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Influence of substrate binding on the mechanical stability of mouse dihydrofolate reductase

Item Type:Article
Title:Influence of substrate binding on the mechanical stability of mouse dihydrofolate reductase
Creators Name:Junker, J.P. and Hell, K. and Schlierf, M. and Neupert, W. and Rief, M.
Abstract: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 approximately 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.
Keywords:Atomic Force Microscopy, Binding Sites, Biophysical Phenomena, Biophysics, Mechanical Stress, Monte Carlo Method, NADP, Protein Binding, Protein Denaturation, Protein Folding, Proteins, Substrate Specificity, Tertiary Protein Structure, Tetrahydrofolate Dehydrogenase, Time Factors, Animals, Mice
Source:Biophysical Journal
ISSN:0006-3495
Publisher:Biophysical Society (U.S.A.)
Volume:89
Number:5
Page Range:L46-L48
Date:November 2005
Additional Information:Authors' reply in: Biophys J 91(5), 2011–2012.
Official Publication:https://doi.org/10.1529/biophysj.105.072066
PubMed:View item in PubMed

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