PMID- 9740740
OWN - NLM
STAT- MEDLINE
DCOM- 19981116
LR  - 20131121
IS  - 1090-7807 (Print)
IS  - 1090-7807 (Linking)
VI  - 134
IP  - 1
DP  - 1998 Sep
TI  - The effects of cryoprotection on the structure and activity of p21 ras: 
      implications for electron spin-echo envelope modulation spectroscopy.
PG  - 142-53
AB  - 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 p21 ras, 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 
      alpha-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 in ras p21.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 by 2H ESEEM in 
      solutions made with d5-glycerol and d7-methyl alpha-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.
CI  - Copyright 1998 Academic Press.
FAU - Halkides, C J
AU  - Halkides CJ
AD  - Department of Chemistry, University of North Carolina at Wilmington, Wilmington, 
      North Carolina, 28403-3297, USA. halkidesc@uncwil.edu
FAU - Farrar, C T
AU  - Farrar CT
FAU - Singel, D J
AU  - Singel DJ
LA  - eng
GR  - 5 T32 GM08313-04/GM/NIGMS NIH HHS/United States
GR  - 5R 37 GM20168/GM/NIGMS NIH HHS/United States
GR  - CA08872/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Magn Reson
JT  - Journal of magnetic resonance (San Diego, Calif. : 1997)
JID - 9707935
RN  - 0 (Cryoprotective Agents)
RN  - 0 (Methylglucosides)
RN  - 2ZD004190S (Threonine)
RN  - 34273-04-6 (Guanylyl Imidodiphosphate)
RN  - 42Z2K6ZL8P (Manganese)
RN  - AR09D82C7G (Deuterium)
RN  - EC 3.6.1.- (GTP Phosphohydrolases)
RN  - EC 3.6.5.2 (Proto-Oncogene Proteins p21(ras))
RN  - PDC6A3C0OX (Glycerol)
SB  - IM
MH  - Cryoprotective Agents
MH  - Deuterium
MH  - Electron Spin Resonance Spectroscopy/*methods
MH  - Freezing
MH  - GTP Phosphohydrolases/metabolism
MH  - Glycerol/pharmacology
MH  - Guanylyl Imidodiphosphate
MH  - Kinetics
MH  - Manganese
MH  - Methylglucosides
MH  - Proto-Oncogene Proteins p21(ras)/*chemistry/*metabolism
MH  - Threonine
MH  - Time Factors
EDAT- 1998/09/19 00:00
MHDA- 1998/09/19 00:01
CRDT- 1998/09/19 00:00
PHST- 1998/09/19 00:00 [pubmed]
PHST- 1998/09/19 00:01 [medline]
PHST- 1998/09/19 00:00 [entrez]
AID - S1090-7807(98)91520-8 [pii]
AID - 10.1006/jmre.1998.1520 [doi]
PST - ppublish
SO  - J Magn Reson. 1998 Sep;134(1):142-53. doi: 10.1006/jmre.1998.1520.