Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR.
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.
Altenbach
Christian
C
Jules Stein Eye Institute, University of California, Los Angeles, California 90095-7008, USA.
Froncisz
Wojciech
W
Hemker
Roy
R
McHaourab
Hassane
H
Hubbell
Wayne L
WL
D017122:Bacteriophage T4 / Q000737:chemistry*; D001703:Biophysics / Q000379:methods*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D015233:Models, Statistical; D009113:Muramidase / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009569:Nitric Oxide / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012997:Solvents / Q000737:chemistry; D013816:Thermodynamics; D013997:Time Factors
D010446:Peptide Fragments; D012997:Solvents; D009569:Nitric Oxide; C026058:EDDA; D004492:Edetic Acid; D009113:Muramidase; D003545:Cysteine; D010100:Oxygen