Motion of Spin-Labeled Side Chains in T4 Lysozyme. Correlation with Protein Structure and Dynamics

Abstract

Thirty single cysteine substitution mutants of T4 lysozyme have been prepared and spin-labeled with a sulfhydryl-specific nitroxide reagent in order to systematically investigate the relationship between nitroxide side-chain mobility and protein structure. The perturbation caused by replacement of a native residue with a nitroxide amino acid was assessed from the resulting changes in biological activity, circular dichroism, and free energy of folding. The nitroxide produced context-dependent changes in stability and activity similar to those observed for substitution with natural amino acids at the same site but had little effect on the circular dichroism spectra. At solvent-exposed sites, the structural perturbation appears to be small at the level of the backbone fold. Nitroxide side-chain mobility faithfully reflects the protein tertiary fold at all sites investigated. The primary determinants of nitroxide side-chain mobility are tertiary interactions and backbone dynamics. Tertiary interactions constrain the side-chain mobility to an extent closely correlated with the degree of interaction. At interhelical loop sites, the side chains have a high mobility, consistent with high crystallographic thermal factors. On the exposed surfaces of alpha-helices, the side-chain mobility is not restricted by interactions with nearest neighbor side chains but appears to be determined by backbone dynamics. An unexpected result is a striking difference between the mobility of residues near the C- and N-termini of helices. These results provide the foundation for another dimension of information in site-directed spin-labeling experiments that can be interpreted in terms of the protein tertiary fold, its equilibrium dynamics and time-dependent conformational changes.

Utilization of Site-Directed Spin Labeling and High-Resolution Heteronuclear Nuclear Magnetic Resonance for Global Fold Determination of Large Proteins with Limited Nuclear Overhauser Effect Data

“…The secondary structure and hydrophilicity patterns should be sufficient for introduction of spin labels with a high level of success. EPR studies have shown that the nitroxide spin label MTSL is readily introduced into all secondary structure elements (particularly helices) and in general does not significantly perturb protein structure (22).…”

Molten globule state of tear lipocalin: ANS binding restores tertiary interactions

“…The line shapes of EPR spectra reflect the dynamic modes of the nitroxide side chain that can be modulated by protein backbone fluctuation, tertiary interaction, etc [24,[30][31][32]. Each EPR spectrum along the sequence provides site-specific dynamic information.…”

Osmolytes modulate conformational exchange in solvent‐exposed regions of membrane proteins

“…The electron paramagnetic resonance (EPR) spectrum of a spin label side chain encodes information regarding the rate and amplitude of label motion, which in turn is dictated by the local structure and tertiary contact made by the spin label side chain. 11 Local protein backbone fluctuations also modulate the motion of spin-labeled side chains, making EPR spectra sensitive to backbone dynamics on the ns timescale 12,13 ; moreover, conformational transitions that result in altered local structure or tertiary contact are easily detected by EPR, as are distinct conformational substates that are in equilibrium.…”