High-Field EPR and ESEEM Investigation of the Nitrogen Quadrupole Interaction of Nitroxide Spin Labels in Disordered Solids: Toward Differentiation between Polarity and Proticity Matrix Effects on Protein Function

Abstract

The combination of high-field electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in revealing subtle changes of the polarity and proticity profiles in proteins enbedded in membranes. This information can be obtained by orientation-selective high-field EPR resolving principal components of the nitroxide Zeeman (g) and hyperfine ( A) tensors of the spin labels attached to specific molecular sites. In contrast to the g- and A-tensors, the (14)N ( I = 1) quadrupole interaction tensor of the nitroxide spin label has not been exploited in EPR for probing effects of the microenvironment of functional protein sites. In this work it is shown that the W-band (95 GHz) high-field electron spin echo envelope modulation (ESEEM) method is well suited for determining with high accuracy the (14)N quadrupole tensor principal components of a nitroxide spin label in disordered frozen solution. By W-band ESEEM the quadrupole components of a five-ring pyrroline-type nitroxide radical in glassy ortho-terphenyl and glycerol solutions have been determined. This radical is the headgroup of the MTS spin label widely used in SDSL protein studies. By DFT calulations and W-band ESEEM experiments it is demonstrated that the Q(yy) value is especially sensitive to the proticity and polarity of the nitroxide environment in H-bonding and nonbonding situations. The quadrupole tensor is shown to be rather insensitive to structural variations of the nitroxide label itself. When using Q(yy) as a testing probe of the environment, its ruggedness toward temperature changes represents an important advantage over the g xx and A(zz) parameters which are usually employed for probing matrix effects on the spin labeled molecular site. Thus, beyond measurenments of g xx and A(zz) of spin labeled protein sites in disordered solids, W-band high-field ESEEM studies of (14)N quadrupole interactions open a new avenue to reliably probe subtle environmental effects on the electronic structure. This is a significant step forward on the way to differentiate between effects from matrix polarity and hydrogen-bond formation.

The Temperature Dependence of Nitroxide Spin–Label Interaction Parameters: a High-Field EPR Study of Intramolecular Motional Contributions

“…1. The R1 radical in the protonated and perdeuterated R1-D 15 (OH) form was synthesized as described previously [26]. Both forms of R1 were kindly provided by Herbert Zimmermann (Max-Planck Institute for Medical Research, Heidelberg).…”

“…The ESEEM data sets were obtained subsequently by stepping the external magnetic field through the nitroxide spectrum (0.2 mT per step). They were evaluated as described previously [26].…”

“…The exact evaluation of the g-and A-tensor values from the cw EPR spectra requires a multi-parameter fit of the theoretical spectra, as obtained from a numerical solution of the spin Hamiltonian, containing the nuclear Zeeman [37] and 14 N quadrupole [26] interaction terms, to the experimental spectra. Fortunately, the Moreover, precise information on A xx and A yy as well as on the 14 N quadrupole interaction tensor can be obtained from an analysis of 3-pulse ESEEM spectra, as it was shown previously [26].…”

Heterogeneity in the Nitroxide Micro-Environment: Polarity and Proticity Effects in Spin-Labeled Proteins Studied by Multi-Frequency EPR

“…The decrease in the root-mean-square deviation in the fitting procedure when going from two to three g xx components did not always allow an unambiguous assignment of the number of components in the spectrum. Thus, to avoid the effects of the A xx hyperfine lines and of the 14 N nuclear quadrupole interaction [10] on the observed shoulders in the g xx region, W-band experiments on 15 N-substituted spin labels were performed on the SRII-V17 protein system (Fig. 2).…”

“…For nitroxide labels, the g anisotropy is already resolved at W-band EPR (95 GHz/3.4 T) and can be further exploited at higher frequency/field settings (e.g., 275 GHz/9.8 T and 360 GHz/12.9 T). Theoretical studies have been performed by several groups to describe the effect of polarity and propensity for H-bond formation on the A-and g-tensor elements at different levels of sophistication and with different focuses [2,[8][9][10][11].…”