Structural effects induced by removal of a disulfide-bridge: the X-ray structure of the C30A/C51A mutant of basic pancreatic trypsin inhibitor at 1.6 A.

The X-ray structure of a variant of basic pancreatic trypsin inhibitor (BPTI) has been analyzed to determine the structural accommodation resulting from removal of a disulfide cross-link in a protein. The disulfide removed, Cys30-Cys51, has been implicated in both the folding pathway of the protein and its overall thermal stability. In the variant studied, C30A/C51A, the disulfide cysteines were replaced by less bulky alanines. The atomic displacements observed for C30A/C51A indicate a set of concerted shifts of two segments of chains, which together significantly diminish a packing defect at the site of the removed cysteine sulfur atoms. The observed structural changes are distributed asymmetrically around the sites of mutation, indicating that the adjacent beta-sheet is more resistant to the perturbation than the alpha-helix on the opposite side of the disulfide bond. The thermal parameters of groups involved in the structural accommodation are not significantly altered. A comparison of the X-ray structures reported for native BPTI determined in three different crystal forms indicates that the magnitude of its conformational variability exceeds that of the structural changes caused by the disulfide removal. This emphasizes the necessity of using isomorphous crystal systems to determine the relatively small effects due to mutation.     

Secretion incompetence of bovine pancreatic trypsin inhibitor expressed in Escherichia coli.

There is increasing evidence that protein folding and protein export are competing processes in prokaryotic cells. Virtually all secretion studies reported to date, however, have employed proteins that are relatively uncharacterized in terms of their folding behavior and three-dimensional structure. In contrast, the structural and biochemical parameters governing the folding of bovine pancreatic trypsin inhibitor (BPTI) and several of its mutants have been studied intensively. We therefore undertook a study of the secretion behavior in Escherichia coli of recombinant BPTI and its mutants. Wild-type BPTI and two well-characterized folding mutants (C14A, C38A)BPTI and (C30A, C51A)BPTI (missing the 14-38 and 30-51 disulfide bonds, respectively), were investigated by analyzing their expression fused to an E. coli signal sequence or to two synthetic IgG-binding domains of staphylococcal protein A. Both disulfide mutants are destabilized relative to wild-type BPTI and exhibit markedly altered folding kinetics: one (C14A, C38A) folds more slowly than wild-type BPTI and the other (C30A, C51A) unfolds more rapidly. Both mutants were observed to be exported 3-10 times more efficiently than the wild-type molecule. Moreover, the levels of unprocessed preprotein in the cytoplasm were severalfold higher for the wild-type fusion than for the fusion to the two folding mutants. Intracellular degradation of the BPTI moiety was also observed. These results are consistent with traffic of intracellular BPTI preproteins on at least three routes along the secretory pathway: (a) facile secretion of unfolded material, (b) intracellular folding leading to secretion blockage, and (c) degradation followed by export of truncated molecules. A novel feature of these findings is the implication that disulfide bonds can form in the bacterial cytoplasm and lead to secretion incompetence.     

Variability of conformations at crystal contacts in BPTI represent true low-energy structures: correspondence among lattice packing and molecular dynamics structures.

The structures of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X-ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the molecule, although the regions that display most variability are associated with the loop structures (residues 14-17 and 24-29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B-factors, although it is not apparent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X-ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H-bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance; the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96-psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X-ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low-energy conformers that have been preferentially selected by crystallization factors.     

Response of dynamic structure to removal of a disulfide bond: normal mode refinement of C77A/C95A mutant of human lysozyme.

In order to investigate the response of dynamic structure to removal of a disulfide bond, the dynamic structure of human lysozyme has been compared to its C77A/C95A mutant. The dynamic structures of the wild type and mutant are determined by normal mode refinement of 1.5-A-resolution X-ray data. The C77A/C95A mutant shows an increase in apparent fluctuations at most residues. However, most of the change originates from an increase in the external fluctuations, reflecting the effect of the mutation on the quality of crystals. The effects of disulfide bond removal on the internal fluctuations are almost exclusively limited to the mutation site at residue 77. No significant change in the correlation of the internal fluctuations is found in either the overall or local dynamics. This indicates that the disulfide bond does not have any substantial role to play in the dynamic structure. A comparison of the wild-type and mutant coordinates suggests that the disulfide bond does not prevent the 2 domains from parting from each other. Instead, the structural changes are characteristic of a cavity-creating mutation, where atoms surrounding the mutation site move cooperatively toward the space created by the smaller alanine side chain. Although this produces tighter packing, more than half of the cavity volume remains unoccupied, thus destabilizing the native state.     

Denaturant-dependent folding of bovine pancreatic trypsin inhibitor mutants with two intact disulfide bonds.

The equilibrium and kinetic behavior of the guanidine hydrochloride (Gdn-HCl) induced unfolding/refolding of four bovine pancreatic trypsin inhibitor (BPTI) mutants was examined by using ultraviolet difference spectroscopy. In three of the mutants, we replaced the buried 30-51 disulfide bond with alanine at position 51 and valine (Val30/Ala51), alanine (Ala30/Ala51), or threonine (Thr30/Ala51) at position 30. For the fourth mutant, the solvent-exposed 14-38 disulfide was substituted by a pair of alanines (Ala14/Ala38). All mutants retained the 5-55 disulfide. Experiments were performed under oxidizing conditions; thus, both the unfolded and folded forms retained two native disulfide bonds. Equilibrium experiments demonstrated that all four mutants were destabilized relative to wild-type BPTI. However, the stability of the 30-51 mutants increased with the hydrophobicity of the residue substituted at position 30. Kinetic experiments showed that all four mutants contained two minor slow refolding phases with characteristics of proline isomerization. The specific behavior of the phases depended on the location of the disulfide bonds. The major unfolding/refolding phase for each of the 30-51 mutants was more than an order of magnitude slower than for Ala14/Ala38 or for BPTI in which the 14-38 disulfide bond was specifically reduced and blocked with iodoacetamide [Jullien, M., & Baldwin, R. L. (1981) J. Mol. Biol. 145, 265-280]. Since this effect is independent of the stability of the protein, it is consistent with a model in which the proper docking of the interior residues of the protein is the rate-limiting step in the folding of these mutants.     

Partially folded bovine pancreatic trypsin inhibitor analogues attain fully native structures when co-crystallized with S195A rat trypsin

Crystal structures, at 1.7 Å resolution, were solved for complexes between each of two chemically synthesized partially folded analogues of bovine pancreatic trypsin inhibitor (BPTI) with the proteolytically inactive rat trypsin mutant S195A. The BPTI analogue termed [14-38]_Abu retains only the disulfide bond between Cys14 and Cys38, while Cys5, Cys30, Cys51, and Cys55 are replaced by isosteric α-amino-n-butyric acid residues. The analogue K26P,A27D[14-38]_Abu contains two further replacements, by statistically favored residues, in the type I β-turn that has been suggested to be a main site for initiation of BPTI folding. As a control, the structure of the complex between S195A trypsin and wild-type BPTI was also solved. Despite significant differences in the degree of structure detected among these three BPTIs in solution by several biophysical techniques, their tertiary folds once bound to S195A trypsin in a crystalline lattice are essentially superimposable.     

Hydrogen exchange in BPTI variants that do not share a common disulfide bond.

Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-51, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-55]Ala and [30-51; 14-38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI folding intermediates containing either the 5-55 or the 30-51 disulfide bond.     

Comparison of solution structures of mutant bovine pancreatic trypsin inhibitor proteins using two-dimensional nuclear magnetic resonance.

Structural perturbations due to a series of mutations at the 30-51 disulfide bond of bovine pancreatic trypsin inhibitor have been explored using NMR. The mutants replaced cysteines at positions 30 and 51 by alanine at position 51 and alanine, threonine, or valine at position 30. Chemical shift changes occur in residues proximate to the site of mutation. NOE assignments were made using an automated procedure, NASIGN, which used information from the wild-type crystal structure. Intensity information was utilized by a distance geometry algorithm, VEMBED, to generate a series of structures for each protein. Statistical analyses of these structures indicated larger averaged structural perturbations than would be expected from crystallographic and other information. Constrained molecular dynamics refinement using AMBER at 900 K was useful in eliminating structural movements that were not a necessary consequence of the NMR data. In most cases, statistically significant movements are shown to be those greater than approximately 1 A. Such movements do not appear to occur between wild type and A30A51, a result confirmed by crystallography (Eigenbrot, C., Randal, M., & Kossiakoff, A.A., 1990, Protein Eng. 3, 591-598). Structural alterations in the T30A51 or V30A51 mutant proteins near the limits of detection occur in the beta-loop (residues 25-28) or C-terminal alpha-helix, respectively.     

NMR structures of two variants of bovine pancreatic trypsin inhibitor (BPTI) reveal unexpected influence of mutations on protein structure and stability

Here we determined NMR solution structures of two mutants of bovine pancreatic trypsin inhibitor (BPTI) to reveal structural reasons of their decreased thermodynamic stability. A point mutation, A16V, in the solvent-exposed loop destabilizes the protein by 20 degrees C, in contrast to marginal destabilization observed for G, S, R, L or W mutants. In the second mutant introduction of eight alanine residues at proteinase-contacting sites (residues 11, 13, 17, 18, 19, 34, 37 and 39) provides a protein that denatures at a temperature about 30 degrees C higher than expected from additive behavior of individual mutations. In order to efficiently determine structures of these variants, we applied a procedure that allows us to share data between regions unaffected by mutation(s). NOAH/DYANA and CNS programs were used for a rapid assignment of NOESY cross-peaks, structure calculations and refinement. The solution structure of the A16V mutant reveals no conformational change within the molecule, but shows close contacts between V16, I18 and G36/G37. Thus, the observed 4.3kcal/mol decrease of stability results from a strained local conformation of these residues caused by introduction of a beta-branched Val side-chain. Contrary to the A16V mutation, introduction of eight alanine residues produces significant conformational changes, manifested in over a 9A shift of the Y35 side-chain. This structural rearrangement provides about 6kcal/mol non-additive stabilization energy, compared to the mutant in which G37 and R39 are not mutated to alanine residues.     

A cold-adapted protease engineered by experimental evolution system.

A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K(m) value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V. The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51. In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.     

Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?

Azurin has a beta-barrel fold comprising eight beta-strands and one alpha helix. A disulfide bond between residues 3 and 26 connects the N-termini of beta strands beta1 and beta3. Three mutant proteins lacking the disulfide bond were constructed, C3A/C26A, C3A/C26I and a putative salt bridge (SB) in the C3A/S25R/C26A/K27R mutant. All three mutants exhibit spectroscopic properties similar to the wild-type protein. Furthermore, the crystal structure of the C3A/C26A mutant was determined at 2.0 A resolution and, in comparison to the wild-type protein, the only differences are found in the immediate proximity of the mutation. The mutants lose the 628 nm charge-transfer band at a temperature 10-22 degrees C lower than the wild-type protein. The folding of the zinc loaded C3A/C26A mutant was studied by guanidine hydrochloride (GdnHCl) induced denaturation monitored both by fluorescence and CD spectroscopy. The midpoint in the folding equilibrium, at 1.3 M GdnHCl, was observed using both CD and fluorescence spectroscopy. The free energy of folding determined from CD is -24.9 kJ.mol-1, a destabilization of approximately 20 kJ.mol-1 compared to the wild-type Zn2+-protein carrying an intact disulfide bond, indicating that the disulfide bond is important for giving azurin its stable structure. The C3A/C26I mutant is more stable and the SB mutant is less stable than C3A/C26A, both in terms of folding energy and thermal denaturation. The folding intermediate of the wild-type Zn2+-azurin is not observed for the disulfide-deficient C3A/C26A mutant. The rate of unfolding for the C3A/C26A mutant is similar to that of the wild-type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction.     

Crystal structure of a Y35G mutant of bovine pancreatic trypsin inhibitor.

The structure of a Y35G mutant of bovine pancreatic trypsin inhibitor (BPTI) was solved by molecular replacement and was refined by both simulated annealing and restrained least-squares at 1.8 A resolution. The crystals belong to the space group P42212, with unit cell dimensions a = b = 46.75 A, c = 50.61 A. The final R-factor is 0.159 and the deviation from ideality for bond distances is 0.02 A. The structure of the mutant differs from that of the native protein, showing an overall root-mean-square (r.m.s.) difference of 1.86 A for main-chain atoms. However, the change is mostly localized in the two loops (respective r.m.s. values of 2.04 A and 3.93 A) and the C terminus (r.m.s. 6.79 A), while the core of the protein is well conserved (r.m.s. 0.45 A). The change in the loop regions can be clearly attributed to the mutation while the difference in the C terminus might be only due to a different crystal packing. Seventy water molecules were included in the model but only seven of them are shared with the native structure. Thermal parameters are showing a good correlation with those for the wild-type of BPTI.     

High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence

A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and structural consequences of truncating the binding loop residues to alanine. In addition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitution at position P(1) and an unrelated Met52Leu change. In spite of the binding loop modification, the affinity for trypsin is only 30 times lower than that of the wild-type protein. At pH 7.5 the protein can be crystallized on the time-scale of hours, yielding very stable crystals of a new (tetragonal) form of BPTI. Conventional source X-ray data collected to 1.4 A at room temperature allowed anisotropic structure refinement characterized by R=0.1048. The structure reveals all 58 residues, including the complete C terminus, which is in a salt-bridge contact with the N terminus. The Cys14-Cys38 disulfide bridge is observed in two distinct chiralities. This bridge, together with an internal water molecule, contributes to the stabilization of the binding loop. The Ala mutations have only an insignificant and localized effect on the binding loop, which retains its wild-type conformation (maximum deviation of loop C(alpha) atoms of 0.7 A at Ala13). Four (instead of the typical three) additional water molecules are buried in an internal cleft and connected to the surface via a sulfate anion. Three more SO(4)(2-) anions are seen in the electron density, one of them located on a 2-fold axis. It participates in the formation of a dimeric structure between symmetry-related BPTI molecules, in which electrostatic and hydrogen bonding interactions resulting from the mutated Lys15Arg substitution are of central importance. This dimeric interaction involves direct recognition loop-recognition loop contacts, part of which are hydrophobic interactions of the patches created by the alanine mutations. Another 2-fold symmetric interaction between the BPTI molecules involves the formation of an antiparallel intermolecular beta-sheet that, together with the adjacent intramolecular beta-hairpin loops, creates a four-stranded structure.     

Crystal structure of recombinant trypsin-solubilized fragment of cytochrome b(5) and the structural comparison with Val61His mutant

The crystal structure of the recombinant trypsin-solubilized fragment of the microsomal cytochrome b(5) from bovine liver has been determined at 1.9 A resolution and compared with the reported crystal structure of the lipase-solubilized fragment of the membrane protein cytochrome b(5). The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16-Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild-type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro-environment of the heme pocket and influencing the stability and the redox potential of the protein.     

Design of the helix-turn-helix motif: nonlocal effects of quaternary structure in DNA recognition investigated by laser Raman spectroscopy

The operator-binding domain of phage lambda repressor provides a model for DNA recognition by the helix-turn-helix (HTH) motif. In the wild-type protein, dimerization is mediated by hydrophobic packing (of the dyad-related helix 5), which serves as an indirect determinant of operator affinity. The mutant repressor, Tyr88----Cys, forms an intersubunit disulfide linkage and exhibits enhancement of both structural stability and operator affinity. Yet the dimer-specific operator affinity of the mutant is 10-fold weaker than that of the wild-type (noncovalent) dimer, suggesting nonlocal effects of the intersubunit disulfide bond on HTH recognition (Sauer et al., 1986). To explore such nonlocal effects, we describe laser Raman studies of the Cys88 mutant repressor and its interaction with operator sites OL1 and OR3. The following results have been obtained: (i) Wild-type and mutant dimers exhibit similar secondary structures, indicated by quantitative comparison of Raman amide I and amide III bands. (ii) The engineered disulfide of the mutant lacks rigorous symmetry; we observe mainly the gauche/gauche/trans CC-S-S-CC rotamer. (iii) Remarkably, distinctive local and nonlocal differences are observed in the mechanisms of DNA recognition by wild-type and mutant repressors. These differences involve specific hydrogen-bonding interactions between the protein and DNA, including guanine N7 sites in the major groove of DNA, and alterations in DNA phosphodiester conformation induced by protein binding. We analyze these differences in relation to crystal structures of the wild-type dimer with and without bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accelerated exchange of a buried water molecule in selectively disulfide-reduced bovine pancreatic trypsin inhibitor

Using magnetic relaxation dispersion (MRD), we have previously shown that the four internal water molecules in bovine pancreatic trypsin inhibitor (BPTI) exchange with bulk water on time scales between 10(-8) and 10(-4) s at room temperature. Because this exchange is controlled by the protein structure, internal water molecules can be used to probe rare conformational fluctuations. Here, we report (2)H and (17)O MRD data at three temperatures for wild-type BPTI and two BPTI variants where the 14-38 disulfide bond has been cleaved by a double Cys --> Ser mutation or by disulfide reduction and carboxamidomethylation. The MRD data show that the internal water molecules are conserved on disulfide cleavage. However, the exchange rate of the water molecule buried near the disulfide bond is enhanced by 2-4 orders of magnitude. The relation of water exchange to other dynamic processes in BPTI is discussed.     

Structural consequences of cysteine substitutions C1977Y and C1977R in calcium-binding epidermal growth factor-like domain 30 of human fibrillin-1

The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29-31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29-30, but not cbEGF30-31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.     

The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin.

Previous crystal structures of thrombin indicate that the 60-insertion loop is a rigid moiety that partially occludes the active site, suggesting that this structural feature plays a decisive role in restricting thrombin's specificity. This restricted specificity is typified by the experimental observation that thrombin is not inhibited by micromolar concentrations of basic pancreatic trypsin inhibitor (BPTI). Surprisingly, a single atom mutation in thrombin (E192Q) results in a 10(-8) M affinity for BPTI. The crystal structure of human thrombin mutant E192Q has been solved in complex with BPTI at 2.3 A resolution. Binding of the Kunitz inhibitor is accompanied by gross structural rearrangements in thrombin. In particular, thrombin's 60-loop is found in a significantly different conformation. Concomitant reorganization of other surface loops that surround the active site, i.e. the 37-loop, the 148-loop and the 99-loop, is observed. Thrombin can therefore undergo major structural reorganization upon strong ligand binding. Implications for the interaction of thrombin with antithrombin and thrombomodulin are discussed.     

Investigating the effects of double mutation C30A/C75A on onconase structure: Studies at atomic resolution

The structure of onconase C30A/C75A double mutant has been determined at 1.12Å resolution. The structure has high structural homology to other onconase structures. The changes being results of mutation are relatively small, distributed asymmetrically around the two mutated positions, and they are observed not only in the mutation region but expanded to entire molecule. Different conformation of Lys31 side chain that influences the hydrogen bonding network around catalytic triad is probably responsible for lower catalytic efficiency of double mutant. The decrease in thermal stability observed for the onconase variant might be explained by a less dense packing as manifested by the increase of the molecular volume and the solvent accessible surface area. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 454–460, 2014.