Structural determinants of nitroxide motion in spin‐labeled proteins: Solvent‐exposed sites in helix B of T4 lysozyme

Abstract

Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain.Keywords: site-directed spin labeling; EPR spectroscopy; side-chain conformation Supplemental material: see www.proteinscience.org Site-directed spin labeling (SDSL) has become a widely used tool to study protein structure, dynamics, and interactions (for reviews, see Hubbell et al. 1996Hubbell et al. , 1998Hubbell et al. , 2000Columbus and Hubbell 2002;Klug and Feix 2004;Fanucci and Cafiso 2006). The SDSL approach requires a cysteine residue to be introduced at a selected site and subsequently modified with a nitroxide reagent to generate a disulfidelinked nitroxide side chain. The most commonly employed spin-label side chain is designated R1 (Fig. 1). The EPR spectra of R1 directly encode information on the motion of the nitroxide, which in turn reflects structure and dynamics of the local protein environment at the labeling site. A primary goal in developing the SDSL method is to enable interpretation of EPR spectra in terms of protein structure and dynamics using well-characterized proteins as models. Success in this regard will allow SDSL to be used to infer these features in poorly characterized proteins that are challenging to other methods.At the present time, simple one-component EPR spectra that arise from R1 on solvent-exposed surfaces of a-helices and in ordered turns are reasonably well understood (Guo et al. 2007). At these sites, the motion of R1 is determined by the internal motion of the side chain and local backbone dynamics (Columbus et al. 2001), and the EPR spectra provide a means for mapping backbone dynamics (Columbus and Hubbell 2004 Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

“…The EPR spectra for the selected sites show little or no change upon photoactivation, as previously reported (12,(37)(38)(39)(40). Because the spectra are sensitive to changes in backbone dynamics (41,42) and local interactions within the protein (29)(30)(31)43), the lack of spectral change upon photoactivation strongly suggests that the conformation of R1, the immediate local environment, and the secondary structure to which it is attached remain the same under rhodopsin activation. Thus, changes in interspin distances between pairs of these nitroxides report relative motion of the structures to which they are attached.…”

“…1), and interspin distances were measured in the inactive (R) and light-activated state (R*), using DEER spectroscopy. A global analysis of the data localized individual spins in the structure, and the results for R are in excellent agreement with the rhodopsin crystal structure from Li et al [Protein Data Bank (PDB) entry 1GZM] (8) and models of R1 based on the known structure of the side chain (29)(30)(31)(32). For R*, the data reveal an outward motion of R1 in TM6 by 5 Å and smaller movements of R1 in TM1, TM7, and in the C-terminal domain following helix H8.…”

“…The use of surface sites is important for two reasons. First, crystallographic studies show that R1 at solvent-exposed surface sites adopts a limited number of rotamers (29)(30)(31)(32). For such sites, the experimental rotamer library (30-32) may be used to build R1 in a structural model of the protein and thus predict interspin distances and distributions for comparison with experimental data, as illustrated here for R (Fig.…”

PELDOR Spectroscopy Distance Fingerprinting of the Octameric Outer‐Membrane Protein Wza from Escherichia coli.

“…These conformers are compatible with a library of MTSSL rotamers derived form MTSSL-labelled T4-lysozyme structures. [11,12] Using the crystallographic locations for the spin labels, an occupancy-weighted distance histogram was calculated, which shows an excellent fit to the PELDOR data reproducing the splitting of the 1-2 peak ( Figure 3C). …”

“…The data are another example of discrete multiple locations of the label [11][12][13] rather than static or so called smooth cone distribution. [14] PELDOR has now been applied to a large highly symmetrical membrane protein oligomer expanding the demonstrated utility of the technique.…”