Long-Range Structural Restraints in Spin-Labeled Proteins Probed by Solid-State Nuclear Magnetic Resonance Spectroscopy

Abstract

Magic-angle spinning NMR studies of 13C,15N-enriched proteins containing a covalently attached nitroxide spin label are presented, with the goal of using paramagnetic relaxation enhancements in the solid state to obtain long-range structural restraints. A 56 aa protein, B1 immunoglobulin-binding domain of protein G (GB1), was used as a model system, with nitroxide spin labels incorporated at residues 28 (α-helix) or 53 (β4-strand) via site-directed spin labeling. The presence of nitroxide spin labels in the GB1 analogues results in negligible pseudocontact shifts and large enhancement of the transverse relaxation rates for a number of residues, as detected by 2D 15N-13Cα correlation spectroscopy. The experimentally observed relaxation rate enhancements are found to be highly correlated with the distance of 1H, 15N, and 13Cα nuclei from the electron spin, with significant relaxation effects observed for nuclei up to ∼20 Å away, thus providing valuable information about the protein fold on length scales inaccessible to traditional solid-state NMR techniques.