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Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
Reprint requests to: Juliette T.J. Lecomte, Department of Chemistry, The Pennsylvania State University, 152 Davey Laboratory, University Park, PA 16802, USA; e-mail: jtl1{at}psu.edu; fax: (814) 863-8403.
We have used a sequence prediction algorithm and a novel sampling method to design protein sequences for the WW domain, a small ß-sheet motif. The procedure, referred to as SPANS, designs sequences to be compatible with an ensemble of closely related polypeptide backbones, mimicking the inherent flexibility of proteins. Two designed sequences (termed SPANS-WW1 and SPANS-WW2), using only naturally occurring L-amino acids, were selected for study and the corresponding polypeptides were prepared in Escherichia coli. Circular dichroism data suggested that both purified polypeptides adopted secondary structure features related to those of the target without the aid of disulfide bridges or bound cofactors. The structure exhibited by SPANS-WW2 melted cooperatively by raising the temperature of the solution. Further analysis of this polypeptide by proton nuclear magnetic resonance spectroscopy demonstrated that at 5°C, it folds into a structure closely resembling a natural WW domain. This achievement constitutes one of a small number of successful de novo protein designs through fully automated computational methods and highlights the feasibility of including backbone flexibility in the design strategy.
Keywords: Protein structure; protein folding; stability and mutagenesis; protein design; WW domain
Abbreviations: BMRB, BioMagResBank • bp, base pair • CD, circular dichroism • DQF-COSY, double-quantum-filtered correlated spectroscopy • DSS, 2,2-dimethyl-2-silapentane-5-sulfonate • EDTA, ethylenediaminetetraacetic acid • GA, genetic algorithm • MALDI, matrix-assisted laser desorption ionization • MOPS, 3–[N-morpholino] propanesulfonic acid • MRE, molar residual ellipticity • NIa, nuclear inclusion a • NOE, nuclear Overhauser effect • NOESY, two-dimensional nuclear Overhauser effect spectroscopy • PAGE, polyacrylamide gel electrophoresis • PCR, polymerase chain reaction • ppm, parts per million • r.m.s.d., root-mean-square deviation • SDS, sodium dodecylsulfate • SPA, sequence prediction algorithm • SPANS, sequence prediction algorithm for numerous states • TFA, trifluoroacetic acid • TOCSY, totally correlated two-dimensional spectroscopy • TPPI, time-proportional phase incrementation • Tris, tris(hydroxymethyl) aminomethane
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