Nucleic Acids Research, 2006, Vol. 34, Web Server issue W235–W238 doi:10.1093/nar/gkl163 RosettaDesign server for protein design Yi Liu and Brian Kuhlman* Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA Received January 30, 2006; Revised February 22, 2006; Accepted March 20, 2006 ABSTRACT The RosettaDesign server identifies low energy amino acid sequences for target protein structures (http:// rosettadesign.med.unc.edu). The client provides the backbone coordinates of the target structure and spe- cifies which residues to design. The server returns to the client the sequences, coordinates and energies of the designed proteins. The simulations are performed using the design module of the Rosetta program (RosettaDesign). RosettaDesign uses Monte Carlo optimization with simulated annealing to search for amino acids that pack well on the target structure and satisfy hydrogen bonding potential. RosettaDesign has been experimentally validated and has been used previously to stabilize naturally occurring pro- teins and design a novel protein structure. INTRODUCTION Recently, there have been many successes in the area of computational protein design. Protein design software has been used to stabilize naturally occurring proteins, perturb protein binding speciﬁcity, design novel biosensors and enzymes and create novel protein structures [for a review see (1–3)]. In most cases, these studies have been performed by laboratories that specialize in computational design and have direct access to the software and the source code (4–6). To make this technology more accessible to the large number of molecular biology laboratories that regularly use amino acid mutagenesis to probe protein structure and func- tion, we have established a web server for protein design that uses the design module of the Rosetta program (Rosetta Design) (7,8). Given a target protein structure or complex, RosettaDesign searches for amino acid sequences that pack well, bury their hydrophobic atoms and satisfy the hydrogen bonding potential of polar atoms. RosettaDesign has been parameterized to return sequences with amino acid frequencies comparable to those found in naturally occurring proteins, and to partition the hydrophobic and polar residues between the surface and the core at naturally occurring frequencies. In general, when redesigning a naturally occurring protein 65% of the residues will mutate. As expected, more sequence variability is seen on the surface of the protein where there are fewer packing con- straints. In the core of the protein 45% of the residues mutate on average. RosettaDesign has been experimentally validated. It has been used to stabilize naturally occurring proteins (9), enhance protein binding afﬁnities (10), design a protein that can switch between 2-folds (11) and create a protein with a novel structure (12). METHOD USED The RosettaDesign server uses the design module of the Rosetta program to perform ﬁxed backbone protein design simulations. The algorithm has been described previously (7,8). Like other protein design programs, RosettaDesign has two primary components: an energy function for eva- luating the relative favorability of a sequence and an optim- ization procedure for searching through sequence space. All atoms in the protein, including hydrogen, are explicitly mod- eled. The energy function consists of (i) a Lennard–Jones potential that favors close packed residues, (ii) the Lazaridis– Karplus implicit solvation model which favors hydrophobic amino acids in the interior of proteins and polar amino acids on the surface (13), (iii) an explicit orientation dependent hydro- gen bonding term (14), (iv) torsion potentials derived from the PDB (15), (v) a unique reference value for each amino acid type and (vi) electrostatic interactions between charged resi- dues are modeled by an additional term that is based on the probability of seeing two amino acid types near each other in the PDB (16). This is a relatively weak term in the energy function. To simplify the optimization procedure and favor low energy designs, amino acid side chains are only allowed to adopt a discrete set of favorable conformations, typically referred to as rotamers. RosettaDesign uses Dunbrack’s back- bone dependent rotamer library (15). To allow for relaxation away from the most preferred side chain conformations, addi- tional rotamers are created for buried residues by varying chi1 and chi2 one standard deviation ( 10 ) away from the most preferred values. Rotamers are also created for the alternate positions hydrogen can adopt on serine, threonine and tyrosine. To ﬁnd low energy sequences, RosettaDesign *To whom correspondence should be addressed. Tel: +1 919 843 0188; Fax: +1 919 966 2852; Email: bkuhlman@email.unc.edu The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. 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