This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.107.114397v1 93/11/3989    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schlierf, M. Articles by Rief, M. Search for Related Content PubMed PubMed Citation Articles by Schlierf, M. Articles by Rief, M.

Direct Observation of Active Protein Folding Using Lock-in Force Spectroscopy

Michael Schlierf ^*, Felix Berkemeier ^* and Matthias Rief ^* 

^* Physik Department E22, Technische Universität München, München, Germany; and Munich Center for Integrated Protein Science, Munich, Germany

Correspondence: Address reprint requests to Matthias Rief, Tel.: 49-89-289-12471; E-mail: mrief{at}ph.tum.de.

Direct observation of the folding of a single polypeptide chain can provide important information about the thermodynamic states populated along its folding pathway. In this study, we present a lock-in force-spectroscopy technique that improves resolution of atomic-force microscopy force spectroscopy to 400 fN. Using this technique we show that immunoglobulin domain 4 from Dictyostelium discoideum filamin (ddFLN4) refolds against forces of 4 pN. Our data show folding of this domain proceeds directly from an extended state and no thermodynamically distinct collapsed state of the polypeptide before folding is populated. Folding of ddFLN4 under load proceeds via an intermediate state. Three-state folding allows ddFLN4 to fold against significantly larger forces than would be possible for a mere two-state folder. We present a general model for protein folding kinetics under load that can predict refolding forces based on chain-length and zero force refolding rate.