help button home button Biophys. J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
Biophys. J. BioFAST: First Published September 23, 2005. doi:10.1529/biophysj.105.072066
© 2005 by the Biophysical Society.

A more recent version of this article appeared on November 1, 2005.
This Article
Right arrow Full Text (Rapid PDF)
Right arrow supplemental
Right arrow All Versions of this Article:
biophysj.105.072066v1
89/5/L46    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Author home page(s):
Jan Philipp Junker
Kai Hell
Michael Schlierf
Walter Neupert
Matthias Rief
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Junker, J. P.
Right arrow Articles by Rief, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Junker, J. P.
Right arrow Articles by Rief, M.

BIOPHYSICAL LETTERS

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

Jan Philipp Junker 1, Kai Hell 2, Michael Schlierf 1, Walter Neupert 2 and Matthias Rief 1*

1 Physik-Department E22, Technische Universitaet Muenchen
2 Institut für Physiologische Chemie, Universitaet Muenchen

* To whom correspondence should be addressed. E-mail: mrief{at}ph.tum.de.

Submitted on August 4, 2005
Revised on August 19, 2005
Accepted on 29 August 2005


  Abstract
We investigated the effect of substrate binding on the mechanical stability of mouse dihydrofolate reductase (DHFR) using single molecule force spectroscopy by AFM. We find that under mechanical forces DHFR unfolds via a metastable intermediate with lifetimes on the millisecond timescale. Based on the 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 (MTX) 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.

Key Words: AFM, DHFR, protein mechanics, protein unfolding, single-molecule force spectroscopy, substrate binding




This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
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]

Home page
 Drug Metab. Dispos.Home page
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]

Home page
 Biophys. JHome page
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]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
Copyright © 2005 by the Biophysical Society.