ProGuide: a flexible framework for modeling global conformational rearrangements in proteins using DEER-derived distance restraints.

Conformational heterogeneity is integral to protein function - ranging from enzyme catalysis to signal transduction - and visualizing distinct conformational states requires experimental techniques capable of providing such structural information. One particularly powerful method, double electron-electron resonance (DEER) spectroscopy, can provide a high-resolution, long-range (~15-80 Å) probability distributions of distances between site-selected pairs of spin labels to resolve intra-protein distance parameters of unique protein conformations, as well as their respective likelihoods within a conformational ensemble. A current frontier in the field of DEER spectroscopy is utilizing this distance information in computational modeling to generate complete structural models of these multiple conformations.

Capturing protein dynamics across timescales with site-directed spin labeling electron paramagnetic resonance spectroscopy.

In the current age of protein structure prediction and determination, resolving the time dependence of structural transitions represents an exciting frontier. Time-resolved biophysical techniques possess the capability to directly observe dynamic structural changes of biomolecules in real time. Here, we review applications of site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy that cover a broad range of protein dynamics, from backbone fluctuations on the ps-ns timescale to protein complex assembly formation on the ms-s timescale.

A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins.

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the timescale of ps-μs using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of μs-ms, corresponding to large-scale protein motions, is inaccessible to those methods.

A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins.

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the time scale of ps-µs using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of µs-ms, corresponding to large-scale protein motions, is inaccessible to those methods.

Closed fumarase C active-site structures reveal SS Loop residue contribution in catalysis.

Fumarase C (FumC) catalyzes the reversible conversion of fumarate to S-malate. Previous structural investigations within the superfamily have reported a dynamic structural segment, termed the SS Loop. To date, active-site asymmetry has raised the question of how SS Loop placement affects participation of key residues during the reaction.