A structural model for the α‐subunit of transducin Implications of its role as a molecular switch in the visual signal transduction mechanism

Hingorani, Vijay N.; Ho, Yee-Kin

doi:10.1016/0014-5793(87)80867-0

Cited by 35 publications

(18 citation statements)

References 30 publications

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“…The EF-Tsbinding ability of the domain II/HI (Fig.5) indicates that the receptor binding function of EF-Tu is probably localized here. This interpretation is in agreement with the investigations of Hingorani and Ho (36). These authors postulated that the receptor binding site of the G-protein, transducin, is also located in its C-terminal domain.…”

Section: Isolated Domain I/iii Does Not Bind Gtp Gdp and Aminoacyl-trnasupporting

confidence: 92%

Interaction of the isolated domain II/III ofThermus thermophiluselongation factor Tu with the nucleotide exchange factor EF-Ts

et al. 1990

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The middle and C-terminal domain (domain II/III) of elongation factor Tu from Thermus thermophilus lacking the GTP/GDP binding domain have been prepared by treating nucleotide-free protein with Staphylococcus aureus V8 protease. The isolated domain II/III of EF-Tu has a compact structure and high resistance against tryptic treatment and thermal denaturation. As demonstrated by circular dichroism spectroscopy, the isolated domain II/III does not contain any alpha-helical structure. Nucleotide exchange factor, EF-Ts, was found to interact with domain II/III, whereas the binding of aminoacyl-tRNA, GDP and GTP to this EF-Tu fragment could not be detected.

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Section: Isolated Domain I/iii Does Not Bind Gtp Gdp and Aminoacyl-trnasupporting

confidence: 92%

Interaction of the isolated domain II/III ofThermus thermophiluselongation factor Tu with the nucleotide exchange factor EF-Ts

et al. 1990

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“…Essentially the same conformational features characterized full-length peptides and their partial sequences, except in one case as will be indicated, and only the former will be shown. Secondary structures predicted in this study for bovine rod transducin a-subunit (bGt 1) synthetic peptides were in general agreement with those expected for these sequences in the parent protein (25)(26)(27).…”

Section: Resultssupporting

confidence: 80%

“…Finally, using the MOTIF program, a computer search was conducted for all tetrapeptides that conform to the putative adhesion motif in the x-subunit of rod transducin (bGt l), the trimeric signal transducing G-protein from the visual system currently best understood in terms of structure and function (23,(25)(26)(27). Accordingly, the first and second positions would be occupied by Lys, Arg, Asp, Asn, Glu or Gln, the third by Asp and the fourth by Ile, Leu or Val.…”

Section: Peptide X : Bgtl(serz24-met239) (Sa Ydmvl Vedde V-nrm-nh2)mentioning

confidence: 99%

Problematic sequences in the synthesis of G‐protein peptides

Steinschneider

1994

International Journal of Peptide and Protein Research

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Peptide synthesis is hampered by amino acid sequence‐dependent aminoacylation (coupling) difficulties that are only partially understood. Analysis of coupling efficiencies in Fmoc‐based, solid‐phase synthesis of G‐protein fragments revealed that several problematic regions included a tetrapeptide structural motif consisting of (in the order of synthesis): (1) an aliphatic amino acid residue, (2) Asp, (3) and (4) a polar, H‐bonding residue each. The results suggest that interference with aminoacylation involved residue‐specific interactions, probably akin to those acting in protein‐protein adhesion, that occurred between functional groups at the reaction center and others located elsewhere in the peptide molecule. Difficult couplings did not correlate in any meaningful way with conformationally based predictive parameters in the literature. The present investigation points towards the occurrence of putative adhesion signals in intact G‐protein α‐subunits where their sequences are highly conserved, suggesting biological function.

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“…A large number of articles has described site-directed mutagenesis of p21, taking as a reference model the tertiary structure of the GDP-binding domain of EF-Tu [9] (and references therein). This model has been used to design three-dimensional models of p21 [16], bacterial initiation factor 2 [I71 and transducin [18]. In initiating a research programme on the structure-function relationships of EF-Tu, the isolation of its GTP-binding domain was obviously one of our first priorities.…”

Section: Methodsmentioning

confidence: 99%

Structure‐function relationships of elongation factor Tu

Jensen

Cool

Mortensen

et al. 1989

European Journal of Biochemistry

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The guanine‐nucleotide‐binding domain (G domain) of elongation factor Tu(EF‐Tu) consisting of 203 amino acid residues, corresponding to the N‐terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141–3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF‐Tu towards thermal denaturation (t50%= 41.3°C vs. 46°C, respectively). Unlike EF‐Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF‐Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH4+, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF‐Tu. K+ and Na+ are the most active followed by NH4+ and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3–5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7–8, similar to that of EF‐Tu. The 70‐S ribosome is the only EF‐Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF‐Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF‐Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain GTP complex and aa‐tRNA. Our results indicate that the isolated G domain of EF‐Tu is a suitable model for studying the basic properties of the guanine nucleotide interaction and the catalytic activity of a guanine‐nucleotide‐binding protein.

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A structural model for the α‐subunit of transducin Implications of its role as a molecular switch in the visual signal transduction mechanism

Cited by 35 publications

References 30 publications

Interaction of the isolated domain II/III ofThermus thermophiluselongation factor Tu with the nucleotide exchange factor EF-Ts

Interaction of the isolated domain II/III ofThermus thermophiluselongation factor Tu with the nucleotide exchange factor EF-Ts

Problematic sequences in the synthesis of G‐protein peptides

Structure‐function relationships of elongation factor Tu

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