This Article Full Text Full Text (PDF) supplemental All Versions of this Article: biophysj.105.072066v1 89/5/L46    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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Junker, J. P. Articles by Rief, M. Search for Related Content PubMed PubMed Citation Articles by Junker, J. P. Articles by Rief, M.

Influence of Substrate Binding on the Mechanical Stability of Mouse Dihydrofolate Reductase

J. P. Junker ^*, K. Hell , M. Schlierf ^*, W. Neupert  and M. Rief ^*

^* Physik-Department E22, Technische Universität München, D-85748 Garching, Germany; and Institut für Physiologische Chemie, Universität München, D-81377 Munich, Germany

Correspondence: Address reprint requests and inquiries to M. Rief, E-mail: mrief{at}ph.tum.de.

We investigated the effect of substrate binding on the mechanical stability of mouse dihydrofolate reductase using single-molecule force spectroscopy by atomic force microscopy. We find that under mechanical forces dihydrofolate reductase unfolds via a metastable intermediate with lifetimes on the millisecond timescale. Based on the measured length increase of 22 nm we suggest a structure for this intermediate with intact substrate binding sites. In the presence of the substrate analog methotrexate and the cofactor NADPH lifetimes of this intermediate are increased by up to a factor of two. Comparing mechanical and thermodynamic stabilization effects of substrate binding suggests mechanical stability is dominated by local interactions within the protein structure. These experiments demonstrate that protein mechanics can be used to probe the substrate binding status of an enzyme.

This article has been cited by other articles:

				A. F. Oberhauser and M. Carrion-Vazquez Mechanical Biochemistry of Proteins One Molecule at a Time J. Biol. Chem., March 14, 2008; 283(11): 6617 - 6621. [Abstract] [Full Text] [PDF]

				T.-T. Kao, K.-C. Wang, W.-N. Chang, C.-Y. Lin, B.-H. Chen, H.-L. Wu, G.-Y. Shi, J.-N. Tsai, and T.-F. Fu Characterization and Comparative Studies of Zebrafish and Human Recombinant Dihydrofolate Reductases--Inhibition by Folic Acid and Polyphenols Drug Metab. Dispos., March 1, 2008; 36(3): 508 - 516. [Abstract] [Full Text] [PDF]

				S. R. K. Ainavarapu, L. Li, and J. M. Fernandez Fingerprinting DHFR in Single-Molecule AFM Studies Biophys. J., September 1, 2006; 91(5): 2009 - 2010. [Full Text] [PDF]