It is generally considered that electrostatic interactions on the protein surface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity. We found that the tenth fibronectin type III domain of human fibronectin (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4. Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a). These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins. Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions.
The tenth fibronectin type III domain of human fibronectin (FNfn10) is a small, monomeric beta-sandwich protein, similar to the immunoglobulins. We have developed small antibody mimics, 'monobodies', using FNfn10 as a scaffold. We initially altered two loops of FNfn10 that are structurally equivalent to two of the hypervariable loops of the immunoglobulin domain. In order to assess the possibility of utilizing other loops in FNfn10 for target binding, we determined the effects of the elongation of each loop on the conformational stability of FNfn10. We found that all six loops of FNfn10 allowed the introduction of four glycine residues while retaining the global fold. Insertions in the AB and FG loops exhibited very small degrees of destabilization, comparable to or less than predicted entropic penalties due to the elongation, suggesting the absence of stabilizing interactions in these loops in wild-type FNfn10. Insertions in the BC, CD and DE loops, respectively, resulted in modest destabilization. In contrast, the EF loop elongation was highly destabilizing, consistent with previous studies showing the presence of stabilizing interactions in this loop. These results suggest that all loops, except for the EF loop, can be used for engineering a binding site, thus demonstrating excellent properties of the monobody scaffold.
The tenth fibronectin type III (FN3) domain of human fibronectin (FNfn10), a prototype of the ubiquitous FN3 domain, is a small, monomeric b-sandwich protein. In this study, we have bisected FNfn10 in each loop to generate a total of six fragment pairs. We found that fragment pairs bisected at multiple loops of FNfn10 show complementation in vivo as tested with a yeast two-hybrid system. The dissociation constant of these fragment pairs determined in vitro were as low as 3 nM, resulting in one of the tightest fragment complementation systems reported so far. Furthermore, we show that the affinity of fragment complementation is correlated with the stability of the uncut parent protein. Exploring this correlation, we screened a yeast two-hybrid library of one fragment and identified mutations that suppress the effect of a destabilizing mutation in the other fragment. One of the identified mutations significantly increased the stability of the uncut wild-type protein, proving that fragment complementation can be used as a novel strategy for the selection of proteins with enhanced stability.Keywords: protein engineering; reconstitution; library screening; b-sheet; surface loops The association of protein fragments into the native fold and concomitant restoration of its function have been reported for a number of proteins Shiba and Schimmel 1992;de Prat Gay and Fersht 1994;Kobayashi et al. 1995;Ostermeier et al. 1999;Berggard et al. 2000;Berggard et al. 2001;Ojennus et al. 2001).This phenomenon of fragment complementation illustrates that an amino acid sequence can be separated without a loss of the information necessary to code for the native structure. Classic examples of restoration of enzyme function by fragment complementation include ribonuclease S-protein (Kato and Anfinsen 1969) and bgalactosidase (Ullmann et al. 1967). Fragment complementation has been used as a tool to understand the mechanism of protein folding (de Prat Gay et al. 1995a,b;Kobayashi et al. 1995;Honda et al. 1999;Jourdan and Searle 2000) and in vivo protein-protein interactions (Johnsson and Varshavsky 1994;Rossi et al. 1997;Karimova et al. 1998;Pelletier et al. 1998;Magliery et al. 2005). Residual structures within the individual fragments may correspond to folding nucleation sites (Dyson et al. 1992a,b;de Prat-Gay 1996). Biophysical studies of such noncovalent complexes from 3 These authors contributed equally to this work. Reprint requests to: Shohei Koide, Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA; e-mail: skoide@uchicago.edu; fax: (773) 702-0439.Abbreviations: CI2, chymotrypsin inhibitor 2; FN3, fibronectin type III domain; FNfn10, tenth FN3 of human fibronectin; GuHCl, guanidine hydrochloride; HSQC, heteronuclear single-quantum correlation; NMR, nuclear magnetic resonance; PCR, polymerase chain reaction.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi
Presently X-ray crystallography of protein-antibody complexes is still the most direct way of identifying B-cell epitopes. The objective of this study was to assess the potential of a computer-based epitope mapping tool (EMT) using antigenic amino acid motifs as a fast alternative in a number of applications not requiring detailed information, e.g. development of pharmaceutical proteins, vaccines and industrial enzymes. Using Gal d 4 as a model protein, the EMT was capable of identifying, in the context of the folded protein, amino acid positions known to be involved in antibody binding. The high sensitivity and positive predictive value of the EMT as well as the relevance of the structural associations suggested by the EMT indicated the existence of amino acid motifs that are likely to be involved in antigenic determinants. In addition, differential mapping revealed that sensitivity and positive predictive value were dependent on the minimum relative surface accessibility (RSA) of the amino acids included in the mapping, demonstrating that the EMTs accommodated for the fact that epitopes are three-dimensional entities with various degrees of accessibility. The comparison with existing prediction scales demonstrated the superiority of the EMT with respect to physico-chemical scales. The mapping tool also performed better than the available structural scales, but the significance of the differences remains to be established. Thus, the EMT has the potential of becoming a fast and simple alternative to X-ray crystallography for predicting structural antigenic determinants, if detailed epitope information is not required.
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