Abstract
Using spin labeling for studying protein macromolecules may reveal their significant dynamical and structural properties. EPR spectrum contains information about protein dynamics and internal motions, but, unfortunately, these are extremely versatile, and spectrum present only ‘digest’ of it. There is is an attractive way of joining Molecular Dynamics (MD) simulation with EPR. MD provides detailed system dynamics, and number of attempts has been made to calculate spectra directly from trajectory data. Still there is no currently reliable algorithm developed to date for this purpose.In this work, the study of complex formation between RNAse Barnase (Bn) and its specific inhibitor Barstar (Bs), is presented. High affinity of Bs to Bn, this protein pair is promising for creating large superstructures with controllable properties. Mutant C40A barstar labeled by C82 with 4-(2-chloromercuriphenyl)-2,2,5,5-tetramethyl-3-imidazoline-D3-1-oxyl, as well as its complex with Bn, was previously studied by X-band EPR to obtain correlation times for macromolecule Brownian diffusion and order parameters for internal dynamics.We built a model of labeled Bs, and BsBn complex, and ran a number of full-atom MD simulations. Both MD and EPR revealed two motional states of the spin label, one highly ordered, and another flexible in free Barstar. Detailed analysis of calculated data showed that difference between these two states was solely due to internal dynamics of the protein. Corresponding azimuthal order parameters calculated from MD trajectories well coincided with experimental ones, obtained from EPR spectra. It was found that formation of BsBn complex leads to complete disappearance of disordered state. Experimental evidence (provided by spin labeling) of key features observed in MD trajectories provide a validation of used parameters and protocols therefore.
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call