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

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 exper

SUBMITTER: Junker JP 

PROVIDER: S-EPMC1366864 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature