W238 Nucleic Acids Research, 2006, Vol. 34, Web Server issue size and characteristics of sequence space compatible with a particular fold. In a few cases, this information has been used to help detect remote homologs (19,20). In general, protein structures and complexes can be stabilized by identifying mutations that increase buried hydrophobic surface area. Towards this end, the RosettaDesign server can be used to search for holes in proteins that can be ﬁlled with larger hydro- phobic residues, or partially buried polar residues that can be replaced by hydrophobic residues. RosettaDesign can be used to search for second-site sup- pressor mutations. In this scenario, the user has a priori know- ledge of a mutation that destabilizes a protein or protein–protein complex. Using a resﬁle, the user can force the destabilizing mutation and use RosettaDesign to search for mutations that will compensate for the ﬁrst mutation. A similar approach was recently used to redesign a protein–protein inter- face so that the redesigned proteins still bind each other, but no longer bind their other naturally occurring binding partners (21). These types of redesigns are useful for probing signal transduction pathways. In cases where a protein can adopt multiple conformations, RosettaDesign can be used to identify sequences that are spe- ciﬁcally optimized for one of the conformations. Mayo and colleagues used this approach to increase the afﬁnity between a receptor protein and its ligand (22). More ambitiously RosettaDesign can be used to help design new protein struc- tures or portions of proteins. In this case, the user must supply the backbone coordinates of the target structure. The challenge is that many arbitrarily chosen protein backbones will not be designable. This is generally reﬂected in poor LJatr and SASApack (see Table 1) values for the redesigned protein. In the future, as our computational resources grow, we plan to modify the RosettaDesign server so that the backbone coordin- ates and the sequence can be optimized simultaneously to allow for tight packing between side chains. ACKNOWLEDGEMENTS This work was supported by a grant from the NIH (1RO1 GM073151-01). Funding to pay the Open Access publication charges for this article was provided by the NIH. Conflict of interest statement. None declared. REFERENCES 1. Pokala,N. and Handel,T.M. (2001) Review: protein design–where we were, where we are, where we’re going. J. Struct. Biol., 134, 269–281. 2. Park,S., Yang,X. and Saven,J.G. (2004) Advances in computational protein design. Curr. Opin. Struct. Biol., 14, 487–494. 3. Butterfoss,G.L. and Kuhlman,B. (2005) Computer-based design of novel protein structures. Annu. Rev. Biophys. Biomol. Struct., 35, 49–65. 4. Dahiyat,B.I. and Mayo,S.L. (1997) De novo protein design: fully automated sequence selection. Science, 278, 82–87. 5. Dwyer,M.A. and Hellinga,H.W. (2004) Periplasmic binding proteins: a versatile superfamily for protein engineering. Curr. Opin. Struct. Biol., 14, 495–504. 6. Harbury,P.B., Plecs,J.J., Tidor,B., Alber,T. and Kim,P.S. (1998) High- resolution protein design with backbone freedom. Science, 282, 1462–1467. 7. Rohl,C.A., Strauss,C.E., Misura,K.M. and Baker,D. (2004) Protein structure prediction using Rosetta. Meth. Enzymol., 383, 66–93. 8. Kuhlman,B. and Baker,D. (2000) Native protein sequences are close to optimal for their structures. Proc. Natl Acad. Sci. USA, 97, 10383–10388. 9. Dantas,G., Kuhlman,B., Callender,D., Wong,M. and Baker,D. (2003) A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins. J. Mol. Biol., 332, 449–460. 10. Eletr,Z.M., Huang,D.T., Duda,D.M., Schulman,B.A. and Kuhlman,B. (2005) E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer. Nature Struct. Mol. Biol., 12, 933–934. 11. Ambroggio,X.I. and Kuhlman,B. (2006) Computational design of a single amino acid sequence that can switch between two distinct protein folds. J. Am. Chem. Soc., 128, 1154–1161. 12. Kuhlman,B., Dantas,G., Ireton,G.C., Varani,G., Stoddard,B.L. and Baker,D. (2003) Design of a novel globular protein fold with atomic-level accuracy. Science, 302, 1364–1368. 13. Lazaridis,T. and Karplus,M. (1999) Effective energy function for proteins in solution. Proteins, 35, 133–152. 14. Kortemme,T., Morozov,A.V. and Baker,D. (2003) An orientation- dependent hydrogen bonding potential improves prediction of specificity and structure for proteins and protein–protein complexes. J. Mol. Biol., 326, 1239–1259. 15. Dunbrack,R.L.Jr and , Cohen,F.E. (1997) Bayesian statistical analysis of protein side-chain rotamer preferences. Protein Sci., 6, 1661–1681. 16. Simons,K.T., Ruczinski,I., Kooperberg,C., Fox,B.A., Bystroff,C. and Baker,D. (1999) Improved recognition of native-like protein structures using a combination of sequence-dependent and sequence-independent features of proteins. Proteins, 34, 82–95. 17. Korkegian,A., Black,M.E., Baker,D. and Stoddard,B.L. (2005) Computational thermostabilization of an enzyme. Science, 308, 857–860. 18. Nauli,S., Kuhlman,B. and Baker,D. (2001) Computer-based redesign of a protein folding pathway. Nature Struct. Biol., 8, 602–605. 19. Pei,J., Dokholyan,N.V., Shakhnovich,E.I. and Grishin,N.V. (2003) Using protein design for homology detection and active site searches. Proc. Natl Acad. Sci. USA, 100, 11361–11366. 20. Saunders,C.T. and Baker,D. (2005) Recapitulation of protein family divergence using flexible backbone protein design. J. Mol. Biol., 346, 631–644. 21. Kortemme,T., Joachimiak,L.A., Bullock,A.N., Schuler,A.D., Stoddard,B.L. and Baker,D. (2004) Computational redesign of protein–protein interaction specificity. Nature Struct. Mol. Biol., 11, 371–379. 22. Shimaoka,M., Shifman,J.M., Jing,H., Takagi,J., Mayo,S.L. and Springer,T.A. (2000) Computational design of an integrin I domain stabilized in the open high affinity conformation. Nature Struct. Biol., 7, 674–678.