Technological advances in site-directed spin labeling of proteins

Abstract

Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion.

Figure 1

Structures of paramagnetic protein labels represented as side chains. (a) The R1 side chain. (b) The cross-linking side chain RX. The cross-link can be formed between i and i± 3 or i± 4 cysteine residues within a regular helix, between i and i± 2 residues in a β strand, or between any elements with properly spaced cysteine residues. (c) The R1p side chain. (d) The TOPP residue introduced by peptide synthesis. Although there may be rotation about individual bonds, the nitroxide is fixed in the same spatial location, because all bonds connecting the nitroxide with the protein are collinear. (e) The ketoxime-linked side chain K1 generated by reaction of a p-acetyl-phenylalanine unnatural amino acid with a hydroxylamine nitroxide reagent. (f) The triazole-linked side chain T1 generated by the reaction of a p-azido-phenylalanine unnatural amino acid with a strained cyclooctyne nitroxide reagent using Cu-free Click chemistry. (g) A disulfide-linked Gd³⁺ chelate side chain. (h) A disulfide-linked TAM spin label.

Figure 2

Characteristic time scale (lifetime of states) for selected protein motions relative to that for X-band EPR spectroscopic methods. The panel above the time line shows cartoons illustrating backbone fluctuations and internal motions of R1 on the ns time scale (left), and conformational exchange on the µs–ms time scale (right). In each case, the red sphere represents the nitroxide of R1. Fast backbone motion adds to the internal motion of R1 on the same time scale and is thus revealed in the CW lineshape (lower panel). Conformational exchange is too slow to affect the CW lineshape, but may be revealed as resolved components in the EPR spectrum, provided that R1 is in a region where the nitroxide experiences distinct environments in each state (upper panel, right). ST-EPR, P-ELDOR, and P-SR can measure conformational exchange kinetics on the µs time scales indicated. For conformational exchange on the time scale of ms and longer, perturbation-relaxation methods are promising. Following a rapid perturbation in an experimental parameter (pressure, temperature, pH, etc.), the relaxation to a new equilibrium is monitored in real time using the EPR spectrum of a judiciously placed spin label. For commercial spectrometers with CW detection, the shortest time that can be measured by relaxation methods is limited by the 100 kHz field modulation frequency to about 50 µs, but has no upper limit.

Figure 3

Mapping backbone and conformational flexibility with SDSL-EPR. (a) The rate (inverse correlation time, τ⁻¹) and order parameter (S) for R1 motion at solvent-exposed helix surface sites in myoglobin are plotted versus the fraction of buried surface area (f _buried) for the helix segment containing R1; f _buried is proportional to the number of contacts the segment makes with the fold. The regular decrease in rate and increase in order of R1 motion with increasing f _buried is consistent with a plausible model in which increased contact of a segment damps motion which is reflected in R1 dynamics. This result strongly supports the contention that the variation in R1 motion from site-to-site at solvent-exposed sites reflects backbone motion rather than local side chain interactions. (b) Regions in conformational exchange (µs or longer, red ribbon), as detected by resolved components in the R1 spectrum and the response to osmotic perturbation, are mapped onto a structural model of myoglobin. The regions identified correspond closely with those obtained by NMR methods [55••]. The figures are adapted from [55••].

Figure 4

Pressure dependence of a protein conformational equilibrium detected by SDSL-EPR. The stack plot of EPR spectra are for the A46R1 mutant of T4 lysozyme as a function of pressure. The two resolved spectral components i and m arise from equilibrium between a folded conformation (i) and a locally unfolded state (m) as illustrated schematically in the cartoon where the colored sphere represents the spin label. The apparent equilibrium constant, K, determined from the spectra, is a function of pressure as shown in the plot of ln[K(P)/K _o] versus pressure. The solid trace is a fit of the data to a model wherein the two conformations have different partial molar volumes as well as compressibilities. The figure is adapted from McCoy and Hubbell [57]. The relative populations of i or m could be directly monitored in time at a fixed field following a pressure jump.