Nucleotide binding and GTP hydrolysis by elongation factor Tu from Thermus thermophilus as monitored by proton NMR.
Proton NMR experiments of the GTP/GDP-binding protein EF-Tu from the extremely thermophilic bacterium Thermus thermophilus HB8 in H2O have been performed paying special attention to the resonances in the downfield region (below 10 ppm). Most of these downfield signals are due to hydrogen bonds formed between the protein and the bound nucleotide. However, three downfield resonances appear even in the nucleotide-free EF-Tu. The middle and C-terminal domain (domain II/III) of EF-Tu lacking the GTP/GDP-binding domain gives rise to an NMR spectrum that hints at a well-structured protein. In contrast to native EF-Tu, the domain II/III spectrum contains no resonances in the downfield region. Several downfield resonances can be used as a fingerprint to trace hydrolysis of protein-bound GTP and temperature effects on the EF-Tu.GDP spectra. NMR studies of the binding of guanosine nucleotide analogues (GMPPNP, GMPPCP) to nucleotide-free EF-Tu have been carried out. The downfield resonances of these complexes differ from the spectrum of EF-Tu.GTP. Protected and photolabile caged GTP was bound to EF-Tu, and NMR spectra before and after photolysis were recorded. The progress of the GTP hydrolysis could be monitored using this method. The downfield resonances have been tentatively assigned taking into account the known structural and biochemical aspects of EF-Tu nucleotide-binding site.
Limmer
S
S
Laboratorium für Biochemie, Universität Bayreuth, FRG.
Reiser
C O
CO
Schirmer
N K
NK
Grillenbeck
N W
NW
Sprinzl
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism*; D010782:Photolysis; D016963:Thermus thermophilus / Q000737:chemistry*
D006150:Guanine Nucleotides; D006153:Guanosine Diphosphate; C011265:guanosine 5'-(beta,gamma-methylene)triphosphate; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu