PEDS Advance Access originally published online on July 25, 2005
Protein Engineering Design and Selection 2005 18(8):389-395; doi:10.1093/protein/gzi047
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Kinetic folding mechanism of PDZ2 from PTP-BL
1Istituto Pasteur–Fondazione Cenci Bolognetti e Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, Università di Roma ‘La Sapienza’, Piazzale A. Moro 5, 00185 Rome, Italy and 2Department of Biophysical Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
3 To whom correspondence should be addressed. E-mail: carlo.travaglini{at}uniroma1.it
PDZ domains represent a large family of protein-interaction modules associated with a variety of unrelated proteins with different functions. We report a complete characterization of the kinetic folding mechanism of a fluorescent variant of PDZ2 from PTP-BL, investigated under a variety of different experimental conditions. For this purpose, we engineered a fluorescent variant of this protein Y43W (called pseudo-wild-type, pWT43). The results suggest the presence of a high-energy intermediate in the folding of PDZ2, as revealed by a pronounced non-linear dependence of the unfolding rate constant on denaturant concentration. Such an intermediate may or may not be detectable depending on the experimental conditions, giving rise to apparent two-state folding under stabilizing conditions (e.g. in the presence of sodium sulfate). Interestingly, even under these conditions, three-state folding can be restored by selectively destabilizing the native-like rate-limiting barrier by one specific mutation (V44A). Finally, we show that data taken on pWT43 under different experimental conditions (e.g. different pH values from 2.1 to 8.0 or in the presence of a stabilizing salt) and also data on a site-directed conservative mutant can be rationalized in terms of a simple reaction scheme involving a single set of intermediates and transition states.
Keywords: chevron plot/Hammond effect/intermediate/protein folding/transition state
Received May 3, 2005; revised June 21, 2005; accepted July 1, 2005.
Edited by Jane Clarke
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