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...

We present the first example of chemo-selective site-specific spin labeling of a monomeric protein with two spectroscopically orthogonal spin labels: a Gadolinium (III) chelate complex and a nitroxide radical. A detailed analysis of the performance of two commercially available Gd(III) ligands in the Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) experiment is reported. A modification of the flip angle of the pump pulse in the Gd(III)-nitroxide DEER experiment is proposed to optimize sensitivity.

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization.

An amphiphilic ABA triblock copolymer was synthesized using poly(2-ethyl-2-oxazoline) (PEtOz) as hydrophilic block [A] and poly(dimethylsiloxane) (PDMS) as hydrophobic block [B]. The cationic ring-opening polymerization of 2-ethyl-2-oxazoline was initiated by benzyl chloride in the presence of NaI. PEtOz-PDMS-PEtOz was characterized in aqueous solution using transmission electron microscopy (TEM). The block copolymers formed vesicles with a [B] block hydrophobic component thickness of 4 nm, which is thin enough for successful reconstitution of proteins. The mean diameters of the vesicles were measured to be in the range of 150-250 nm, with a narrow distribution. The electrochemical properties of planar PEtOz-PDMS-PEtOz membrane films spread across a Teflon aperture were investigated by electrochemical impedance spectroscopy (EIS). The impedance data showed an increase of planar membrane capacitance (C MEM ) from 2.58 × 10 −7 to 2.71 × 10 −7 F cm −2 and a decrease of membrane resistance (R MEM ) from 12 to 10.8 cm 2 . The increase of C MEM over time in buffer solution can be explained by an increase of the dielectric constant as a result of membrane electrolyte incorporation and/or by the formation and growth of defect in the free-standing films. In contrast to the formation of thin-walled vesicles, the spread triblock copolymer formed a free-standing membrane with a 9 nm thickness. Based on the two possible conformations (bridge midblock conformation and loop midblock conformation) that the triblock copolymer can have, we can conclude that PEtOz-PDMS-PEtOz formed 4 nm thick polymer vesicles with intercalated loop midblock structure in aqueous solution, while 9 nm thick free-standing polymer films with bilayer loop midblock conformation or with bridge midblock conformation were formed by aperture spreading.