The Effects of Cryoprotection on the Structure and Activity of p21ras:Implications for Electron Spin-Echo Envelope Modulation Spectroscopy

Abstract

Electron spin-echo envelope modulation (ESEEM) spectroscopy is widely used to investigate the active sites of biological molecules in frozen solutions. Various cryoprotection techniques, particularly the addition of co-solvents, are commonly employed in the preparation of such samples. In conjunction with ESEEM studies of Mn(II) guanosine nucleotide complexes of p21ras,we have investigated the effects of cryoprotection on the spectroscopy, the structure, and the activity of this protein. Echo decay times, which typically govern ESEEM spectral resolution, were found to vary linearly with the concentration of glycerol or methyl α-D-glucopyranoside (MG), with both additives equally effective on a per-mole basis. The effect of glycerol and MG on the ESEEM amplitudes of various protein nucleiwas studied inrasp21·Mn(II)·5′guanylylimido-diphosphate(p21·Mn(II)-GMPPNP) complexes: these additives did not alter the distances of these nuclei from the Mn(II) ion. In particular, in p21 incorporating [2H-3]Thr, the Mn(II)-[2H-3]Thr35 distance was found to be unaffected by the concentration of cryoprotectant or the rate of freezing. The proximity of the cryoprotectants to the Mn(II) ion was probed by2H ESEEM in solutions made with d5-glycerol and d7-methyl α-D-glucopyranoside (d7-MG). In p21·Mn(II)GMPPNP, the large deuterium modulations from the d5-glycerol exhibit saturation behavior with increasing d5-glycerol concentration, implying that glycerol, a widely used cryoprotectant, replaces the aquo ligands of the Mn(II) ion. The interaction between the Mn(II) ion of p21 and MG, however, is less intimate: the deuterium ESEEM amplitudes are much smaller for samples prepared with d7-MG than with d5-glycerol. Several polyhydroxylic compounds were found to have essentially no effect on the ability of the guanosine 5′-triphosphate (GTP) hydrolysis activating protein, GAP334, to catalyze hydrolysis of p21·guanosine 5′-triphosphate. This observation implies that the introduction of cryoprotectant does not significantly perturb the structure of p21 and gives insight into the mechanism of the GTPase reaction.