Molecular dynamics simulations.

Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy has emerged as an efficient tool to elucidate the structure and conformational dynamics of biomolecules under native-like conditions. This article summarizes the basics as well as recent progress of site-directed spin labeling. Continuous wave EPR spectra analyses and pulse EPR techniques are reviewed with special emphasis on applications to the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis and the photosynthetic reaction center from Rhodobacter sphaeroides R26.

Spin labeling EPR

Recent developments including pulse and multi-frequency techniques make the combination of site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy an attractive approach for the study of protein-protein or protein-oligonucleotide interaction. Analysis of the spin label side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin label side chains allow the modeling of protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. Structural changes can be detected with millisecond time resolution. Inter- and intra-molecular distances are accessible in the range from approximately 0.5 to 8 nm by the combination of continuous wave and pulse EPR methods. Recent applications include the study of transmembrane substrate transport, membrane channel gating, gene regulation and signal transfer.

Inter- and intra-molecular distances determined by EPR spectroscopy and site-directed spin labeling reveal protein-protein and protein-oligonucleotide interaction.

https://www.cell.com/biophysj/pdf/S0006-3495(06)71978-6.pdf

A Structure-Based Simulation Approach for Electron Paramagnetic Resonance Spectra Using Molecular and Stochastic Dynamics Simulations

ESR spectroscopy of site-directed spin-labeled biomolecules (Site-Directed Spin Labeling, SDSL) has emerged as a powerful method for studying the structure and conformational dynamics of proteins and nucleic acids under conditions relevant to function (for reviews see, e.g., Feix and Klug 1998; Hubbell et al. 1996; Hubbell et al. 1998, 2002). In this technique a spin-label side chain is introduced at a selected site via cysteine substitution mutagenesis followed by modification of the unique sulfhydryl group with a specific paramagnetic nitroxide reagent. The continuous wave (cw) ESR spectrum yields information about the nitroxide side chain mobility, solvent accessibility, the polarity of its immediate environment, and the distance between the nitroxide and another paramagnetic center in the protein. Hence, ESR data analysis of a series of spin-labeled variants of a given protein allows defining elements of secondary structure, including their solvent exposure, to characterize protein topography and to determine orientations of individual segments of the protein. A complete analysis allows modeling of protein structures with a spatial resolution at the level of the backbone fold (Hubbell et al. 1998, 2000; Koteiche and Mchaourab 1999; Mchaourab and Perozo 2000; Perozo et al. 1998; Wegener et al. 2001a). This method is applicable to any protein with a cloned gene that can be expressed. In particular, it has been shown to be very useful in studying large membrane proteins or protein complexes that are not amenable to NMR methods or do not crystallize.

Matthias Müller

Papers

Molecular dynamics simulation and EPR spectroscopy of nitroxide side chains in bacteriorhodopsin