Preparation of Escherichia coli elongation factor tu-guanosine 5′-triphosphate analogs

Delaria, Katherine; Jurnak, Frances

doi:10.1016/0003-2697(89)90038-9

Cited by 8 publications

(3 citation statements)

References 25 publications

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“…We suspect that the difference band at 661 cm"1 is somewhat sharper for GDP in p21 than in EF-Tu because the range of angles from an equilibrium C2' endo ribose pucker angle is somewhat larger in EF-Tu than in p21. This conjecture, as well as the extra sharpness of the p21 spectrum compared to the EF-Tu spectrum generally, is in agreement with the somewhat greater binding affinity of p21 for GDP (K¿ = 2 X 10"11 M; John et al, 1990) than that of EF-Tu (Ká = 2 X 10'9 M; Delaria & Jurnak, 1989). point must be synthesized before the transcript can self-cleave in the ternary complex whereas RNA freed from the complex by heating can cleave with only 3 or more nucleotides present beyond the cleavage site.…”

Section: Resultssupporting

confidence: 80%

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21

et al. 1991

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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.

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Section: Resultssupporting

confidence: 80%

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21

et al. 1991

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“…Such GTP analogs were also used in functional assays, for example, the study of in vitro polypeptide synthesis (Shorey et al" 1971;Girbes et al, 1976; Karim & Thompson, 1986) and of single steps of this process, such as the translocation reaction (Moazed et al 1988), ternary complex formation (Thompson & Karim, 1982;Delaria et al 1991;Nazarenko et al 1994), and nucleotide binding relative to that of GTP (Delaria & Jumak, 1989). All these investigations revealed that the function of the protein is affected by modification of the nucleotide structure.…”

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confidence: 99%

Interaction of Guanosine Nucleotides and Their Analogs with Elongation Factor Tu from Thermus thermophilus

Wagner¹,

Simón²,

Sprinzl³

et al. 1995

Biochemistry

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Transient kinetic experiments on the interaction of nucleotide-free EF-Tu from Thermus thermophilus with nucleotides using intrinsic protein fluorescence, extrinsic nucleotide fluorescence and fluorescence resonance energy transfer show that nucleotide binding is in general at least a two-step process. The first step is a weak initial binding, which is followed by a relatively slow isomerization of the protein-nucleotide complex in which changes of both intrinsic and extrinsic fluorescence, as well as energy transfer, occur. The values obtained for the equilibrium and kinetic constants confirm the earlier observation that EF-Tu has a higher affinity for GDP than GTP. This is mainly due to a lower dissociation rate constant for GDP, in combination with a somewhat higher effective association rate constant. Modifications of the triphosphate moiety of GTP are quite well tolerated by EF-Tu, with GTP gamma S displaying the same affinity as GTP and with GppNHp and GppCH2p being only ca. 2-3-fold less strongly bound. Caged GTP is bound about 6-fold more weakly than GTP. These results suggest that the binding of GppNHp and GppCH2p is likely to be similar to that of GTP. The photolytic protecting group of caged GTP (or the loss of one of the negative charges on the gamma-phosphate group) appears to interfere to a certain extent with the interaction with the protein, but the affinity is high enough to permit generation of 1:1 complexes for dynamic structural studies. Discrimination between GDP and ADP is dramatic, with a difference of 6 orders of magnitude in affinity.

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“…(Commercial preparations of GDP are often contaminated with GMP, GTP, ppGpp and/or other contaminants. A procedure for GDP purification is described in [15].) The fact that under saturating conditions, GDP forms an equimolar complex with intact EF-Tu makes the filter-binding assay a suitable tool for the determination of percentage of active molecules of EF-Tu and its variants in a preparation.…”

Section: Assay Methods For Ef-tumentioning

confidence: 99%

Bacterial elongation factors EF-Tu, their mutants, chimeric forms, and domains: Isolation and purification

Jonák

2007

Journal of Chromatography B

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Preparation of Escherichia coli elongation factor tu-guanosine 5′-triphosphate analogs

Cited by 8 publications

References 25 publications

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21

Interaction of Guanosine Nucleotides and Their Analogs with Elongation Factor Tu from Thermus thermophilus

Bacterial elongation factors EF-Tu, their mutants, chimeric forms, and domains: Isolation and purification

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