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 experiments demonstrate that protein mechanics can be used to probe the substrate binding status of an enzyme.

FIGURE 1

(A) Schematic representation of the Ddfilamin-DHFR protein construct, stretched between a gold surface and a gold-coated cantilever tip. (B) Proposed structure of the intermediate state (dark green). The C-terminal part shown in light green unfolds during the first unfolding event. (C) Typical force versus extension curve. The unfolding pattern of the Ddfilamin domains is shown in blue (and in red for domain 4), whereas the unfolding event of DHFR is shown in green. The inset shows a magnification in which the intermediate state is clearly visible. (D) Increase in contour length for the complete unfolding of DHFR. (E) Increase in contour length for the unfolding of DHFR from the native state to the intermediate state.

FIGURE 2

(A, B) Force versus time curves for DHFR without substrate (A) and with both substrates (B). The arrows mark the unfolding force and intermediate lifetime; panels C–E show the lifetimes of the intermediate state for different substrate conditions. The blue markers represent the experimental data with N^1/2-error bars, whereas the green lines are Monte Carlo simulations of the experiment. Unfolding force distributions for the native state are shown as insets.