Examination of Protein G stability and Binding Characteristics Using Four Body Nearest Neighbor Contact Potentials

Transcription

1 Examination of Protein G stability and Binding Characteristics Using Four Body Nearest Neighbor Contact Potentials Abstract Gregory M. Reck George Mason University A computational geometry technique employing Delaunay tessellation to derive a statistical residue contact potential has been used to study the folding/unfolding stability and binding characteristics of a number of reported mutants of the B domains of protein G. Protein G is a multidomain bacterial cell wall protein that functions as a binding unit to several proteins including immunoglobulin G (IgG). The B domains of this protein are small thermostable folding units that have been used as model systems to study the interactions of residues participating in beta-sheet secondary structures. A virtual mutagenesis technique using Delaunay tessellation is used to compute the four-body nearest-neighbor contact potentials of single and multiple point mutations of these domains. The total contact potentials are then compared with the IgG binding affinity measurements and with values of DG determined from protein stability measurements. The total contact potential for mutations of residues near the IgG binding site correlates with measured dissociation constants, and mutations in or around the hydrophobic core show a positive correlation with DG. Stability correlations are evident, but not as strong for mutations in the central region of the solvent-exposed face of the beta sheet. Introduction Delauney tessellation is a novel technique for analyzing protein structural data that can provide unique information regarding the neighborhood of each residue (1). This approach provides insight into protein systems by coupling a three-dimensional nearestneighbor characterization of the protein structure derived through statistical geometry methods with compositional analysis of the relationships among the residues in the folded structure. The method can be used to examine topological features of the protein, to conduct statistical geometric analysis of the protein structure, and to explore protein attributes related to its composition (2). In particular, the method can produce a fourbody statistical (or contact) potential for each residue, that indicates the likelihood of that residue to be involved with its neighbors based on a large reference set of proteins (3). The 1-D contact potential profiles built from amino acid sequences can help identify residues in environments such as specific fold families, binding sites or active regions. The total contact potential (sum of the potential for each residue in the sequence) can be related to the stability of the protein (4). While statistical potentials can not be directly related to Boltzman parameters, changes in contact potential due to mutations should correlate with resulting changes in free energy (DDG) or in melting temperatures. The effects of mutagenesis can be simulated by changing the appropriate residue in the native sequence to the new (mutated) residue, computing a new potential profile, and comparing the mutant profile with the native profile. Protein G is a multi-domain globular protein that was initially isolated from a human group G streptococcal strain (5). It drew immediate attention as a potential immunological reagent because of its binding affinity to IgG from a broad range of mammals (6), and was quickly cloned, sequenced and expressed (7). The N-terminal region of the protein contains domains for binding sites to albumin, while the C-terminal region

2 2 includes the B domains that are associated with binding to the constant fragment (Fc) region of IgG. Structural characterization of the B region revealed 2 and in some strains 3 homologous domains (8-12). Each of these domains represents a stable folding unit, identified as B1, B2 or B3 with greater than 90% identity between the domains. NMR analysis of the 56 residues in the B1 domain (8) revealed a 4-stranded beta sheet structure with the 2 central beta strands in parallel configuration and the outer strands in antiparallel configuration. The outer strands are connected by a 4-turn alpha helix that spans the beta sheet and each outer strand then connects to one of the inner strands. Each of the 2 central beta strands then leads to a terminus of the domain. The B2 subunit differs from B1 by 6 residues (10,11) and exhibits the same overall structure, but shows some variation in melting temperature and binding affinity to Fc. Physicochemical characterization of B1 shows a stable globular folding unit with unusually high thermal stability: a melting temperature of 87 C and completely reversible thermal denaturation (13). With no disulfide bridges or bound ligands, this stability is attributed in large part to a very compact hydrophobic core. An initial folding analysis computed free energy difference DG (unfolding) for both B1 and B2and compared the values. It was observed that changes in the free energy of unfolding resulting from mutations would likely cause relatively large changes in melting temperature (13). These features have led to the use of B1 as a model folding unit for mutagenesis studies of the structure and energetics of beta sheet formation in proteins. Studies include the influence of side-chain interactions on the stability of the beta sheet, both for adjacent betastrands in the interior of the sheet (14,15) and also for the edge strand residues at solvent-exposed positions (16,17). Another mutagenesis effort studied the effect of core residue mutations on protein stability (18) Yet another effort explored whether entropic Figure 1. The structure of GB1 binding protein. factors (limiting the number of conformations accessible to a residue) dominate in the propensity of B1 to form beta-sheets (19). Mutagenesis has also been employed in efforts to change the characteristics of the B domains. The B2 unit was modified in a successful effort to improve the alkaline stability for applications in protein-based affinity chromatography (20). Another study has demonstrated a significant increase in thermal stability of B1 through multiple mutations guided by computer-based design (21,22). Stability data provided by these studies on the mutant proteins ( D DG) can be used to demonstrate the utility of Delaunay tessellation to interpret and predict the effects of these variations on stability. Previous studies have also focused on the functional role of the B subunits by exploring the nature of the binding interaction with the Fc region. Early characterization of the B1-Fc complex (12) indicated that the binding site is dominated by side-chain interactions. Specific B1 residues directly involved with the binding site were confirmed with alanine scanning mutagenesis (23), by systematically modifying each amino acid in the region and measuring the dissociation constant of the mutant. A key

3 3 feature of the binding site is a double knobin-hole interface. On the immunoglobulin molecule, the Fc binding site is located in a hinge region that is described as adaptive, exposed, nonpolar, and energetically important (24). This study examines the use of DT by computing the contact potential for GB1 mutants from a number of stability and binding studies employing mutagenesis that have been reported in the literature. a number of as well as the GB2 domain. The differences in contact potential between wild type and mutants are then compared to the measured D DG values and melting temperatures of the mutations and also to dissociation rate data for binding of the mutants to IgG Fc. A comparison is also made between the GB1 and GB2 domains. Computational Methods The Delaunay tessellation method begins by tessellating the entire protein structure using the three dimensional spatial coordinates of only the alpha-carbon atoms in the protein chain (data on the specific residue associated with each alpha carbon is stored). The tessellation consists of two parts, first a Voronoi tessellation partitions the volume of the protein into a set of polyhedra such that each contains a single alpha-carbon atom and all points in the interior of the polyhedra are closer to that atom than any other alphacarbon atom. Examination of these results shows that four polyhedra always meet at a common vertex. In the second step, the Voronoi polyhedra are used to create a set of Delaunay simplices by linking together each group of four carbon atoms whose polyhedra share a single common vertex. This process is illustrated in 2D in figure 2 where the vertices of the Voronoi polyhedra link 3 carbon atoms, thus the Delaunay simplices are always triangles. In 3D, the Delaunay simplices are always tetrahedra, with an alpha-carbon at each vertex and they are space-filling (i.e., they tile the entire interior volume of the protein). Our interest is in the Delaunay simplices that represent the ensemble of neighboring alpha-carbon atoms. This approach to defining nearest neighbors is uniquely objective since it is independent of an arbitrary definition of nearest-neighbor distance criteria. While the Delaunay simplices can provide considerable geometric and topological information regarding the protein structure, the tessellation can be extended to include a statistical analysis of the amino acid composition of each of the Delaunay simplices in the structure. Since each simplex is defined by four alpha-carbon atoms, the specific amino acids associated with each of the carbons define a compositional quadruplet for each simplex. This quadruplet can than be associated with a statistical potential for each simplex. Given the standard set of amino acids and order independence, there are 8855 possible combinations of residues in quadruplets. Using a Figure 2. Voronoi/Delaunay tessellation in 2D space. (Voronoi polyhedra dashed lines, Delaunay simplices--solid lines). representative non-redundant set of highresolution protein structures from PDB, the observed frequency of occurrence has been determined for each possible quadruplet. This observed frequency can be compared with the frequency of random occurrence to give the log-likelihood factor (or statistical

4 4 potential) for each quadruplet as follows: q ijkl = log f ijkl. p ijkl The log-likelihood factors can be summed for all of the simplices in the protein to give a total statistical potential score for the protein. In addition, the q factors can be summed for all of the simplices in which a given alpha-carbon participates (a residue may participate in a number of simplices, core residues typically participate in more simplices than surface residues), and if this is done for each residue in a protein sequence, a 3D-1D potential profile can be obtained for the entire sequence. The DT potential represents a statistical frequency or knowledge-based potential that is similar in many respects to the equilibrium representation of free energies, since it represents the equilibrium between observed frequencies in a representative set and purely random frequencies (46). The starting point for the DT method is a high-resolution structural coordinate file. The X-ray structures for both B1 (12) and B2 (9) (resolution 1.66 A) have been archived in PDB and are satisfactory for tessellation and analysis. Two structures are available for B1 (PDB entries 1pga and 1pgb) based on two crystal forms of the protein (resolutions of 1.92 and 2.07 A) and the a-c positions vary by 0.25A rms Results Comparison of the B1 and B2 domains Figure 3 shows a comparison of the contact potential profile for B1 compared with the profile for the B2 domain. The red arrows indicate the location of the 6 residues that vary between the two domains and the colored bars at the bottom of the chart identify the extents of a-helix and b-strand secondary structures. Since the PDB file for B2 includes additional residues at both the N terminal and C terminal ends, these residues were excluded from the coordinate file before tessellation in order to provide a more direct comparison of contact potential. The two domains vary by only 0.5 A rms, but the variations in residue at the six Figure 3. Comparison of contact potential profiles for proteins B1 and B2. Red arrows identify the 6 residue positions that differ from B1 to B2 as follows: I6V, L7I, E19K, A24E, V29A, and E42V. Colored bars at bottom of chart identify range of b-strand (yellow) and a-helix (green).

5 positions alter the compositions of the simplices at a number of positions along the sequence resulting in differences in residue contact potential and also in the total contact potential for the overall protein. The total potential for B1 is 2.24, compared to a total potential for the truncated B2 sequence of The overall features of the profiles are quite similar for the two proteins. For example, the high positive potential values at several positions (5, 7, 26, 30, 52 and 54) coincide with hydrophobic residues at core locations, although several surface sites also have comparably positive values (28, 46, 35). It is apparent that the central regions of the first, third and fourth b-strands as well as the central region of the a-helix have residue potentials that are either positive or near zero. Many residues in these regions are involved in the interaction between the b- sheet and the a-helix and are contributors to the tightly packed hydrophobic core of this protein. The mid-portion of the fourth b- strand has the highest grouping of positive values. Residue 53 at the center of this group has been a target site for several mutagenesis studies of b-sheet side chain interactions (14,15). Residues that have been identified as contributors to the binding between GB1 and IgG are located at several points between positions 27 near the middle of the helix and position 43 at the start of the third beta strand. The potential profile shows considerable variability throughout this region that may indicate unusual topological features contributing to binding activity. Protein G Binding to IgG-Fc An X-ray structure of protein G complexed with the Fc fragment of human IgG has been acquired (12), and has provided insight into the nature of the protein-protein interface and the key residues in the Fc binding interaction with the GB1 domain. The GB1 binding site was characterized as utilizing charged and polar residues in hydrogen bonds and salt bridges compared to protein A stabilization with IgG Fc through hydrophobic effects with fewer polar interactions. The site also incorporates two knob-inhole interactions, one with a residue on the GB1 surface that extends into a pocket on Fc, and vice versa. A subsequent study used alanine scanning mutagenesis to further investigate the influence of individual residues on IgG Fc binding (23). This study used the X-ray structure of the GB1-Fc fragment complex to identify residues located in the interface region between the two proteins. Ten residues were identified based on a calculation of the decrease of static solvent accessible surface between unbound and bound structures. One of these residues was mutated to cysteine to accommodate a fluorophore and all measurements were made relative to this variant of the wild type. Nine additional mutants were created by substituting alanine for the wild type residue at each of the remaining nine locations (in addition to the cysteine substitution), and the dissociation binding constants of each of the mutants was measured. The resulting data showed considerable variability ranging from no effect to a > 4000 increase in dissociation rate. Five of the mutants showed substantial decreases in binding affinity (> 10-fold increase in dissociation rate constant). The relationship between four-body contact potential and protein GB1 binding to IgG was investigated by analyzing the mutagenesis data provided in reference 23. The total contact potential relative to wild type was computed for seven of the ten mutants and is correlated with the measured dissociation constants in fig. 4. The mutant used as a site for the fluorophore was not included since the effect of the fluorophore could not be appropriately accommodated in the tessellation. Also, two of the mutated residues were not identified in reference 18 as contributing to the binding site, one showed no effect on binding and the second was not identified as a critical residue in ref. 23. The correlation is strongly influenced

6 Figure 4. Correlation of dissociation constant data from ref. 23 with contact potential for mutations of core residues in protein GB1. Mutations include: E27A, K28A, K31A, N35A, D40A, E42A, and W43A. The inset shows a different linear correlation excluding the data point for E27A (Kd > 1000) with R 2 = by the dissociation constant measurement in excess of 1000 mm for mutant E27A. The glutamate at position 27 is the knob that extends into a cavity on Fc, and since alanine-scanning mutagenesis effectively removes side chains, the binding contribution of this residue was lost and the binding interaction was too weak to be quantified. While the data in figure 4 suggest a strong correlation (R 2 =0.87), there is a concern regarding the inclusion of cysteine in each of the mutants. Quadruplets containing cysteine typically have extreme values and the inclusion of cysteine in the region of study may distort the contact potential data. Stability A significant amount of mutagenesis data has been reported in the literature and can be used to explore the relationship between total contact potential and protein stability. However, several factors must be considered in selecting and analyzing data sets, including reference state modifications and multisite mutations. Each of the mutagenesis data sets incorporated in this study include one or several reference state or background mutations that were necessary or desirable in order to accomplish the scientific objectives of the original study. For example, many studies include a mutation from threonine to glutamine at the second position in the GB1 sequence. This substitution eliminates a processing issue in which a portion of the E. coli GB1 protein product lacks the N- terminal methionine. This could destabilize the protein by up to 1 kcal/mol (18), but this is not an issue in a specific study if the change is baselined. Another example is the creation of a desirable host environment for a systematic mutagenesis study at a fixed guest site. This may involve modification of one or several adjacent residues in order to isolate or create the necessary environment for evaluating mutated residues at the guest location. In the binding study described above, a Q32C mutation was baselined to provide a covalent attachment site for a fluorophore for reporting during titration. In each case, the changes are used consistently, and have little or no influence on the study objective. However, multiple mutations introduce uncertainty in the tessellation computation and make comparisons between data sets more difficult.

7 7 It is most desirable to have high resolution coordinate data available for both the w.t. and the mutant proteins in order to compute an accurate contact potential. However, mutant coordinate data is not typically available. Since tessellation is based on the coordinates of only the a-carbons rather than the side-chain conformations, the assumption is typically made that the position of the backbone carbons will not change significantly as a result of mutation. This may be acceptable for single point mutations, but as the number of substitutions increases some changes are expected. The problem will be exacerbated if mutations are adjacent or concentrated in a local area. Data is not currently available on practical limits associated with this effect. A number of the data sets examined in this study include multiple mutations. Previous efforts have demonstrated a significant relationship between total contact potential and protein stability for hydrophobic core mutations of well-characterized protein systems (4). While several mutagenic studies of the GB1 system have included a limited number of core residues, one study has focused exclusively on the stability and structural integrity of core mutants of protein G (18). In this effort, five sites were selected on the b-sheet, four on the internal side facing the a-helix with no solvent accessible surface, and one control site on the solvent exposed surface of the b- sheet. A randomization strategy at each of the selected sites was used to generate a large number of transformants which were subjected to a screening process eventually leading to 7 mutants. Most of the mutants included substitutions at more than one of the five sites, two had changes at 3 sites, two had changes at 4 sites, and one included changes at all 5 positions. Each of the mutants also included the T2Q background modification described earlier. Figure 5. Correlation of free energy of unfolding from ref. 18 for mutations of b-strand residues that interface with the hydrophobic core of protein GB1. The structural stability of the mutants was assessed by NMR, and equilibrium unfolding was measured in guanidinium chloride at ph 5.4 and 25 C. The slopes of the unfolding curves were converted to free energy and the DG values are shown in fig. 5 versus the total contact potential for each mutant. The data show a correlation with an R 2 correlation coefficient of Since nearly half of the mutants had substitutions at 5 or more sites, the correlation is reasonable considering the uncertainties associated with multi-site mutations. Another study which involved modifications to interior residues was focused on increasing the stability of GB1 in an effort to demonstrate protein design methodologies and algorithms (21). This approach employs a computational strategy to simultaneously evaluate a number of factors that contribute to protein stability coupled with a combinatorial optimization algorithm to select the residues and structures that minimize an energy function. For GB1, the effort focused on a set of ~8 boundary residues that lie in the region between the hydrophobic core residues (~10) and the solvent-exposed surface. The program also began with three core mutants resulting from a previous optimization study. The result of the effort

8 8 was a 7-fold mutant with a melting temperature in excess of 100 C (compared to 83 C for w.t.). In this study, an NMR structure of the most stable mutant was determined reducing the concern that the extensive mutations might significantly alter the structure of the protein. Experimental thermodynamic data was reported for several mutants representing intermediate steps in the transformation to the final thermostable protein. These data are shown in fig. 6 and show a high level of correlation to the computed contact potential with an R 2 coefficient of Previous studies have shown a relationship between the contact potential of hydrophobic core mutants and the associated change in DG, but the relationship is less clear for surface residues where the influence of adjacent side chains is diminished and solvent interactions are introduced. Several studies have used site-specific mutagenesis of b- sheet residues in GB1 to systematically examine side chain interactions and residue propensities to form b-sheets by measuring the effect Figure 6. Correlation of free energy of unfolding from ref. 21 with difference in contact potential for thermostable mutants of GB1 (DDG values are referenced to GB1). of the mutation on the stability of the protein. Typically, a region on the solvent exposed b-sheet surface was modified to provide an unbiased host environment for a guest or mutation position in either a central b-strand (15,16), or an edge b-strand of the sheet (17) or both (14). The contact potential for each of the mutants in these studies was computed and compared with measured values of the melting temperature for the mutants. In each case, the data were sorted with respect to the type of residue: hydrophobic, polar or charged. Figure 7. Correlation of melting temperature data from ref. 15 with difference in contact potential for mutations of the b-sheet surface residue at central strand position 53. The data in figure 7 display the data from ref. 15 which involved substitution of guest residues at position 53, a solvent exposed position in the mid-portion of one of the central strands. Two of the host residues surrounding this site were modified to provide a computed solvent accessibility (15) at this site of 68% for A, 75% for I and 71% for F. The data indicate that stability does correlate with contact potential for this solvent exposed site. There does appear to be additional effects associated with residue type reflecting the propensity of different types to form beta sheets as well as the influence of solvent accessibility. The stability with glycine is significantly lower, and this data point (as well as proline) was not included in the determination of the correlation coefficient.

9 9 The data shown in fig. 8 are derived from a second mutagenesis study (17) that systematically examined the effect of different residues at the same guest position (53) used in the previous effort. In this case, a different host environment was established surrounding the guest site by replacing 2 adjacent threonine residues with serine. There were also other differences in background modifications, but the efforts were quite similar. The data are quite similar, especially with respect to the residue type, reinforcing the correlation with contact potential. While stability changes for single site mutations on the solvent-facing side of an edge strand in a beta sheet did not correlate with contact potentials, another study that modified pairs of residues on the surface of the sheet (14) did lead to the correlation shown in figure 9. In this study, both site 44 on the edge strand and site 53 on the central strand served as guest sites for pairs of mutants. The hydrophobic-polar combinations were each examined in two separate mutants with the residues switched. The features of the correlation in figure 9 are similar to the correlations for single residue variations at the central position 53 shown earlier. However, these data include interactions between the residue pairs that may contribute or detract from overall protein stability. In general, the stability or melting temperature of these mutants is higher than the single point mutants shown in fig. 6 and 7. The R 2 correlation coefficients are similar for all three data sets shown. Figure 8. Correlation of melting temperature from ref. 17 for residue mutations at position 53, a central strand location. Another mutagenesis study (16) focused on the effects of changes in residues at a single guest position (44) on a beta strand at the edge of the beta-sheet. The three adjacent side chains in the host environment on the same side of the sheet were modified to alanine. While solvent accessibility is higher at this site, interaction with adjacent residues will be significantly reduced compared to positions on the central strands. Thus the stability associated with favorable side chain interactions for some residues will not occur. The data from this study failed to show any significant correlation with contact potential and are not shown. Figure 9. Correlation of melting temperature data from ref. 14 with difference in total contact potential for paired mutations of b- sheet surface residues. Discussion Protein GB1, a small thermostable protein with substantial beta sheet secondary struc-

10 10 ture has been selected as a model protein system to demonstrate the use of Delaunay tessellation in protein stability and binding studies. Using data from the literature on reported mutagenesis studies of GB1, contact potentials have been derived for the mutants and then compared with measured values of dissociation constant and DG or melting temperature. Positive correlations have been shown with binding affinity for mutants of key residues in the binding interface with IgG. Correlations have also been shown with thermal stability for mutations of the compact hydrophobic core in GB1 between the interior beta sheet face and the helix. A strong correlation was determined for several core mutants that were engineered for increased thermal stability. For mutants on the solvent-exposed central surface of the GB1 beta sheet, the correlations were not as strong as for the core, but clearly showed a relationship with thermal stability. Mutations of individual residues on the edge strands of the beta sheet did not show a relationship between stability and contact potential, but when paired with central strand residues, the correlation was evident. Thus DT four-body contact potential can aid in protein analysis and understanding and could be particularly useful in guiding future mutagenesis efforts. References 1. Singh, R.K., Tropsha, A. and Vaisman, I.I. (1995) Delaunay tessellation of proteins: Four body nearest neighbor propensities of amino acid residues, J. Comput. Biol., 3:2, Zheng,W., Cho, S.J., Vaisman, I.I. and Tropsha, A. (1997) A new approach to protein fold recognition based on Delaunay tessellation of protein structure, Biocomputing 97, eds. R.B. Altman, et.al., World Scientific, 1997, Vaisman, I.I., Tropsha, A. and Zheng,W. (1998) Compositional preferences in quadruplets of nearest neighbor residues in protein structures: statistical geometry analysis, IEEE Symposia on Intelligence and Systems, Carter, C.W., LeFebvre, B.C., Cammer, S.A., Tropsha, A. and Edgell, M.H. (2001) Four-body potentials reveal protein-specific correlations to stability changes caused by hydrophobic core mutations. J. Mol. Biol., 311, Bjorck, L. and Kronvall, G. (1984). Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol., 133:2, Akerstrom, G. and Bjorck, L. (1986). A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties. J. Biol. Sci., 261:22, Fahnestock, S.R., Alexander, P., Nagle, J. and Filpula,D. (1986). Gene for an immunoglobulin-binding protein from a group G streptococcus. J. Bacteriol., 167:3, Gronenborn, A.M., Filpula, D.R., Essig, N.Z., Achari, A. Whitlow, M., Wingfield, P.T. and Clore, G.M. (1991). A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science, 253, Achari, A., Hale, S.P., Howard, A.J., Clore, G.M., Gronenborn, A.M., Hardman, K.D., and Whitlow, M. (1992) Angstrom x-ray structure of the B2 immunoglobulin-binding domain of streptococcal protein-g and comparison to the NMR structure of the B1 domain, Biochem., 31:43, Orban, J., Alexander, P. and Bryan, P. (1992). Sequence-specific 1H NMR assignments and secondary structure of the streptococcal protein G B2 domain, Biochem., 31, Gallagher, T., Alexander, P., Bryan, P. and Gilliland, G.L. (1994) Two crystal structures of the B1 immunoglobulinbinding domain of streptococcal protein G and comparison with NMR. Biochem., 33, Sauer-Eriksson, A.E., Kleywegt, G.J., Uhlen, M. and Jones, T.A. (1995) Crystal structure of the C2 fragment of streptococcal protein G in complex with

11 11 the Fc domain of human IgG, Structure, 3, Alexander, P., Fahnestock, S., Lee, T., Orban, J. and Bryan, P. (1992) Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: Why small proteins tend to have high denaturation temperatures. Biochem., 31, Smith, C.K. and Regan, L. (1995). Guidelines for protein design: the energetics of B-sheet side chain interactions. Science, 270, Smith, C.K., Withka, J.M. and Regan, L., (1994) A thermodynamic scale for the B-sheet forming tendencies of the amino acids, Biochem., 33: Minor, D.L. and Kim, P.S. (1994,). Context is a major determinant of B- sheet propensity. Nature, 371, Minor, D.L. and Kim, P.S. (1994) Measurement of the b-sheet-forming propensities of amino acids, Nature, 367, Gronenborn, A.M., Frank, M.K. and Clore, G.M. (1996) Core mutants of the immunoglobulin binding domain of streptococcal protein G: stability and structural integrity. FEBS Lett., 398: Stone, M.J., Gupta, S., Snyder, N. and Regan, L. (2001) Comparison of protein backbone entropy and B-sheet stability: NMR-derived dynamics of protein G domain mutants. J. Am. Chem. Soc., 123, Gulich, S., Linhult, M., Stahl, S. and Hober, S. (2002) Engineering streptococcal protein G for increased alkaline stability. Prot. Eng., 15(10): Malakauskas, S.M. and Mayo, S.L. (1998) Design, structure and stability of a hyperthermophilic protein variant. Nat. Struct. Biol., 5(6): Strop, P., Marinescu, A.M. & Mayo, S.L. (2000) Structure of a protein G helix variant suggests the importance of helix propensity and helix dipole interactions in protein design, Protein Sci., 9: Sloan, D.J. and Hellinga, H.W. (1999) Dissection of the protein G B1 domain binding site for human IgG Fc fragment. Protein Sci., 8: DeLano, W.L., Ultsch, M.H., Abraham, M. and Wells, J.A. (2000) Convergent solutions to binding at a protein-protein interface. Science, 287: