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Protein Science (2004), 13:847-853. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Fast and faster: A designed variant of the B-domain of protein A folds in 3 µsec

Pooja Arora1, Terrence G. Oas1,2 and Jeffrey K. Myers3

1 Department of Chemistry and
2 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA
3 Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8725, USA

(RECEIVED December 2, 2003; FINAL REVISION December 15, 2003; ACCEPTED December 15, 2003)



Abstract

We have introduced the mutation glycine 29 to alanine, designed to increase the rate of protein folding, into the B-domain of protein A (BdpA). From NMR lineshape analysis, we find the G29A mutation increases the folding rate constant by threefold; the folding time is 3 µsec. Although wild-type BdpA folds extremely fast, simple-point mutations can still speed up the folding; thus, the folding rate is not evolutionarily maximized. The short folding time of G29A BdpA (the shortest time yet reported) makes it an attractive candidate for an all-atom molecular dynamics simulation that could potentially show a complete folding reaction starting from an extended chain. We also constructed a fluorescent variant of BdpA by mutating phenylalanine 13 to tryptophan, allowing fluorescence-based time-resolved temperature-jump measurements. Temperature jumps and NMR complement each other, and give a very complete picture of the folding kinetics.

Keywords: rapid protein folding; mutation; denaturant; helix propensity; dynamic NMR

Reprint requests to: Jeffrey K. Myers, Department of Biochemistry, Vanderbilt University Medical Center, 5140 MRB III, 465 21st Avenue South, Nashville, TN 37232-8725, USA; e-mail: myers{at}structbio.vanderbilt.edu; fax: (615) 936-2211.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03541304.


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