An Isotope Edited Classical Raman Difference Spectroscopic Study of the Interactions of Guanine Nucleotides with Elongation Factor Tu and H-ras p21

Danny Manor, Gezhi Weng, Hua Deng, Robert Callender, Sharon Cosloy, Cun xiang Chen, Valeria Balogh-Nair, Kathy Delaria, Frances Jurnak

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)10914-10920
Number of pages7
JournalBiochemistry
Volume30
Issue number45
DOIs
StatePublished - Nov 1 1991

ASJC Scopus subject areas

  • Biochemistry

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