Helix-capping interaction in λ cro protein: A free energy simulation analysis

The stability mutant Tyr-26 → Asp was studied in the Cro protein from bacteriophage λ using free energy molecular dynamics simulations. The mutant was calculated to be more stable than the wild type by 3.0 ± 1.7 kcal/mol/monomer, in reasonable agreement with experiment (1.4 kcal/mol/monomer). Moreover, the aspartic acid in the mutant was found to form a capping interation with the amino terminus of the third α-helix of Cro. The simulations were analyzed to understand better the source of the stability of this helix-capping interaction and to examine the results in light of previous explanations of stabilizing helix caps-namely, a model of local unsatisfied hydrogen bonds at the helix termini and the helix macro dipole model. Analysis of the simulations shows that the stabilizing effect of this charged helical cap is due both to favorable hydrogen bonds with backbone NH groups at the helix terminus and to favorable electrostatic interactions (but not hydrogen bonds) with their carbonyls (effectively the next row of local dipoles in the helix). However, electrostatic interactions are weak or negligible with backbone dipolar groups in the helix further away from the terminus. Moreover, the importance of other local electrostatic interactions with polar side chains near the helix terminus, which are neglected in most treatments of this effect, are shown to be important. Thus, the results support a model that is intermediate between the two previous explanations: both unsatisfied hydrogen bonds at the helix terminus and other, local preoriented dipolar groups stabilize the helix cap. These findings suggest that similar interactions with preoriented dipolar groups may be important for cooperativity in other charge–dipole interactions and may be employed to advantage for molecular design. © 1994 Wiley-Liss, Inc.     

Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions

Hydrophobic substitutions at solvent-exposed positions in two alpha-helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild-type Arc, with a rough correlation between the number of polar-to-hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar-to-hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a t(m) 6 degrees C higher than wild-type. By contrast, a helix A mutant with three similar polar-to-hydrophobic substitutions was 23 degrees C less stable than wild-type. Also, one set of three polar-to-hydrophobic substitutions in helix B was tolerated when introduced into the wild-type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues.     

Structure of a protein G helix variant suggests the importance of helix propensity and helix dipole interactions in protein design

Six helix surface positions of protein G (Gbeta1) were redesigned using a computational protein design algorithm, resulting in the five fold mutant Gbeta1m2. Gbeta1m2 is well folded with a circular dichroism spectrum nearly identical to that of Gbeta1, and a melting temperature of 91 degrees C, approximately 6 degrees C higher than that of Gbeta1. The crystal structure of Gbeta1m2 was solved to 2.0 A resolution by molecular replacement. The absence of hydrogen bond or salt bridge interactions between the designed residues in Gbeta1m2 suggests that the increased stability of Gbeta1m2 is due to increased helix propensity and more favorable helix dipole interactions.     

Analysis of the interaction between charged side chains and the alpha-helix dipole using designed thermostable mutants of phage T4 lysozyme

It was shown previously that the introduction of a negatively charged amino acid at the N-terminus of an alpha-helix could increase the thermostability of phage T4 lysozyme via an electrostatic interaction with the "helix dipole" [Nicholson, H., Becktel, W. J., & Matthews, B. W. (1988) Nature 336, 651-656]. The prior report focused on the two stabilizing substitutions Ser 38----Asp (S38D) and Asn 144----Asp (N144D). Two additional examples of stabilizing mutants, T109D and N116D, are presented here. Both show the pH-dependent increase in thermal stability expected for the interaction of an aspartic acid with an alpha-helix dipole. Control mutants were also constructed to further characterize the nature of the interaction with the alpha-helix dipole. High-resolution crystal structure analysis was used to determine the nature of the interaction of the substituted amino acids with the end of the alpha-helix in both the primary and the control mutants. Control mutant S38N has stability essentially the same as that of wild-type lysozyme but hydrogen bonding similar to that of the stabilizing mutant S38D. This confirms that it is the electrostatic interaction between Asp 38 and the helix dipole, rather than a change in hydrogen-bonding geometry, that gives enhanced stability. Structural and thermodynamic analysis of mutant T109N provide a similar control for the stabilizing replacement T109D. In the case of mutant N116D, there was concern that the enhanced stability might be due to a favorable salt-bridge interaction between the introduced aspartate and Arg 119, rather than an interaction with the alpha-helix dipole. The additivity of the stabilities of N116D and R119M seen in the double mutant N116D/R119M indicates that favorable interactions are largely independent of residue 119. As a further control, Asp 92, a presumed helix-stabilizing residue in wild-type lysozyme, was replaced with Asn. This decreased the stability of the protein in the manner expected for the loss of a favorable helix dipole interaction. In total, five mutations have been identified that increase the thermostability of T4 lysozyme and appear to do so by favorable interactions with alpha-helix dipoles. As measured by the pH dependence of stability, the strength of the electrostatic interaction between the charged groups studied here and the helix dipole ranges from 0.6 to 1.3 kcal/mol in 150 mM KCl. In the case of mutants S38D and N144H, NMR titration was used to measure the pKa's of Asp 38 and His 144 in the folded structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrostatic stabilization in four-helix bundle proteins.

Charge substitutions generated by site-directed mutagenesis at the termini of adjacent anti-parallel alpha-helices in a four-helix bundle protein were used to determine a precise value for the contribution of indirect charge-charge interactions to overall protein stability, and to simulate the electrostatic effects of alpha-helix macrodipoles. Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti-parallel helices in the four-helix bundle cytochrome b562 from Escherichia coli. Previously, theoretical calculations of helix macrodipole interactions using modeled four-helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol. Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges. At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti-parallel helix pair. Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude. The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect. Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti-parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.     

Exhaustive mutagenesis of six secondary active-site residues in Escherichia coli chorismate mutase shows the importance of hydrophobic side chains and a helix N-capping position for stability and catalysis

Secondary active-site residues in enzymes, including hydrophobic amino acids, may contribute to catalysis through critical interactions that position the reacting molecule, organize hydrogen-bonding residues, and define the electrostatic environment of the active site. To ascertain the tolerance of an important model enzyme to mutation of active-site residues that do not directly hydrogen bond with the reacting molecule, all 19 possible amino acid substitutions were investigated in six positions of the engineered chorismate mutase domain of the Escherichia coli chorismate mutase-prephenate dehydratase. The six secondary active-site residues were selected to clarify results of a previous test of computational enzyme design procedures. Five of the positions encode hydrophobic side chains in the wild-type enzyme, and one forms a helix N-capping interaction as well as a salt bridge with a catalytically essential residue. Each mutant was evaluated for its ability to complement an auxotrophic chorismate mutase deletion strain. Kinetic parameters and thermal stabilities were measured for variants with in vivo activity. Altogether, we find that the enzyme tolerated 34% of the 114 possible substitutions, with a few mutations leading to increases in the catalytic efficiency of the enzyme. The results show the importance of secondary amino acid residues in determining enzymatic activity, and they point to strengths and weaknesses in current computational enzyme design procedures.     

Positional dependence of the effects of negatively charged Glu side chains on the stability of two-stranded α-helical coiled-coils

The effects on protein stability of negatively charged Glu side chains at different positions along the length of the α-helix were investigated in the two-stranded α-helical coiled-coil. A native coiled-coil has been designed which consists of two identical 35 residue polypeptide chains with a heptad repeat Q_gV_aG_bA_cL_dQ_eK_f and a Cys residue at position 2 to allow the formation of an interchain 2-2′ disulphide bridge. This coiled-coil contains no intra- or interchain electrostatic interactions and served as a control for peptides in which Glu was substituted for Gln in the e or g heptad positions. The effect of the substitutions on stability was determined by urea denaturation at 20°C with the degree of unfolding monitored by circular dichroism spectroscopy. A Glu substituted for Gln near the N-terminus in each chain of the coiled-coil stabilizes the coiled-coil at pH 7, consistent with the charge–helix dipole interaction model. This stability increase is modulated by pH change and the addition of salt (KCl or guanidine hydrochloride), confirming the electrostatic nature of the effect. In contrast, Glu substitution in the middle of the helix destabilizes the coiled-coil because of the lower helical propensity and hydrophobicity of Glu compared with Gln at pH 7. Taking the intrinsic differences into account, the apparent charge–helix dipole interaction at the N-terminus is approximately 0.35 kcal/mol per Glu substitution. A Glu substitution at the C-terminus destabilizes the coiled-coil more than in the middle owing to the combined effects of intrinsic destabilization and unfavourable charge–helix dipole interaction with the negative pole of the helix dipole. The estimated destabilizing charge–helix dipole interaction of 0.08 kcal/mol is smaller than the stabilizing interaction at the N-terminus. The presence of a 2-2′disulphide bridge appears to have little influence on the magnitude of the charge–helix dipole interactions at either end of the coiled-coil. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge charge interactions

Computational design of surface charge-charge interactions has been demonstrated to be an effective way to increase both the thermostability and the stability of proteins. To test the robustness of this approach for proteins with predominantly beta-sheet secondary structure, the chicken isoform of the Fyn SH3 domain was used as a model system. Computational analysis of the optimal distribution of surface charges showed that the increase in favorable energy per substitution begins to level off at five substitutions; hence, the designed Fyn sequence contained four charge reversals at existing charged positions and one introduction of a new charge. Three additional variants were also constructed to explore stepwise contributions of these substitutions to Fyn stability. The thermodynamic stabilities of the variants were experimentally characterized using differential scanning calorimetry and far-UV circular dichroism spectroscopy and are in very good agreement with theoretical predictions from the model. The designed sequence was found to have increased the melting temperature, DeltaT (m) = 12.3 +/- 0.2 degrees C, and stability, DeltaDeltaG(25 degrees C) = 7.1 +/- 2.2 kJ/mol, relative to the wild-type protein. The experimental data suggest that a significant increase in stability can be achieved through a very small number of amino acid substitutions. Consistent with a number of recent studies, the presented results clearly argue for a seminal role of surface charge-charge interactions in determining protein stability and suggest that the optimization of surface interactions can be an attractive strategy to complement algorithms optimizing interactions in the protein core to further enhance protein stability.     

Effects of charged amino acids at b and c heptad positions on specificity and stability of four-chain coiled coils

An understanding of the balance of chemical forces responsible for protein stability and specificity of structure is essential for the success of efforts in protein design. Specifically, electrostatic interactions between charged amino acids have been explored extensively to understand the contribution of this force to protein stability. Much research on the importance of electrostatic interactions as specificity and stability determinants in two-stranded coiled coils has been done, but there remains significant controversy about the magnitude of the attractive forces using such systems. We have developed a four-stranded coiled-coil system with charged residues incorporated at b and c heptad positions to explore the role of charge interactions. Here, we test quantitatively the effects of varying sidechain length on the magnitude of such electrostatic interactions. We synthesized peptides containing either aspartate or ornithine at both b and c heptad positions and tested their ability to self-associate and to hetero-associate with one another and with peptides containing glutamate or lysine at the same positions. We find that interactions between glutamate and either lysine or ornithine are more favorable than the corresponding interactions involving aspartate. In each case, charged interactions provide additional stability to coiled coils, although helix propensity effects may play a significant role in determining the overall stability of these structures.     

Effect of salt on the urea-unfolded form of barstar probed by m value measurements

To probe for residual structure present in the urea-unfolded form of the small protein barstar, to determine how salt might modulate such structure, and to determine how such structure might affect the stability of the protein, mutant variants that display m values different from that of the wild-type protein have been studied. The mutant proteins were obtained by site-directed mutagenesis at residue positions located on the surface of the folded protein. The m value, which represents the preferential free energy of interaction of urea with the unfolded form in comparison to that with the folded state, was determined from equilibrium urea-induced unfolding curves. Mutant proteins for which the m values were significantly greater than (m(+) mutant forms), significantly smaller than (m(-) mutant forms), or similar to (m(0) mutant forms) the m value determined for the wild-type protein were studied. The unfolded forms of the m(0), m(+) and m(-) mutant proteins represent different components within the unfolded form ensemble, which differ from each other in their solvent-exposed surface areas. Hence, the m value has been used as a measure of residual structure in the unfolded form. To further understand the nature of structures present in the unfolded form ensemble, the effects of the salt KCl on the stabilities of the wild-type and the mutant proteins, as well as on the structures present in the unfolded form ensemble, were also studied. It was found that the m values of the m(0), m(+) and m(-) mutant proteins all converge to the wild-type m value in the presence of KCl. This result indicates that the salt modulates residual structure in the unfolded form by screening electrostatic interactions that maintain compact and expanded components in the unfolded protein ensemble. The use of free energy cycles has allowed the effect of salt on the structure and free energy of the unfolded protein to be related to the stability of the protein.     

Stabilizing ionic interactions in a full-consensus ankyrin repeat protein

Full-consensus designed ankyrin repeat proteins (DARPins), in which randomized positions of the previously described DARPin library have been fixed, are characterized. They show exceptionally high thermodynamic stabilities, even when compared to members of consensus DARPin libraries and even more so when compared to naturally occurring ankyrin repeat proteins. We determined the crystal structure of a full-consensus DARPin, containing an N-capping repeat, three identical internal repeats and a C-capping repeat at 2.05 Å resolution, and compared its structure with that of the related DARPin library members E3_5 and E3_19. This structural comparison suggests that primarily salt bridges on the surface, which arrange in a network with almost crystal-like regularity, increase thermostability in the full-consensus NI₃C DARPin to make it resistant to boiling. In the crystal structure, three sulfate ions complement this network. Thermal denaturation experiments in guanidine hydrochloride directly indicate a contribution of sulfate binding to the stability, providing further evidence for the stabilizing effect of surface-exposed electrostatic interactions and regular charge networks. The charged residues at the place of randomized residues in the DARPin libraries were selected based on sequence statistics and suggested that the charge interaction network is a hidden design feature of this protein family. Ankyrins can therefore use design principles from proteins of thermophilic organisms and reach at least similar stabilities.     

Role of the charge-charge interactions in defining stability and halophilicity of the CspB proteins

Charge-charge interactions on the surface of native proteins are important for protein stability and can be computationally redesigned in a rational way to modulate protein stability. Such computational effort led to an engineered protein, CspB-TB that has the same core as the mesophilic cold shock protein CspB-Bs from Bacillus subtilis, but optimized distribution of charge-charge interactions on the surface. The CspB-TB protein shows an increase in the transition temperature by 20 degrees C relative to the unfolding temperature of CspB-Bs. The CspB-TB and CspB-Bs protein pair offers a unique opportunity to further explore the energetics of charge-charge interactions as the substitutions at the same sequence positions are done in largely similar structural but different electrostatic environments. In particular we addressed two questions. What is the contribution of charge-charge interactions in the unfolded state to the protein stability and how amino acid substitutions modulate the effect of increase in ionic strength on protein stability (i.e. protein halophilicity). To this end, we experimentally measured the stabilities of over 100 variants of CspB-TB and CspB-Bs proteins with substitutions at charged residues. We also performed computational modeling of these protein variants. Analysis of the experimental and computational data allowed us to conclude that the charge-charge interactions in the unfolded state of two model proteins CspB-Bs and CspB-TB are not very significant and computational models that are based only on the native state structure can adequately, i.e. qualitatively (stabilizing versus destabilizing) and semi-quantitatively (relative rank order), predict the effects of surface charge neutralization or reversal on protein stability. We also show that the effect of ionic strength on protein stability (protein halophilicity) appears to be mainly due to the screening of the long-range charge-charge interactions.     

Protein stability and electrostatic interactions between solvent exposed charged side chains

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.     

Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alphavirus nucleocapsid protein contains a putative coiled coil alpha-helix important for core assembly

The alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. This protein consists of a poorly conserved N-terminal region of unknown function and a C-terminal conserved autoprotease domain with a major role in virion formation. In this study, an 18-amino-acid conserved region, predicted to fold into an alpha-helix (helix I) and embedded in a low-complexity sequence enriched with basic and Pro residues, has been identified in the N-terminal region of the alphavirus capsid proteins. In Sindbis virus, helix I spans residues 38 to 55 and contains three conserved leucine residues, L38, L45, and L52, conforming to the heptad amino acid organization evident in leucine zipper proteins. Helix I consists of an N-terminally truncated heptad and two complete heptad repeats with beta-branched residues and conserved leucine residues occupying the a and d positions of the helix, respectively. Complete or partial deletion of helix I, or single-site substitutions at the conserved leucine residues (L45 and L52), caused a significant decrease in virus replication. The mutant viruses were more sensitive to elevated temperature than wild-type virus. These mutant viruses also failed to accumulate cores in the cytoplasm of infected cells, although they did not have defects in protein translation or processing. Analysis of these mutants using an in vitro assembly system indicated that the majority were defective in core particle assembly. Furthermore, mutant proteins showed a trans-dominant negative phenotype in in vitro assembly reactions involving mutant and wild-type proteins. We propose that helix I plays a central role in the assembly of nucleocapsid cores through coiled coil interactions. These interactions may stabilize subviral intermediates formed through the interactions of the C-terminal domain of the capsid protein and the genomic RNA and contribute to the stability of the virion.     

The effects of ionic strength on protein stability: the cold shock protein family

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins.     

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.     

Position-dependent interactions between cysteine residues and the helix dipole

A protein model was developed for studying the interaction between cysteine residues and the helix dipole. Site-directed mutagenesis was used to introduce cysteine residues at the N-terminus of helix H in recombinant sperm whale myoglobin. Based on the difference in thiol pK(a) between folded proteins and an unfolded peptide, the energy of interaction between the thiolate and the helix dipole was determined. Thiolates at the N1 and N2 positions of the helix were stabilized by 0.3 kcal/mole and 0.7 kcal/mole, respectively. A thiolate at the Ncap position was stabilized by 2.8 kcal/mole, and may involve a hydrogen bond. In context with other studies, an experimentally observed helix dipole effect may be defined in terms of two distinct components. A charge-dipole component involves electrostatic interactions with peptide bond dipoles in the first two turns of the helix and affects residues at all positions of the terminus; a hydrogen bond component involves one or more backbone amide groups and is only possible at the capping position due to conformational restraints elsewhere. The nature and magnitude of the helix dipole effect is, therefore, position-dependent. Results from this model system were used to interpret cysteine reactivity in rodent hemoglobins and the thioredoxin family.     

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.