A Spirocyclohexyl Nitroxide Amino Acid Spin Label for Pulsed EPR Spectroscopy Distance Measurements

Abstract

Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy offer accurate, sensitive tools for the characterization of structure and function of macromolecules and their assemblies. A new rigid spin label, spirocyclohexyl nitroxide α-amino acid and its N-(9-fluorenylmethoxycarbonyl) (Fmoc) derivative, has been synthesized that exhibit slow enough spin echo dephasing to permit accurate distance measurements by pulse EPR at temperatures up to 125 K in 1:1 water:glycerol and at higher temperatures in matrices with higher glass transition temperatures. Distance measurements in the liquid nitrogen temperature range are less expensive than those that require liquid helium, which will greatly facilitate applications of pulsed EPR to the study of structure and conformation for peptides and proteins.

Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

“…By contrast, the echo decays for methyl-containing 1 at temperatures above 80 K fit best with a simple exponential that occurs when a dynamic process dominates the decay (11). The difference in the shapes of the echo decays for labels 1 and 3 supports the assertion that the longer values of T m for 3 than for 1 are due to replacement of the methyl groups by spirocyclohexyls and elimination of the contribution of methyl rotation to the echo dephasing.…”

“…Replacing the gem-dimethyl groups with spirocyclohexyl groups eliminates the methyl group effect and decreases 1/T m dramatically at temperatures T > 80 K, as previously illustrated for the spirocyclohexyl analog of TOAC, 2 (11), and other structurally related spirocyclohexyl nitroxides (12). The spirocyclohexyl nitroxides also are more resistant to bioreduction than MTSL-like nitroxides (11,13,14). In glassy or frozen solvents softening of the matrix with increasing temperature permits increasing motional modulation and averaging of g and A anisotropy, which also increases 1/T m .…”

“…Spirocyclohexyl piperidone (11,19) was subjected to reductive amination (20) to convert carbonyl to primary amine, which was then converted to an amide using chloroacetyl chloride (21). With the primary amine group protected as an amide, the secondary amine within the piperidine ring was oxidized to nitroxide using meta-chloroperoxybenzoic acid.…”

“…FIGURE 3 Temperature dependence of log(1/T m ) at X-band for 1, 2, and 3 in glassy mixtures. Data for 1 and 2 in water/glycerol are reproduced from (11). To see this figure in color, go online.…”

Development and Application of Spin Traps, Spin Probes, and Spin Labels

“…The replacement of the gem-methyl groups with spirocyclohexyl groups removes a major spin echo dephasing mechanism and makes T m long enough to allow PELDOR measurements at T > 80 K eliminating the need for expensive liquid helium ( Fig. 8; Rajca et al, 2010). Moreover, due to high resistance to bioreduction the spirocyclohexane-substituted nitroxides are potentially suitable for in-cell PELDOR measurements, where fast reduction of spin labels is the main experimental obstacle.…”