High-Resolution Measurement of Distance and Orientation in Myosin: EPR of a Bifunctional Spin Label

Abstract

We present a method for obtaining high-resolution information on protein backbone structure and dynamics using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) and molecular modeling. Two complementary EPR techniques were employed to measure protein orientation (conventional EPR) and intra-protein distances (dipolar electron-electron resonance, DEER). BSL attaches at Cys positions i and i+4 on a helix, greatly reducing probe mobility relative to the peptide backbone, compared to monofunctional labels. Accurate modeling of BSL provides the coordinates required to directly relate spectroscopic data to backbone structure (both orientation and distance), and dynamics (rotational motion). In the current work, the motor protein Dictyostelium myosin II was used to demonstrate this approach. We measured nucleotide-dependent structural transitions of two key helices within the myosin catalytic domain (CD). Two double-Cys sites were engineered, with one Cys pair located on the relay helix, and the other on a stable helix in the upper 50kD domain. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin. BSL on a construct with two pairs was used to measure interprobe distances. The effect of ADP binding on both orientation and distance was clearly detected with BSL, but not with a monofunctional label. The significance of this work is twofold: (1) A structural transition in the relay helix upon ADP binding was clearly defined with high resolution. (2) BSL spectra demonstrate superior resolution, compared to monofunctional spin labels, making it possible to directly translate spectroscopic data to protein structure and dynamics. This work was funded by grants from NIH (R01 AR32961, T32 AR07612, P30 AR0507220).