¹⁵ N- and ² H-substituted maleimide spin labels: Improved sensitivity and resolution for biological EPR studies

Abstract

The resolution and sensitivity of electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) for biological applications are greatly improved by deuteration and substitution of 15N for 14N in the spin-labeled probe N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (MSL We have shown that the sensitivity and resolution of electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy were substantially improved for biological studies by deuteration of the frequently used maleimide spin label N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (MSL) which binds covalently to proteins (1, 2). Significant gains in detectability and resolution were observed with [2H]MSL in both the fast motion limit for freely tumbling spin label (rc 10 psec) and in the slow to very slow correlation time range exhibited by labeled proteins such as bovine serum albumin and hemoglobin in viscous media (0.1 ,usec s rT ' 1 msec). The improvements resulted from a reduction in the inhomogeneous line broadening due to the weaker superhyperfine interactions of the unpaired electron with deuterium than with hydrogen.To further advance our capabilities for interpretation of spectra of labeled proteins, we prepared the doubly substituted that 15N or deuterium substitution increased sensitivity for a freely tumbling spin label and that '5N simplified the spectrum by reduction of the number of nuclear manifolds from three to two (4-9). However, a doubly substituted probe with '5N and deuterium had not been prepared previously. The potential increase in sensitivity and resolution of such a probe seemed particularly advantageous in biological systems in which the substances to be spin labeled are often present in low concentrations and are macromolecules with slow correlation times in the range appropriate for analysis by ST-EPR techniques. (11,12) and spin labeled with a given maleimide analogue at a ratio of one spin label per tetramer as described by Beth et al. (13). GAPDHase activity was 28% inhibited by MSL and its two analogues.

In vivo EPR oximetry using an isotopically-substituted nitroxide: Potential for quantitative measurement of tissue oxygen

“…Therefore, nitroxide 1 is more O 2 -sensitive than nitroxide 3 and should be able to resolve smaller changes in O 2 . As expected, at 1 GHz, the spectral characteristics of nitroxides 1 and 3 , and their relative responsiveness to O 2 are comparable to our previous findings and for other similar spin labels at 9.5 GHz [26,27,30]. …”

A Comparison of Resolution-Enhancement Methods in Saturation-Transfer EPR

“…The x(+ 1, -1), y(+ 1, -1), and all three turning points for the ml = 0 spin-state overlap in the center of the spectrum for the '4N-Mal-6 system. Thus, as noted by Beth et al (27), all turning points for the two spin states of the '5N-Mal-6 system are fairly well resolved.…”

“…GHz, and for the out-of-phase dispersion (UD) display at 35 GHz. X-band V'2 spectral parameters for a '5N-Mal-6 system have recently been described in (27,28) and will not be discussed here further. Three spectral ratios, A'/A, B'/B and E'/E, which appear rather sensitive to changes in motional rates are shown for the 35 GHz V'2 spectra in the center of Fig.…”

“…Two ways to enhance the resolution of ST-EPR for complex motions have recently been suggested: (a) increasing the observational frequency to 35 GHz (Q-band) to increase g anisotropy and spread out the spin states (26), and (b) substituting the '5N isotope for the naturally occurring '4N in the spin label nitroxide to decrease the number of spin states from three (ml = -1, 0, +1) to two (mIn = -',4, +1/2) (27,28). In the present communication we describe the first comparison of these two approaches for resolution enhancement in ST-EPR.…”

Molecular distances from dipolar coupled spin-labels: the global analysis of multifrequency continuous wave electron paramagnetic resonance data

“…For this reason, perdeuterated spin-labels have been used in this work to reduce the extent of inhomogeneous broadening of spectral features, thereby enabling resolution of smaller dipolar couplings than would be possible using normal isotope spin-labels (Beth et al, 1984;Park and Trommer, 1989). As a general guide, perdeuteration of the nitroxide ring reduces the width of the low field line from -4.5 Gauss to 2 Gauss in the rigid limit at X-band microwave frequency (Beth et al, 1981). Eq.…”