An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21.
We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family. In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site. A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange. We find that surprisingly good Raman spectra of bound ligands can be obtained with this method and that the method can be easily generalized to other systems. The data show that the guanine amino group of the nucleotide interacts differently with both EF-Tu and p21 than it does with water, showing a change in hydrogen-bonding properties upon binding. On the other hand, no change in hydrogen bonding is observed at guanine's N7. The data strongly suggest that the conformation of the nucleotide when bound to EF-Tu and that p21 is the C2' endo pucker of the ribose ring and anti about the glycosidic bond. These results are compared to previous structural and chemical studies.
Manor
D
D
Department of Physics, City College, City University of New York, New York 10031.
Weng
G Z
GZ
Deng
H
H
Cosloy
S
S
Chen
C X
CX
Balogh-Nair
V
V
Delaria
K
K
Jurnak
F
F
Callender
R
R
D000595:Amino Acid Sequence; D003903:Deuterium; D004926:Escherichia coli / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007700:Kinetics; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D013059:Spectrum Analysis, Raman / Q000379:methods; D014316:Tritium
D014316:Tritium; D006153:Guanosine Diphosphate; D003903:Deuterium; D010444:Peptide Elongation Factor Tu; D016283:Proto-Oncogene Proteins p21(ras)