Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy

Abstract

A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i þ 3 or i and i þ 4 in an α-helix, at i and i þ 2 in a β-strand, or with cysteine residues in adjacent strands in a β-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.pulsed EPR | protein dynamics S ite-directed spin labeling (SDSL) is a general method for characterizing protein topography, local and global structure, and dynamics by electron paramagnetic resonance (EPR) spectroscopy (1-5). SDSL can be applied to both soluble and membrane proteins of arbitrary molecular weight under physiological conditions. In traditional SDSL, a unique cysteine residue is introduced into a recombinant protein via site-directed mutagenesis, and subsequently reacted with a sulfhydryl-specific nitroxide reagent to generate a paramagnetic side chain. In addition, an SDSL strategy based on a genetically encoded unnatural amino acid was recently reported, a method that allows labeling in the presence of native thiols (6).The most widely used spin label for SDSL studies is a disulfidelinked side chain designated R1, in part because its inherent flexibility allows for introduction at virtually any site within a protein-even buried ones-typically with no more energetic cost than a natural amino acid substitution at that site (7,8). Indeed, R1 has proven useful for determining protein topology (2, 9), characterizing nanosecond backbone motions (4,10,11), mapping structural changes (3,(12)(13)(14), and predicting protein structure de novo (15). Although the amplitude of nanosecond internal motion and the number of preferred rotamers of R1 are limited (16), these features nevertheless make it problematic t...

Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label

“…1B) indicates that the label is strongly immobilized on the nanosecond time scale, i.e. that it is rigidly fixed to the protein, as expected for the bifunctional attachment of BSL (1,28,29,35).…”

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

“…Fig. 4B shows DEER data in the WT′ and cavity mutant for the four pairs involving 128R1, 131R1, and 132R1, the last two of which monitor the center of helix H. In all cases, the distance distributions in the WT′ protein are multimodal, with modes that could, in principle, be accounted for by rotamers of R1 (27) but could also be due to distinct states of the host helices. Under any condition, the most probable distances are in reasonable agreement with those estimated from modeling the R1 side chain in the crystal structure of the WT protein [Protein Data Bank (PDB) ID code 3LZM] (Table S3).…”

Conformational dynamics and distribution of nitroxide spin labels

“…1). This label can assume energetically relaxed conformations when two cysteine sites are introduced at residues i and i+3 or i+4 in an -helix, at positions i and i+1 in a -strand or at the nearest position in an adjacent -strand [37]. Labeling with bisMTSL can potentially result in the misleading attachment of two distinct spin labels to two adjacent reactive thiol groups in a side reaction, which does however appear to be much slower.…”