Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR

Abstract

In site-directed spin labeling, the relative solvent accessibility of spin-labeled side chains is taken to be proportional to the Heisenberg exchange rate (W(ex)) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of W(ex) are determined by continuous wave power saturation methods and expressed as a proportional and dimensionless parameter Pi. In the experiments presented here, NiEDDA is characterized as a paramagnetic reagent for solvent accessibility studies, and it is shown that absolute values of W(ex) can be determined from Pi, and that the proportionality constant relating them is independent of the paramagnetic reagent and mobility of the nitroxide. Based on absolute exchange rates, an accessibility factor is defined (0 < rho < 1) that serves as a quantitative measure of side-chain solvent accessibility. The accessibility factors for a nitroxide side chain at 14 different sites in T4 lysozyme are shown to correlate with a structure-based accessibility parameter derived from the crystal structure of the protein. These results provide a useful means for relating crystallographic and site-directed spin labeling data, and hence comparing crystal and solution structures.

FIGURE 1

The reaction of the spin label with a cysteine to generate the nitroxide side chain R1.

FIGURE 2

Line broadening of 4-cyano-2,2,5,5-tetramethylpryollidine-1-oxyl in solution by 3 mM NiEDDA as a function of viscosity. Line broadening at constant ambient temperature was measured directly as ΔΔH_pp from the first-derivative EPR spectra, and viscosity was varied by addition of glycerol.

FIGURE 3

(A) Lorentzian broadening of 4-cyano-2,2,5,5-tetramethylpryollidine-1-oxyl due to Heisenberg exchange at different concentrations of NiEDDA. The EPR spectrum is shown in gray. Each spectrum was fitted to a convolution of the spectrum in the absence of NiEDDA and a single Lorentzian line as described in Materials and Methods. The best fit is shown in black and the corresponding Lorentzian as a dotted line. (B) The exchange frequency is calculated directly from the best-fit Lorentzian in panels A and C, and plotted versus the concentration of NiEDDA. (C) Lorentzian broadening of T4L 44R1 due to NiEDDA. The EPR spectrum is shown in gray. Each spectrum was fitted to a convolution of the spectrum in the absence of NiEDDA and a single Lorentzian line as described in Materials and Methods. The best fit is shown in black and the corresponding Lorentzian as a dotted line.

FIGURE 4

Ribbon model of T4L showing the location of the sites investigated as spheres on the corresponding α-carbon.

FIGURE 5

EPR spectra of T4L mutants containing the R1 nitroxide side chain at the designated position. All spectra are recorded in buffer at ambient temperature.

FIGURE 6

The accessibility parameter Π versus concentration of NiEDDA for residues 44R1, 65R1, and 74R1.

FIGURE 7

Correlation of W_ex determined by CW saturation and by saturation recovery EPR. (A) W_ex for NiEDDA (black) and O₂ (gray) determined by SR and CW saturation are plotted for R1 residues along the sequence 128–135. SR data is taken from Pyka et al. (19) and shown as dotted lines with the same color scheme. The right axis shows the same data in units of ρ. (B) W_ex determined from SR is plotted versus W_ex from CW saturation. Open gray circles are data for O₂, and solid circles for NiEDDA. The dashed line has a slope of 1, passing through the origin.

FIGURE 8

Correlation between experimental measures of R1 accessibility (k_ex(Oxygen), k_ex(NiEDDA)), and the structure-based parameter f_V.