Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location

Abstract

Phase memory relaxation times ( T M or T 2) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels ( N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T M and x. T M values of seven positions are between 1.6 μs for the most buried residue (L79C) and 4.7 μs for a residue at the protein surface (W245C). In deuteriated buffer, longer T M are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T M as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.