EPR and saturation transfer EPR studies on glyceraldehyde 3-phosphate dehydrogenase

Abstract

Electron paramagnetic resonance (EPR) and saturation transfer–EPR (ST–EPR) techniques were employed to investigate the hydrodynamic properties of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Both apo- and holoenzyme were spin-labeled at the active site cysteine-149 residue with N- (1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) - maleimide. The apo- and holoenzymes were observed to have the same hydrodynamic structure and the spectroscopic results were consistent with these complexes behaving as spheres with hydrated radii of 41 Å. The environment of the paramagnetic electron was significantly more polar in the spin-labeled holoenzyme than in the spin-labeled apoenzyme, suggesting that either ionic residues are positioned closer to the active site in the holoenzyme or that ionic segments of coenzyme nicotinamide adenine dinucleotide (NAD+) itself may interact with the paramagnetic electron of the maleimide spin label. The dependence of the phase quadrature second harmonic absorption ST-EPR signal upon microwave and Zeeman modulation field intensities was examined with particular attention directed to effects of the variation of these fields over samples of finite dimensions. Such considerations are crucial when comparing data obtained employing different microwave resonators or different sample geometries within the same resonator. The problem of accurately establishing phase nulls for phase quadrature measurements was examined as was the problem of estimating phase resolution. Thus, the present work represents both a specific application and a generalization of the ST-EPR methodology proposed by Thomas, Dalton, and Hyde [J. Chem. Phys. 65, 3006 (1976)].

Comparing continuous wave progressive saturation EPR and time domain saturation recovery EPR over the entire motional range of nitroxide spin labels

“…The fitting protocol consisted of starting with data that was phased instrumentally for absorption and dispersion and with Zeeman phase determined by the low power quadrature nulling condition [9]. The phase nulling condition for quadrature signals gives the correct phase condition to within 12° [9,38]. For the regime studied here, the quadrature signals were an order of magnitude smaller than the in-phase signals, making even a 0.5°phasing error significant.…”

“…Parameters individual to a cluster of data sets, for example microwave amplitude, microwave phase, and modulation phase were similarly linked within the data cluster. The fitting protocol consisted of starting with data that was phased instrumentally for absorption and dispersion and with Zeeman phase determined by the low power quadrature nulling condition [9]. The phase nulling condition for quadrature signals gives the correct phase condition to within 12° [9,38].…”

“…Site specific incorporation of spin labels into DNA and RNA has been accomplished with spin labels either rigidly incorporated or attached through a flexible linker [4][5][6][7][8]. Spin-labeled biological materials are generally studied with continuous wave (CW) EPR techniques such as progressive power saturation and saturation transfer EPR (ST-EPR) [9]. The power saturation method has been the approach of choice for determining collision-induced relaxation of spin-labeled proteins in environments containing spin relaxants, and has provided structural and functional information [10,11].…”

Non-linear electron spin resonance study of archaebacteria lipid dynamics

“…The use of Thomas et al [22] calibration curves requires an accurate spectrometer setting, in order to work in their same instrumental conditions. Indeed, since the line shape of ST-ESR spectra is critically dependent upon the extent and distribution of the microwave magnetic field and the modulation amplitude [34][35][36], an exact calibration of these parameters has proved to be necessary.…”

Interação de pequenas moléculas com proteínas: um estudo usando métodos convencionais e transferência de saturação de R.P.E.

“…I~.. (32,33) c) I H 1 t T, »1 -Eeglao de mOVlmento lento (43) experimental Em medidas de tempo de correlação no intervalo de ( 47) e a matriz A2 pode ser diagonalizada por um processo idêntico ao descrito acima ( 48) onde RI~a matriz dos cossenos diretores do sistema cristalográfico abc, em relaçâo ao sistema de eixos principais xyz, fixo na mol~cula do marcador.…”