Hinz, H.-J., Flossdorf, J., Cesareni, G., Kokkinidis, M. Biochemistry 32:3867-3876, 1993), L⁴¹V, and L⁴¹A, respectively. The conformational stabilities at 25°C expressed by the standard Gibbs energies of denaturation, ΔG, are 71.7, 61.1, and 46.1 kJ/(mol of dimmer). The corresponding transition enthalpies have been obtained from extrapolation using the c(T)and c(T) functions. Their values at 25°C are 176.3, 101.9, and 141.7 kJ/(mol of dimmer) for wild-type ROP, L⁴¹V, and L⁴¹A, respectively. When the stability perturbation resulting from the cavity creating mutations is referred to the exchange of 1 mol of CH₂ group, the average ΔΔG value is ?5.0 ± 1 kJ/(mol of CH₂ group). This decrease in conformation stability suggests that dimeric ROP exhibits the same susceptibility to Leu → Yal and Leu → Ala exchanges as small monomeric proteins. Careful determinations of the partial specific heat capacities of wild-type and mutated protein solutions suggest that the mutational effects are predominantly manifested in the native rather than the unfolded state. © 1995 Wiley-Liss, Inc.     

Relationship of sidechain hydrophobicity and α-helical propensity on the stability of the single-stranded amphipathic α-helix

The aim of the present investigation is to determine the effect of α-helical propensity and sidechain hydrophobicity on the stability of amphipathic α-helices. Accordingly, a series of 18-residue amphipathic α-helical peptides has been synthesized as a model system where all 20 amino acid residues were substituted on the hydrophobic face of the amphipathic α-helix. In these experiments, all three parameters (sidechain hydrophobicity, α-helical propensity and helix stability) were measured on the same set of peptide analogues. For these peptide analogues that differ by only one amino acid residue, there was a 0.96 kcal/mole difference in α-helical propensity between the most (Ala) and the least (Gly) α-helical analogue, a 12.1-minute difference between the most (Phe) and the least (Asp) retentive analogue on the reversed-phase column, and a 32.3°C difference in melting temperatures between the most (Leu) and the least (Asp) stable analogue. The results show that the hydrophobicity and α-helical propensity of an amino acid sidechain are not correlated with each other, but each contributes to the stability of the amphipathic α-helix. More importantly, the combined effects of α-helical propensity and sidechain hydrophobicity at a ratio of about 2:1 had optimal correlation with α-helix stability. These results suggest that both α-helical propensity and sidechain hydrophobicity should be taken into consideration in the design of α-helical proteins with the desired stability.     

Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T(m)), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta Delta G(d)(50 degrees C) = -4 kcal/mole; Delta T(m) = -22 degrees C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (Delta Delta G(d)(50 degrees C) = -3 kcal/mole; Delta T(m) = -11 degrees C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (Delta Delta G(d)(50 degrees C) = -1 kcal/mole; Delta T(m) = +2 degrees C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (Delta Delta G(d)(50 degrees C) = +1 kcal/mole; Delta T(m) = +17 degrees C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.     

Molecular dynamics as a tool to detect protein foldability. A mutant of domain B1 of protein G with non-native secondary structure propensities

The usefulness of molecular dynamics to assess the structural integrity of mutants containing several mutations has been investigated. Our goal was to determine whether molecular dynamics would be able to discriminate mutants of a protein having a close-to-wild-type fold, from those that are not folded under the same conditions. We used as a model the B1 domain of protein G in which we replaced the unique central alpha-helix by the sequence of the second beta-hairpin, which has a strong intrinsic propensity to form this secondary structure in solution. In the resulting protein, one-third of the secondary structure has been replaced by a non-native one. Models of the mutants were built based on the three-dimensional structure of the wild-type GB1 domain. During 2 ns of molecular dynamics simulations on these models, mutants containing up to 10 mutations in the helix retained the native fold, while another mutant with an additional mutation unfolded. This result is in agreement with our circular dichroism and NMR experiments, which indicated that the former mutants fold into a structure similar to the wild-type, as opposed to the latter mutant which is partly unfolded. Additionally, a mutant containing six mutations scattered through the surface of the domain, and which is unfolded, was also detected by the simulation. This study suggests that molecular dynamics calculations could be performed on molecular models of mutants of a protein to evaluate their foldability, prior to a mutagenesis experiment.     

Electrostatics significantly affect the stability of designed homeodomain variants

The role of electrostatic interactions in determining the stability of designed proteins was studied by constructing and analyzing a set of designed variants of the Drosophila engrailed homeodomain. Computational redesign of 29 surface positions results in a 25-fold mutant with moderate stability, similar to the wild-type protein. Incorporating helix dipole and N-capping considerations into the design algorithm by restricting amino acid composition at the helix termini and N-capping positions yields a ninefold mutant of the initial design (a 23-fold mutant of wild-type) that is over 3 kcal mol(-1) more stable than the protein resulting from the unbiased design. Four additional proteins were constructed and analyzed to isolate the effects of helix dipole and N-capping interactions in each helix. Based on the results of urea-denaturation experiments and calculations using the finite difference Poisson-Boltzmann method, both classes of interaction are found to increase the stability of the designed proteins significantly. The simple electrostatic model used in the optimization of rotamers by iterative techniques (ORBIT) force-field, which is similar to the electrostatic models used in other protein design force-fields, is unable to predict the experimentally determined stabilities of the designed variants. The helix dipole and N-capping restrictions provide a simple but effective method to incorporate two types of electrostatic interactions that impact protein stability significantly.     

Proline in alpha-helix: stability and conformation studied by dynamics simulation

Free-energy simulations have been used to estimate the change in the conformational stability of short polyalanine alpha-helices when one of the alanines is replaced by a proline residue. For substituting proline in the middle of the helix the change in free energy of folding (delta delta G degrees) was calculated as 14 kJ/mol (3.4 kcal/mol), in excellent agreement with the one available experimental value. The helix containing proline was found to be strongly kinked; the free energy for reducing the angle of the kink from 40 degrees to 15 degrees was calculated, and found to be small. A tendency to alternate hydrogen bonding schemes was observed in the proline-containing helix. These observations for the oligopeptide agree well with the observation of a range of kink angles (18-35 degrees) and variety of hydrogen bonding schemes, in the rare instances where proline occurs in helices in globular proteins. For substituting proline at the N-terminus of the helix the change in free energy of folding (delta delta G degrees) was calculated as -4 kJ/mol in the first helical position (N1) and +6 kJ/mol in the second helical position (N2). The observed frequent occurrence of proline in position N1 in alpha-helices in proteins therefore has its origin in stability differences of secondary structure. The conclusion reached here that proline may be a better helix former in position N1 than (even) alanine, and thus be a helix initiator may be testable experimentally by measurements of fraction helical conformation of individual residues in oligopeptides of appropriate sequence. The relevance of these results in regards to the frequent occurrence of proline-containing helices in certain membrane proteins is discussed.     

Contributions of a hydrogen bond/salt bridge network to the stability of secondary and tertiary structure in lambda repressor.

In the N-terminal domain of lambda repressor, the Asp 14 side chain forms an intrahelical, hydrogen bond/salt bridge with the Arg 17 side chain and a tertiary hydrogen bond with the Ser 77 side chain. By measuring the stabilities to urea denaturation of the wild-type N-terminal domain and variants containing single, double, and triple alanine substitutions at positions 14, 17, and 77, the side-chain interaction energies, the coupling energy between interactions, and the intrinsic effects of each wild-type side chain on protein stability have been estimated. These studies indicate that the Asp 14-Arg 17 and Asp 14-Ser 77 interactions are stabilizing by roughly 0.8 and 1.5 kcal/mol, respectively, but that Asp 14, by itself, is destabilizing by roughly 0.9 kcal/mol. We also show that a peptide model of alpha-helix 1, which contains Asp 14 and Arg 17, forms a reasonably stable, monomeric helix in solution and responds to alanine mutations at positions 14 and 17 in the fashion expected from the intact protein studies. These studies suggest that it is possible to view the stability effects of mutations in intact proteins in a hierarchical fashion, with the stability of units of secondary structure being distinguishable from the stability of tertiary structure.     

Effects of alanine substitutions in alpha-helices of sperm whale myoglobin on protein stability.

The peptide backbones in folded native proteins contain distinctive secondary structures, alpha-helices, beta-sheets, and turns, with significant frequency. One question that arises in folding is how the stability of this secondary structure relates to that of the protein as a whole. To address this question, we substituted the alpha-helix-stabilizing alanine side chain at 16 selected sites in the sequence of sperm whale myoglobin, 12 at helical sites on the surface of the protein, and 4 at obviously internal sites. Substitution of alanine for bulky side chains at internal sites destabilizes the protein, as expected if packing interactions are disrupted. Alanine substitutions do not uniformly stabilize the protein, either in capping positions near the ends of helices or at mid-helical sites near the surface of myoglobin. When corrected for the extent of exposure of each side chain replaced by alanine at a mid-helix position, alanine replacement still has no clear effect in stabilizing the native structure. Thus linkage between the stabilization of secondary structure and tertiary structure in myoglobin cannot be demonstrated, probably because of the relatively small free energy differences between side chains in stabilizing isolated helix. By contrast, about 80% of the variance in free energy observed can be accounted for by the loss in buried surface area of the native residue substituted by alanine. The differential free energy of helix stabilization does not account for any additional variation.     

Folding of a designed simple ankyrin repeat protein

Ankyrin repeats (AR) are 33-residue motifs containing a beta-turn, followed by two alpha-helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5 degrees C both follow monoexponential kinetics with k(folding) = 28 sec(-1) and k(unfolding) = 0.9 sec(-1). Kinetic and equilibrium unfolding parameters at 5 degrees C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.     

Insights into thermal resistance of proteins from the intrinsic stability of their α-helices

To investigate the role of α helices in protein thermostability, we compared energy characteristics of α helices from thermophilic and mesophilic proteins belonging to four protein families of known three-dimensional structure, for at least one member of each family. The changes in intrinsic free energy of α-helix formation were estimated using the statistical mechanical theory for describing helix/coil transitions in peptide helices [Munoz, V., Serrano, L. Nature Struc. Biol. 1:399–409, 1994; Munoz, V., Serrano, L. J. Mol. Biol. 245:275–296, 1995; Munoz, V., Serrano, L. J. Mol. Biol. 245:297–308, 1995]. Based on known sequences of mesophilic and thermophilic RecA proteins we found that (1) a high stability of α helices is necessary but is not a sufficient condition for thermostability of RecA proteins, (2) the total helix stability, rather than that of individual helices, is the factor determining protein thermostability, and (3) two facets of intrahelical interactions, the intrinsic helical propensities of amino acids and the side chain–side chain interactions, are the main contributors to protein thermostability. Similar analysis applied to families of L-lactate dehydrogenases, seryl-tRNA synthetases, and aspartate amino transferases led us to conclude that an enhanced total stability of α helices is a general feature of many thermophilic proteins. The magnitude of the observed decrease in intrinsic free energy on α-helix formation of several thermoresistant proteins was found to be sufficient to explain the experimentally determined increase of their thermostability. Free energies of intrahelical interactions of different RecA proteins calculated at three temperatures that are thought to be close to its normal environmental conditions were found to be approximately equal. This indicates that certain flexibility of RecA protein structure is an essential factor for protein function. All RecA proteins analyzed fell into three temperature-dependent classes of similar α-helix stability (ΔG_int = 45.0 ± 2.0 kcal/mol). These classes were consistent with the natural origin of the proteins. Based on the sequences of protein α helices with optimized arrangement of stabilizing interactions, a natural reserve of RecA protein thermoresistance was estimated to be sufficient for conformational stability of the protein at nearly 200°C. Proteins 29:309–320, 1997. © 1997 Wiley-Liss, Inc.     

Chemical cross-linking between lysine groups in vimentin oligomers is dependent on local peptide conformations

Previous studies have shown that cytoplasmic intermediate filaments, other than the keratins, are each constructed from a single type of polypeptide chain. Studies involving chemical crosslinking between lysine groups have shown that assembly of the filaments begins with the formation of dimers in which the peptide chains are parallel and in exact register, and that these dimers further associate in antiparallel patterns having specific degrees of overlap. In the present study, molecular modeling of the conformations of vimentin molecules indicated that lysine side chains in identical positions in regions of α-helix in parallel chains might be unable to be linked because they are on opposite sides of the coiled coil hydrophobic core. Examination of published data on chemical crosslinking of lysines in vimentin confirmed that there were no instances of linkage within dimers between the nine pairs of identical lysines that lie more than one position within α-helical regions in parallel chains. Even among linkages that apparently were between dimers, only one of the 11 linkage products identified involved lysines that were both within an α-helical region. In 10 of the 11 identified linkages between dimers, one or both of the linked lysines were in regions of random coil conformation. These results of molecular modeling indicate that relative motion between polypeptide chains in oligomers of intermediate filament proteins is not sufficient to overcome an orientation of lysine groups that is unfavorable for their chemical linkage. This finding supports the interpretations of keratin cross-linking data indicating that parallel homodimers are the basis for keratin intermediate filament assembly. © 1996 Wiley-Liss, Inc.     

Knowledge-based protein secondary structure assignment

We have developed an automatic algorithm STRIDE for protein secondary structure assignment from atomic coordinates based on the combined use of hydrogen bond energy and statistically derived backbone torsional angle information. Parameters of the pattern recognition procedure were optimized using designations provided by the crystallographers as a standard-of-truth. Comparison to the currently most widely used technique DSSP by Kabsch and Sander (Biopolymers 22:2577-2637, 1983) shows that STRIDE and DSSP assign secondary structural states in 58 and 31% of 226 protein chains in our data sample, respectively, in greater agreement with the specific residue-by-residue definitions provided by the discoverers of the structures while in 11% of the chains, the assignments are the same. STRIDE delineates every 11th helix and every 32nd strand more in accord with published assignments. © 1995 Wiley-Liss, Inc.     

Thermodynamic model of secondary structure for α-helical peptides and proteins

A thermodynamic model describing formation of α-helices by peptides and proteins in the absence of specific tertiary interactions has been developed. The model combines free energy terms defining α-helix stability in aqueous solution and terms describing immersion of every helix or fragment of coil into a micelle or a nonpolar droplet created by the rest of protein to calculate averaged or lowest energy partitioning of the peptide chain into helical and coil fragments. The α-helix energy in water was calculated with parameters derived from peptide substitution and protein engineering data and using estimates of nonpolar contact areas between side chains. The energy of nonspecific hydrophobic interactions was estimated considering each α-helix or fragment of coil as freely floating in the spherical micelle or droplet, and using water/cyclohexane (for micelles) or adjustable (for proteins) side-chain transfer energies. The model was verified for 96 and 36 peptides studied by ¹H-nmr spectroscopy in aqueous solution and in the presence of micelles, respectively ([set I] and [set 2]) and for 30 mostly α-helical globular proteins ([set 3]). For peptides, the experimental helix locations were identified from the published medium-range nuclear Overhauser effects detected by ¹H-nmr spectroscopy. For sets 1, 2, and 3, respectively, 93, 100, and 97% of helices were identified with average errors in calculation of helix boundaries of 1.3, 2.0, and 4.1 residues per helix and an average percentage of correctly calculated helix—coil states of 93, 89, and 81%, respectively. Analysis of adjustable parameters of the model (the entropy and enthalpy of the helix—coil transition, the transfer energy of the helix backbone, and parameters of the bound coil), determined by minimization of the average helix boundary deviation for each set of peptides or proteins, demonstrates that, unlike micelles, the interior of the effective protein droplet has solubility characteristics different from that for cyclohexane, does not bind fragments of coil, and lacks interfacial area. © 1997 John Wiley & Sons, Inc. Biopoly 42: 239–269, 1997     

Physical reasons for secondary structure stability: alpha-helices in short peptides

It was recently found that some short peptides (including C- and S-peptide fragments of RNase A) can have considerable helicity in solution, ^1–12 which was considered to be surprising. Does the observed helicity require a new explanation, or is it consistent with previous understanding? In this work we show that this helicity is consistent with the physical theory of secondary structure^12–19 based on an extension of the conventional Zimm-Bragg model.²⁰ Without any special modifications, this theory explains reasonably well almost all the experimentally observed dependencies of helicity on pH, temperature, and amino acid replacements. We conclude that the observed “general level” of helicity of C- and S-peptides (5–30% at room temperature and 10–50% near 0°C) is “normal” for short peptides consisting mainly of helix-forming and helix-indifferent residues. The helicity is modified by a multitude of weak specific side chain interactions, many of which are taken into account by the present theory;^13–19 some discrepancies between the theory and experiment can be explained by weak side-chain-side chain interactions that were neglected. A reasonable coincidence of the theory with experiment suggests that it had been used to investigate the role of local interactions in the formation of α-helical “embryos” in unfolded protein chains.     

Alpha helix capping in synthetic model peptides by reciprocal side chain–main chain interactions: Evidence for an N terminal “capping box”

A significant fraction of the amino acids in proteins are alpha helical in conformation. Alpha helices in globular proteins are short, with an average length of about twelve residues, so that residues at the ends of helices make up an important fraction of all helical residues. In the middle of a helix, H-bonds connect the NH and CO groups of each residue to partners four residues along the chain. At the ends of a helix, the H-bond potential of the main chain remains unfulfilled, and helix capping interactions involving bonds from polar side chains to the NH or CO of the backbone have been proposed and detected. In a study of synthetic helical peptides, we have found that the sequence Ser-Glu-Asp-Glu stabilizes the alpha helix in a series of helical peptides with consensus sequences. Following the report by Harper and Rose, which identifies SerXaaXaaGlu as a member of a class of common motifs at the N termini of alpha helices in proteins that they refer to as “capping boxes,” we have reexamined the side chain–main chain interactions in a varient sequence using ¹H NMR, and find that the postulated reciprocal side chain-backbone bonding between the first Ser and last Glu side chains and their peptide NH partners can be resolved: Deletion of two residues N terminal to the Ser-Glu-Asp-Glu sequence in these peptides has no effect on the initiation of helical structure, as defined by two-dimensional (2D) NMR experiments on this variant. Thus the capping box sequence Ser-Glu-Asp-Glu inhibits N terminal fraying of the N terminus of alpha helix in these peptides, and shows the side chain–main chain interactions proposed by Harper and Rose. It thus acts as a helix initiating signal. Since normal a helix cannot propagate beyond the N terminus of this structure, the box acts as a termination signal in this direction as well. © 1994 John Wiley & Sons, Inc.     

Continuum electrostatics of the C-peptide: anatomy of the problem.

A computational study of the role of all ionizable groups of the C-peptide in its helix-coil transition is performed within the framework of continuum electrostatics. The method employed in our computations involves a numeric solution of the Poisson equation with the Boundary Element Method. Our calculations correctly predict the experimentally observed trends in the helix-coil equilibrium of the C-peptide, and suggest that the mechanisms involved are more complex than usually presumed in the literature. Our results suggest that electrostatic interactions in the unfolded conformation are often more important than in the helix, total electrostatic contribution to the helix-coil transition due to the side chains of the C-peptide destabilizes the helix, changes in the helix stability produced by the changes in the ionization state of the side chains are dominated by side chain effects, the effect of the helix dipole on the energetics of the helix-coil transition of the C-peptide is either minor or similar to other contributions in magnitude; while the formation of a salt bridge is electrostatically favorable, formation of the hydrogen bond between a charged and a polar side chains is not. Factors limiting the accuracy of the computations are discussed.     

Selection and analyses of variants of a designed protein suggest importance of hydrophobicity of partially buried sidechains for protein stability at high temperatures

Computationally designed proteins of high stability provide specimen in addition to natural proteins for the study of sequence‐structure stability relationships at the very high end of protein stability spectrum. The melting temperature of E_1r26, a protein we previously designed using the A Backbone‐based Amino aCid Usage Survey (ABACUS) sequence design program, is above 110 °C, more than 50 °C higher than that of the natural thioredoxin protein whose backbone (PDB ID 1R26 ) has been used as the design target. Using an experimental selection approach, we obtained variants of E_1r26 that remain folded but are of reduced stability, including one whose unfolding temperature and denaturing guanidine concentration are similar to those of 1r26. The mutant unfolds with a certain degree of cooperativity. Its structure solved by X‐ray crystallography agrees with that of 1r26 by a root mean square deviation of 1.3 Å, adding supports to the accuracy of the ABACUS method. Analyses of intermediate mutants indicate that the substitution of two partially buried hydrophobic residues (isoleucine and leucine) by polar residues (threonine and serine, respectively) are responsible for the dramatic change in the unfolding temperature. It is suggested that the effects of mutations located in rigid secondary structure regions, but not those in loops, may be well predicted through ABACUS mutation energy analysis. The results also suggest that hydrophobic effects involving intermediately buried sidechains can be critically important for protein stability at high temperatures.     

Prediction of protein folding class from amino acid composition

An empirical relation between the amino acid composition and three-dimensional folding pattern of several classes of proteins has been determined. Computer simulated neural networks have been used to assign proteins to one of the following classes based on their amino acid composition and size: (1) 4α-helical bundles, (2) parallel (α/β)₈ barrels, (3) nucleotide binding fold, (4) immunoglobulin fold, or (5) none of these. Networks trained on the known crystal structures as well as sequences of closely related proteins are shown to correctly predict folding classes of proteins not represented in the training set with an average accuracy of 87%. Other folding motifs can easily be added to the prediction scheme once larger databases become available. Analysis of the neural network weights reveals that amino acids favoring prediction of a folding class are usually over represented in that class and amino acids with unfavorable weights are underrepresented in composition. The neural networks utilize combinations of these multiple small variations in amino acid composition in order to make a prediction. The favorably weighted amino acids in a given class also form the most intramolecular interactions with other residues in proteins of that class. A detailed examination of the contacts of these amino acids reveals some general patterns that may help stabilize each folding class. © 1993 Wiley-Liss, Inc.     

Stabilization of myoglobin by multiple alanine substitutions in helical positions.

We have carried out a series of multiple Xaa-->Ala changes at nonadjacent surface positions in the sequence of sperm whale myoglobin. Although the corresponding single substitutions do not increase the thermal stability of the protein, multiple substitutions enhance the stability of the resulting myoglobins. The effect observed is an increase in the observed Tm (midpoint unfolding temperature) relative to that predicted from assuming additivity of the free energy changes corresponding to single mutations. The stabilization occurs in the presence of urea, as measured by the dependence of the unfolding temperature on urea concentration. The sites that have been altered occur in different helices and are not close in sequence or in the native structure of myoglobin. The observed effect is consistent with a role of multiple alanines in residual interactions in the unfolded state of the mutant proteins.