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.

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR

“…The agreement of experimental and simulated solvent accessibility was much better when the spin label was included in the simulation (Fig. 4), as observed previously for spin-labeled T4 lysozyme (27). Experimental distance measurements are in better agreement with computational simulations not only when the spin-label is included in place of the native side chain (22) but also when the backbone atoms are unrestrained (Fig.…”

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

“…EPR environmental parameters were determined for both the closed and pH-activated desensitized conformations (referred to as the desensitized state from here on). Changes in probe dynamics were evaluated from line shape differences (the inverse of the width of the central resonant line, ⌬H o Ϫ1 ), and the accessibility to either membrane soluble molecular O 2 (⌸O 2 ) or watersoluble NiEdda (⌸NiEdda) was determined from power saturation experiments (31,33).…”

“…First derivative absorption spectra were recorded at an incident microwave power of 2.0 milliwatt, a modulation frequency of 100 kHz, and a modulation amplitude of 1.0 gauss. Our analyses were centered on three types of dynamic EPR structural information (32,33). The first is the mobility of the spin probe, calculated as the inverse of the central line width of the first derivative absorption spectra (⌬H o…”

Structure, Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

“…They are also consistent with the conclusion of Chen et al (17) that the crystal structure of apo EmrE in this detergent is likely to be misfolded. Accessibility Profiles Identify Transmembrane Segments and Water-accessible Regions-The orientation and boundaries of EmrE transmembrane helices were deduced from measurement of collision frequency of each spin-labeled site with paramagnetic probes that are either lipid-soluble (molecular oxygen) or water-soluble (NiEDDA) (24,33). For a TM helix, the O 2 concentration gradient in the bilayer leads to a signature accessibility pattern consisting of 3.6 periodicity with increasing values at maxima near the middle of the membrane.…”