FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1994855870> ?p ?o ?g. }

• W1994855870 endingPage "23253" @default.
• W1994855870 startingPage "23244" @default.
• W1994855870 abstract "Most G-proteins require a guanine nucleotide exchange factor (GEF) to regulate a variety of critical cellular processes. Interestingly, a small number of G-proteins switch between the active and inactive forms without a GEF. Translation elongation factor 1A (eEF1A) normally requires the GEF eEF1Bα to accelerate nucleotide dissociation. However, several mutant forms of eEF1A are functional independent of this essential regulator in vivo. GEF-independent eEF1A mutations localize close to the G-protein motifs that are crucial for nucleotide binding. Kinetic analysis demonstrated that reduced GDP affinity correlates with wild type growth and high translation activities of GEF-independent mutants. Furthermore, the mutant forms show an 11–22-fold increase in rates of GDP dissociation from eEF1A compared with the wild type protein. All mutant forms have dramatically enhanced stability at elevated temperatures. This, coupled with data demonstrating that eEF1A is also more stable in the presence of nucleotides, suggests that both the GEF and nucleotide have stabilizing effects on eEF1A. The biochemical properties of these eEF1A mutants provide insight into the mechanism behind GEF-independent G-protein function. Most G-proteins require a guanine nucleotide exchange factor (GEF) to regulate a variety of critical cellular processes. Interestingly, a small number of G-proteins switch between the active and inactive forms without a GEF. Translation elongation factor 1A (eEF1A) normally requires the GEF eEF1Bα to accelerate nucleotide dissociation. However, several mutant forms of eEF1A are functional independent of this essential regulator in vivo. GEF-independent eEF1A mutations localize close to the G-protein motifs that are crucial for nucleotide binding. Kinetic analysis demonstrated that reduced GDP affinity correlates with wild type growth and high translation activities of GEF-independent mutants. Furthermore, the mutant forms show an 11–22-fold increase in rates of GDP dissociation from eEF1A compared with the wild type protein. All mutant forms have dramatically enhanced stability at elevated temperatures. This, coupled with data demonstrating that eEF1A is also more stable in the presence of nucleotides, suggests that both the GEF and nucleotide have stabilizing effects on eEF1A. The biochemical properties of these eEF1A mutants provide insight into the mechanism behind GEF-independent G-protein function. GTPases regulate a variety of cellular functions with a conserved mechanism of nucleotide binding and hydrolysis. Signal transduction, control of cell cycle and differentiation during cell division, protein biosynthesis, vesicular trafficking, and translocation of membrane proteins are key cellular processes where GTPases play critical roles. Based on their functional roles, domain structures, or sizes, the superfamily of GTPases can be divided into many families, including small G proteins (Ras GTPase superfamily), heterotrimeric G proteins, and the translation factor family (1Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1394) Google Scholar). All GTPases share a G-domain with conserved sequence elements, such as switch I and II, the P-loop (phosphate binding loop), and the NKXD element (2Dever T.E. Glynias M.J. Merrick W.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1814-1818Crossref PubMed Scopus (467) Google Scholar). Guanine nucleotides form specific interactions with these sites, which are modulated by G-protein accessory factors to create the switch mechanism between the active and inactive forms (1Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1394) Google Scholar, 3Sprang S.R. Coleman D.E. Cell. 1998; 95: 155-158Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). GTPase activation factors stimulate GTP hydrolysis, resulting in the inactive GDP-bound form. Guanine nucleotide exchange factors (GEFs) 2The abbreviations used are:GEFguanine nucleotide exchange factoreEF1eEF1A, eEF1B, and eEF2, translation elongation factor 1, 1A, 1B, and 2, respectivelyeEFSecselenocysteine-specific elongation factoreRF3eukaryotic release factor 3eIF5Beukaryotic initiation factor 5BFRETfluorescence resonance energy transfermant2′-(or 3′)-O-N-methylanthraniloyl. catalyze GDP release by reducing the nucleotide affinity (1Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1394) Google Scholar). This allows G-proteins to rebind GTP due to a higher cellular concentration of GTP and thus switch to their active form. The GEFs interact with the switch I and II regions while inserting residues close to or into the P-loop and Mg2+ binding site. The insertion of the GEF residues perturbs the interaction surface in the phosphate binding region, resulting in the release of phosphate groups, which in turn causes dissociation of the nucleotide. In contrast to the mechanism of exchange and the G-proteins themselves, GEFs show little to no conservation in sequence or structure (3Sprang S.R. Coleman D.E. Cell. 1998; 95: 155-158Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). guanine nucleotide exchange factor eEF1A, eEF1B, and eEF2, translation elongation factor 1, 1A, 1B, and 2, respectively selenocysteine-specific elongation factor eukaryotic release factor 3 eukaryotic initiation factor 5B fluorescence resonance energy transfer 2′-(or 3′)-O-N-methylanthraniloyl. The crystal structures of the majority of G-proteins solved in the presence of nucleotides show that the β-phosphate of the nucleotide interacts with the P-loop, and this interaction is considered to be the most important element for the tight binding of nucleotide (1Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1394) Google Scholar). Another important contributor to binding affinity and specificity is the NKXD element, which interacts with the nucleotide base (2Dever T.E. Glynias M.J. Merrick W.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1814-1818Crossref PubMed Scopus (467) Google Scholar). Until recently, the order of events that leads to nucleotide release and the region of the nucleotide released first were unclear. However, a sequence of interactions was proposed at least for some G-proteins. According to the model, upon binding of the GEF to the G-protein, the phosphate groups are released first, and then the base of the entering nucleotide binds to the NKXD motif and displaces the GEF (4Bos J.L. Rehmann H. Wittinghofer A. Cell. 2007; 129: 865-877Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar). Although most of the GTPases require a GEF, some G-proteins can function efficiently without an exchange factor. Typically, GEFs accelerate the nucleotide release from G-proteins, which is normally a slow process. A G-protein without a GEF probably allows nucleotide release rapid enough for cell survival. G-proteins could maintain rapid nucleotide release rates that lead to GEF independence in several ways. Lower affinity of GDP for the G-protein could allow a G-protein to function without a GEF. In addition, the G-protein may not require the separate level of regulation of activity typically performed by the GEF. The eukaryotic proteins translation elongation factor 2 (eEF2), release factor 3 (eRF3), initiation factor 5B (eIF5B), selenocysteine-specific elongation factor (eEFSec) and the eEF1A-like GTPases Hbs1p and Guf1p apparently function independently of a GEF. This is mostly explained by reduced GDP affinity or higher dissociation rate constants for GDP release from the G-protein in the absence of GEF. For example, the rate constants for GDP dissociation from both eIF5B and SelB (bacterial EFSec) are higher than other translation factors (5Pisareva V.P. Hellen C.U. Pestova T.V. Biochemistry. 2007; 46: 2622-2629Crossref PubMed Scopus (14) Google Scholar, 6Thanbichler M. Bock A. Goody R.S. J. Biol. Chem. 2000; 275: 20458-20466Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and eEFSec has lower affinity for GDP (7Tujebajeva R.M. Copeland P.R. Xu X.M. Carlson B.A. Harney J.W. Driscoll D.M. Hatfield D.L. Berry M.J. EMBO Rep. 2000; 1: 158-163Crossref PubMed Scopus (245) Google Scholar, 8Fagegaltier D. Hubert N. Yamada K. Mizutani T. Carbon P. Krol A. EMBO J. 2000; 19: 4796-4805Crossref PubMed Scopus (245) Google Scholar). This, coupled with the higher level of GTP over GDP in the cell, allows spontaneous regeneration of the active form of the protein (6Thanbichler M. Bock A. Goody R.S. J. Biol. Chem. 2000; 275: 20458-20466Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 8Fagegaltier D. Hubert N. Yamada K. Mizutani T. Carbon P. Krol A. EMBO J. 2000; 19: 4796-4805Crossref PubMed Scopus (245) Google Scholar, 9Merrick W.C. Nyborg J. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 89-126Google Scholar). Some studies suggest that the ribosome acts as a GEF for prokaryotic RF-3 and EF-G (10Zavialov A.V. Hauryliuk V.V. Ehrenberg M. J. Biol. (Bronx N. Y.). 2005; 4: 9Google Scholar). The variety for the GEF requirement and the sequence and structural diversity of GEFs implies that GEF proteins might have gained different functions, which may be specific to the GTPase·GEF complex, cell type or the organism. One of the G-proteins in the translation family is eukaryotic translation elongation factor (eEF1A). It binds and recruits aminoacyl-tRNAs to the A-site of the ribosome. eEF1A is part of the eEF1 complex, including the eEF1B subunits. eEF1B is composed of the α and γ subunits in fungi, and a third β subunit is present in metazoans. The eEF1Bα subunit performs the catalytic GEF function for eEF1A. The γ subunit is probably a regulatory subunit, since the eEF1Bαγ complex has a proposed role in the oxidative stress response pathway (11Olarewaju O. Ortiz P.A. Chowdhury W. Chatterjee I. Kinzy T.G. RNA Biol. 2004; 1: 12-17Crossref Scopus (40) Google Scholar). The crystal structure of Saccharomyces cerevisiae eEF1A with the catalytic C terminus of eEF1Bα shows that one face of eEF1Bα interacts with domain I, whereas the other interacts with domain II (12Andersen G.R. Pedersen L. Valente L. Chatterjee I. Kinzy T.G. Kjeldgaard M. Nyborg J. Mol. Cell. 2000; 6: 1261-1266Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Domain I contains the nucleotide and Mg2+ binding site, whereas domain II is the proposed aminoacyl-tRNA binding site of eEF1A. In addition to its established role as a GEF for eEF1A and accelerating the rate of GDP dissociation by 700-fold (13Pittman Y. Valente L. Jeppesen G.R. Andersen G.R. Patel S. J. Biol. Chem. 2006; 281: 19457-19468Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), eEF1Bα also affects translational fidelity (14Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar). Based on the crystal structures of bacterial and eukaryotic EF-Tu/eEF1As, mammalian eEF1A has evolved from bacterial EF-Tu by the insertion of about 70 amino acids into the loop regions between the domains (9Merrick W.C. Nyborg J. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 89-126Google Scholar). S. cerevisiae eEF1A has 81% identity and 89% similarity to human eEF1A (15Schirmaier F. Phillipson P. EMBO J. 1984; 3: 3311-3315Crossref PubMed Scopus (99) Google Scholar, 16Anand M. Valente L. Carr-Schmid A. Munshi R. Olarewaju O. Ortiz P. Kinzy T.G. Symp. Quant. Biol. 2001; 66: 439-448Crossref PubMed Scopus (14) Google Scholar). These results suggest that although the structure and the function of eEF1A are well conserved, the GEF for eEF1A has gained more complexity and perhaps more functions throughout evolution. In S. cerevisiae, the TEF5 gene encoding eEF1Bα is essential in vivo (17Hiraga K. Suzuki K. Tsuchiya E. Miyakawa T. FEBS Lett. 1993; 316: 165-169Crossref PubMed Scopus (46) Google Scholar). Interestingly, the requirement for the TEF5 gene can be suppressed by the presence of excess substrate, eEF1A. Such an eEF1Bα-deficient strain, however, shows defects in growth and translation (18Kinzy T.G. Woolford Jr., J.L. Genetics. 1995; 141: 481-489Crossref PubMed Google Scholar). Two independent, unbiased genetic screens performed to isolate suppressors of the eEF1Bα requirement in vivo yielded only eEF1A mutations. The mutant forms of eEF1A that function as suppressors of the eEF1Bα deficiency allow growth similar to a wild type eEF1A strain. In order to analyze the effect of suppressor mutations independent of any other eEF1 components, strains lacking eEF1Bα and both chromosomal eEF1A genes and thus expressing only the mutant form of eEF1A were prepared. Surprisingly, these strains show no growth defects and little to no reduction in total translation (19Ozturk S.B. Vishnu M.R. Olarewaju O. Starita L.M. Masison D.C. Kinzy T.G. Genetics. 2006; 174: 651-663Crossref PubMed Scopus (11) Google Scholar). Interestingly, all mutations map to the nucleotide-binding domain of eEF1A. Each mutation is in very close proximity to at least one of the conserved sequence elements of the G-protein, which suggests that nucleotide affinity to eEF1A might be affected, creating an open conformation to allow accelerated GDP release without eEF1Bα. In order to determine the mechanism of the bypass suppression of an essential GEF, we analyzed the effect of mutant forms of eEF1A on nucleotide binding by fluorescence resonance energy transfer (FRET) from hydrophobic residues of eEF1A to fluoresecently 2′-(or 3′)-O-N-methylanthraniloyl (mant)-labeled nucleotides. The equilibrium dissociation constants (Kd) for mant-GDP binding to eEF1A mutant forms increased up to 37-fold compared with that of wild type eEF1A, indicating reduced GDP affinity. Using stopped-flow kinetics, the mutant forms of eEF1A displayed increased GDP dissociation rates up to 22-fold compared with the wild type protein. However, the Kd values for mant-GMPPNP, a nonhydrolyzable homolog of GTP, were essentially unchanged, indicating that the selective pressure reduces GDP but not GTP binding. Although the mutations do not cause a fundamental change in the native state of the protein as observed by CD spectroscopy, the mutant forms showed dramatically increased stability. Enhanced stability was also observed when eEF1A was bound to guanine nucleotides. Thus, this study demonstrates that the GEF eEF1Bα as well as the distribution of the nucleotide-bound state of eEF1A within the cell probably contribute to stabilizing the protein. The consequences of these eEF1A mutations on the specificity of effects on GDP versus GTP binding raises the questions of evolutionary development of GEF function and independence as well as G-protein complexity. Yeast Techniques and Mutant Preparation—S. cerevisiae strains used in this study are listed in Table 1. Escherichia coli DH5α was used for plasmid preparation. Standard yeast genetic methods were employed (20Burke D. Dawson D. Stearns T. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000Google Scholar). Yeast cells were grown in YEPD (1% Bacto yeast extract, 2% peptone, 2% dextrose) as the carbon source (21Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1992: 13.0.1-13.14.17Google Scholar). The R164K, T22S, A112T, and A117V eEF1A mutants were prepared in pTKB754 (TEF1 URA3) by PCR mutagenesis using the QuikChange method (Stratagene). The resulting plasmids were transformed into MC213 (TEF2 TRP1), loss of wild-type eEF1A plasmid was monitored by growth on 5-fluoroanthranilic acid (22Toyn J.H. Gunyuzlu P.L. White W.H. Thompson L.A. Hollis G.F. Yeast. 2000; 16: 553-560Crossref PubMed Scopus (93) Google Scholar), and the recovered strains TKY961, TKY963, TKY964, and TKY965, respectively, were used for the purification of the mutant proteins.TABLE 1S. cerevisiae strainsStrainGenotypeReferenceMC213MATα ura3-52 leu2-3,112 trp1-Δ1 lys2-20 met2-1 his4-713 tef1::LEU2 tef2Δ tef5::TRP1 pTEF1 URA319Ozturk S.B. Vishnu M.R. Olarewaju O. Starita L.M. Masison D.C. Kinzy T.G. Genetics. 2006; 174: 651-663Crossref PubMed Scopus (11) Google ScholarTKY368MATα ura3-52 leu2-Δ1 trp1-Δ101 lys2-801 met2-1 his4-713 tef5::TRP1 pTEF5 LEU213Pittman Y. Valente L. Jeppesen G.R. Andersen G.R. Patel S. J. Biol. Chem. 2006; 281: 19457-19468Abstract Full Text Full Text PDF PubMed Scopus (29) Google ScholarTKY961MATα ura3-52 leu2-3,112 trp1-Δ1 lys2-20 met2-1 his4-713 tef1::LEU2 tef2Δ tef5::TRP1 pTEF1 URA3 (R164K)19Ozturk S.B. Vishnu M.R. Olarewaju O. Starita L.M. Masison D.C. Kinzy T.G. Genetics. 2006; 174: 651-663Crossref PubMed Scopus (11) Google ScholarTKY963MATα ura3-52 leu2-3,112 trp1-Δ1 lys2-20 met2-1 his4-713 tef1::LEU2 tef2Δ tef5::TRP1 pTEF1 URA3 (T22S)19Ozturk S.B. Vishnu M.R. Olarewaju O. Starita L.M. Masison D.C. Kinzy T.G. Genetics. 2006; 174: 651-663Crossref PubMed Scopus (11) Google ScholarTKY964MATα ura3-52 leu2-3,112 trp1-Δ1 lys2-20 met2-1 his4-713 tef1::LEU2 tef2Δ tef5::TRP1 pTEF1 URA3 (A112T)19Ozturk S.B. Vishnu M.R. Olarewaju O. Starita L.M. Masison D.C. Kinzy T.G. Genetics. 2006; 174: 651-663Crossref PubMed Scopus (11) Google ScholarTKY965MATα ura3-52 leu2-3,112 trp1-Δ1 lys2-20 met2-1 his4-713 tef1::LEU2 tef2Δ tef5::TRP1 pTEF1 URA3 (A117V)19Ozturk S.B. Vishnu M.R. Olarewaju O. Starita L.M. Masison D.C. Kinzy T.G. Genetics. 2006; 174: 651-663Crossref PubMed Scopus (11) Google Scholar Open table in a new tab Protein Purification—eEF1A was purified as described (13Pittman Y. Valente L. Jeppesen G.R. Andersen G.R. Patel S. J. Biol. Chem. 2006; 281: 19457-19468Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 23Carvalho M.G. Carvalho J.F. Merrick W.C. Arch. Biochem. Biophys. 1984; 234: 603-611Crossref PubMed Scopus (68) Google Scholar) from strains TKY368, TKY961, TKY963, TKY964, and TKY965 (Table 1) with the following modifications. The eluted and dialyzed material from the protein solution was applied to CM-52 cation exchanger, pre-equilibrated with buffer 1 (20 mm Tris, pH 7.5, 0.1 mm EDTA, pH 8.0, 25% glycerol, 1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, and 1 μg/ml aprotonin). The CM-52 column was washed with buffer 1 with 50 mm KCl and eluted using a 50–300 mm KCl salt gradient of buffer 1. eEF1A-containing fractions were dialyzed overnight against 10 volumes of buffer 1 with 100 mm KCl and stored in aliquots at -80 °C. Protein fractions used for assays were >95% pure, as determined by SDS-PAGE. Mant-labeled Guanine Nucleotide Binding Assay—Mant-GMPPNP and mant-GDP were both purchased from Molecular Probes and purified with a 1-ml Hi-Trap DEAE-Sepharose Fast Flow column (Amersham Biosciences) using an AKTA fast protein liquid chromatography system (Amersham Biosciences). Nucleotide-containing fractions were combined, lyophilized using a speed vacuum, resuspended in 100 μl of distilled H2O, and stored at -20 °C (13Pittman Y. Valente L. Jeppesen G.R. Andersen G.R. Patel S. J. Biol. Chem. 2006; 281: 19457-19468Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The binding affinity for mant-GDP or mant-GMMPNP to wild type or the mutant forms of eEF1A was measured by a fluorimetric titration assay using a FluoroMax-3 spectrofluorimeter (Horiba Jobin Yvon Inc.). All assays were performed at 25 °C. Wild type or mutant forms of eEF1A (1 μm) in 2.5 ml of binding buffer (10% glycerol, 50 mm Tris-HCl, pH 8.0, 50 mm KCl, and 5 mm MgCl2) were placed in a 10 × 10 × 40-mm quartz cuvette with a magnetic stirring bar. Increasing concentrations of mant-nucleotide were added with continuous stirring for 3 min. Fluorescence changes of the mant-nucleotides (Fobs) were monitored upon indirect excitation via FRET. FRET excited tryptophans or tyrosines of eEF1A at a wavelength of 280 nm, and the emission wavelength of 440 nm was for the mant moiety of the nucleotides. The slit widths were 1.05 nm for both wavelengths. The protein and nucleotide complex-dependent fluorescence values (Fem) were obtained by correcting for titration volume and inner filter effect using the equation Fem = Fobs × (Vf/Vo) × 10(0.5(Abex(280) + Abem(440))), plotted against mant-GMPPNP or mant-GDP concentrations, and fit to the equations Fem = C + fEbEb + fMMt and Eb = (Kd + Mt + Et - (((Kd + Mt + Et)2 - 4MtEt0.5)))/2, where Vf is the final volume, Vo is the initial volume, Abex(280) is the excitation absorbance of mant-nucleotide, and Abem(440) is the emission absorbance of mant-nucleotide, C is background fluorescence, fM is the fluorescence coefficient of free mant-nucleotide, fEb is the fluorescence coefficient of mant-nucleotide bound to eEF1A, Et is the total eEF1A protein concentration, Mt is the total concentration of mant-nucleotide, the concentration of eEF1A bound to mant-nucleotide is Eb, and Kd is the mant-GMPPNP or mant-GDP dissociation constant. Fluorescence Stopped-flow Kinetic Experiments—Stopped-flow experiments using mant-GDP (Molecular Probes) were done in a SF-2001 (KinTek Corp.) stopped-flow spectrophotometer equipped with a photomultiplier detection system as described in Ref. 13Pittman Y. Valente L. Jeppesen G.R. Andersen G.R. Patel S. J. Biol. Chem. 2006; 281: 19457-19468Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar. CD Measurements—CD measurements were made using a circular dichroism spectrometer (model 400; Aviv Biomedical Inc.). The protein concentrations of the purified fractions were determined from the measurement of their different spectra in 6 m guanidine HCl between pH 12.5 and pH 6.0. The molar concentration (MC) of protein in the cuvette was calculated using the formula MC = A/(2.357Y + 830W), where A is the absorbance at 294 nm, Y is the number of tyrosines, and W is the number of tryptophans. The stock solutions were diluted to 0.2–0.4 mg/ml in purification buffer 1 (20 mm Tris, pH 7.5, 0.1 mm EDTA, pH 8.0, 25% glycerol, 1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, and 1 μg/ml aprotonin) to a total volume of 300 μl in cells with a 0.1-cm path wavelength. The wavelength was set to 200–260 nm with 0.5-nm intervals. The wavelength measurements were carried out at 25 °C. After the temperature scan at 222 nm to obtain the melting curve, the wavelength scan was measured at 70 °C and 25 °C again after the samples were cooled down to test the refolding properties of the proteins. The CD spectra were analyzed using neural network analysis programs to determine α-helical, antiparallel, and parallel β-structure, turns, and the remainder content of the proteins (24Bohm G. Muhr R. Jaenicke R. Protein Eng. 1992; 5: 191-195Crossref PubMed Scopus (1022) Google Scholar, 25Unneberg P. Merelo J.J. Chacon P. Moran F. Proteins. 2001; 42: 460-470Crossref PubMed Scopus (81) Google Scholar). The Aviv Macro Editor program was used to collect the wavelength spectra as a function of temperature. The cells were equilibrated for 2 min at 5 °C integrals, and the spectra were accumulated three times at a given temperature. The convex constraint algorithm was used to deconvolute the spectra to obtain the minimum number of basis spectra needed to fit the data (26Perczel A. Hollosi M. Tusnady G. Fasman G.D. Protein Eng. 1991; 4: 669-679Crossref PubMed Scopus (328) Google Scholar, 27Greenfield N.J. Nature Protocols. 2006; 1: 2527-2535Crossref PubMed Scopus (579) Google Scholar). All CD data were reported in degrees cm2 dmol-1 ([θ]). Interactions of Guanine Nucleotides with eEF1A Are Well Represented by the Change in Mant-nucleotide Fluorescence—In order to determine the base-line guanine nucleotide binding characteristics of eEF1A, FRET from tryptophans and tyrosines of eEF1A to the mant fluorophore of GDP or GMPPNP was determined. Enhanced mant-nucleotide fluorescence with eEF1A (Figs. 1B and 2B) compared with mant-nucleotides alone (Figs. 1A and 2A) is the indication of FRET between eEF1A and the mant-nucleotides. In order to determine guanine nucleotide binding affinity of eEF1A, Kd values of eEF1A for mant-GDP and mant-GMPPNP were determined by a fluorimetric titration assay. The Kd for mant-GDP binding to eEF1A was determined as 0.095 μm (Fig. 1B). This value is in agreement with a Kd of 1 μm for unlabeled GDP and eEF1A (28Saha S.K. Chakraburtty K. J. Biol. Chem. 1986; 261: 12599-12603Abstract Full Text PDF PubMed Google Scholar) and our previously published Kd of 0.18 μm for mant-GDP binding to eEF1A determined by stopped-flow kinetics (13Pittman Y. Valente L. Jeppesen G.R. Andersen G.R. Patel S. J. Biol. Chem. 2006; 281: 19457-19468Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To determine GTP binding affinity of eEF1A, the mant-labeled nonhydrolyzable analog of GTP, mant-GMPPNP, was used. The previously published Kd of the eEF1A·GTP complex is 0.7 μm (28Saha S.K. Chakraburtty K. J. Biol. Chem. 1986; 261: 12599-12603Abstract Full Text PDF PubMed Google Scholar). The Kd for mant-GMPPNP binding to eEF1A was measured as 0.52 μm (Fig. 2B), indicating that mant-GMPPNP binding to eEF1A is a good representation of unmodified GTP binding to eEF1A.FIGURE 2GEF-independent mutants of eEF1A do not affect GMPPNP binding. The Kd values for the wild type and mutant forms of eEF1A and mant-GMPPNP were measured. Aliquots of mant-GMPPNP were added to binding buffer without (A) or with 1 μm of wild type (B) or mutant forms (C–E) of eEF1A. The fluorescence was measured and plotted as in Fig. 1. The Kd values are measured as 0.47 (T22S) (C), 0.41 (R164K) (D), 1.01 (A112T) (E), and 1.09 μm (A117V) (F). Residuals for the fits are shown in the lower panels to detect the experimental error for the fitted data sets.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mutations of eEF1A That Suppress the Requirement for eEF1Bα Reduce GDP Affinity up to 37-fold—All of the mutations that allow eEF1A to function without its guanine nucleotide exchange factor eEF1Bα cluster in the GTP-binding domain (19Ozturk S.B. Vishnu M.R. Olarewaju O. Starita L.M. Masison D.C. Kinzy T.G. Genetics. 2006; 174: 651-663Crossref PubMed Scopus (11) Google Scholar). In order to determine if these mutations affect nucleotide affinity in the absence of the exchange factor, we analyzed the GDP binding to the mutant forms of eEF1A by the mant-GDP fluorescence assay. Upon binding of the nucleotide to the eEF1A mutants, an increase in fluorescence was observed, and the data were fitted to determine Kd values for each mutant. The values obtained were 3.54 (T22S), 2.66 (R164K), 1.80 (A112T), and 1.18 μm (A117V) (Fig. 1, C–F, and Table 2), which represent a 12–37-fold increase in Kd as compared with wild type eEF1A (0.095 μm; Fig. 1B). Thus, one reason for the suppression of the requirement for the guanine nucleotide exchange factor, eEF1Bα, is the reduced affinity of the mutant forms to GDP compared with the wild type eEF1A.TABLE 2Kd values for mGDP and GTP, koff for mGDPKd for mGDPKd for mGMPPNPkoff for mGDPμmμms–1Wild type0.095 ± 0.010.52 ± 0.020.17T22S3.54 ± 0.540.47 ± 0.191.88R164K2.66 ± 00.41 ± 0.402.54A112T1.80 ± 0.091.01 ± 0.311.96A117V1.18 ± 0.291.09 ± 0.093.89 Open table in a new tab GEF-independent Mutations of eEF1A Do Not Affect GTP Binding—eEF1Bα catalyzes the release of GDP to allow the inactive (GDP-bound) form of eEF1A to recycle back to its active (GTP-bound) form. In order to determine whether these eEF1A mutations reduce binding of both GDP and GTP or specifically GDP affinity, the Kd of the eEF1A mutant proteins for mant-GMPPNP were obtained. The change in fluorescence values was plotted against the increasing concentration of mant-GMPPNP and fitted to the equations to give Kd values of 1.01 (A112T), 1.09 (A117V), 0.47 (T22S), and 0.41 μm (R164K) (Fig. 2, C–F, and Table 2). The fact that the Kd values of the mutant forms are very similar to the Kd value of wild type eEF1A (0.52 μm; Fig. 2B) indicates that genetic selection during the isolation of the suppressors for the requirement of eEF1Bα targets GDP but not GTP affinity. GEF-independent Mutants of eEF1A Dissociate GDP at a Higher Rate—To determine if the mutations cause rapid spontaneous GDP dissociation from eEF1A in the absence of its GEF, we measured the dissociation rate constant of the mant-GDP·eEF1A complex using stopped-flow kinetics. Mutant forms of eEF1A were prebound to mant-GDP and then rapidly mixed with an excess of nonfluorescent GDP. The rate of mantnucleotide release was obtained by monitoring the decrease in fluorescence over time. The fluorescent intensity decayed exponentially on the order of seconds. The time course of mant-GDP displacement from the binary complex of each mutant by excess GDP was fitted by a single-exponential term, yielding dissociation rate constants (koff) of 3.89 (A117V), 2.54 (R164K), 1.96 (A112T), and 1.88 s-1 (T22S) (Fig. 3, A–D, and Table 2). The average dissociation rate constant of wild type eEF1A is 0.17 s-1 (13Pittman Y. Valente L. Jeppesen G.R. Andersen G.R. Patel S. J. Biol. Chem. 2006; 281: 19457-19468Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These data show that the nucleotide dissociation rate constants were stimulated 11–22-fold in the GEF-independent forms of eEF1A. These results support the contributions of higher GDP release rates in addition to reduced nucleotide affin" @default.
• W1994855870 created "2016-06-24" @default.
• W1994855870 creator A5026504401 @default.
• W1994855870 creator A5054710367 @default.
• W1994855870 date "2008-08-01" @default.
• W1994855870 modified "2023-09-26" @default.
• W1994855870 title "Guanine Nucleotide Exchange Factor Independence of the G-protein eEF1A through Novel Mutant Forms and Biochemical Properties" @default.
• W1994855870 cites W1484592475 @default.
• W1994855870 cites W1493968530 @default.
• W1994855870 cites W1520541928 @default.
• W1994855870 cites W1553703599 @default.
• W1994855870 cites W1905058380 @default.
• W1994855870 cites W1976944760 @default.
• W1994855870 cites W1981843090 @default.
• W1994855870 cites W1993735775 @default.
• W1994855870 cites W2003879700 @default.
• W1994855870 cites W2009668088 @default.
• W1994855870 cites W2015642465 @default.
• W1994855870 cites W2020305960 @default.
• W1994855870 cites W2024484334 @default.
• W1994855870 cites W2025938842 @default.
• W1994855870 cites W2027629550 @default.
• W1994855870 cites W2031064526 @default.
• W1994855870 cites W2050957868 @default.
• W1994855870 cites W2052749821 @default.
• W1994855870 cites W2059402338 @default.
• W1994855870 cites W2066507549 @default.
• W1994855870 cites W2077506852 @default.
• W1994855870 cites W2080364507 @default.
• W1994855870 cites W2081764750 @default.
• W1994855870 cites W2085267287 @default.
• W1994855870 cites W2091807611 @default.
• W1994855870 cites W2093821259 @default.
• W1994855870 cites W2100256775 @default.
• W1994855870 cites W2110158147 @default.
• W1994855870 cites W2116433369 @default.
• W1994855870 cites W2119111999 @default.
• W1994855870 cites W2148047088 @default.
• W1994855870 cites W2156571424 @default.
• W1994855870 cites W2157391924 @default.
• W1994855870 cites W2163895770 @default.
• W1994855870 cites W341703637 @default.
• W1994855870 doi "https://doi.org/10.1074/jbc.m801095200" @default.
• W1994855870 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2517005" @default.
• W1994855870 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18562321" @default.
• W1994855870 hasPublicationYear "2008" @default.
• W1994855870 type Work @default.
• W1994855870 sameAs 1994855870 @default.
• W1994855870 citedByCount "13" @default.
• W1994855870 countsByYear W19948558702012 @default.
• W1994855870 countsByYear W19948558702013 @default.
• W1994855870 countsByYear W19948558702014 @default.
• W1994855870 countsByYear W19948558702017 @default.
• W1994855870 countsByYear W19948558702019 @default.
• W1994855870 countsByYear W19948558702020 @default.
• W1994855870 countsByYear W19948558702022 @default.
• W1994855870 crossrefType "journal-article" @default.
• W1994855870 hasAuthorship W1994855870A5026504401 @default.
• W1994855870 hasAuthorship W1994855870A5054710367 @default.
• W1994855870 hasBestOaLocation W19948558701 @default.
• W1994855870 hasConcept C104317684 @default.
• W1994855870 hasConcept C105795698 @default.
• W1994855870 hasConcept C126619667 @default.
• W1994855870 hasConcept C143065580 @default.
• W1994855870 hasConcept C185592680 @default.
• W1994855870 hasConcept C207332259 @default.
• W1994855870 hasConcept C2778301229 @default.
• W1994855870 hasConcept C33923547 @default.
• W1994855870 hasConcept C35651441 @default.
• W1994855870 hasConcept C512185932 @default.
• W1994855870 hasConcept C54355233 @default.
• W1994855870 hasConcept C55493867 @default.
• W1994855870 hasConcept C86803240 @default.
• W1994855870 hasConceptScore W1994855870C104317684 @default.
• W1994855870 hasConceptScore W1994855870C105795698 @default.
• W1994855870 hasConceptScore W1994855870C126619667 @default.
• W1994855870 hasConceptScore W1994855870C143065580 @default.
• W1994855870 hasConceptScore W1994855870C185592680 @default.
• W1994855870 hasConceptScore W1994855870C207332259 @default.
• W1994855870 hasConceptScore W1994855870C2778301229 @default.
• W1994855870 hasConceptScore W1994855870C33923547 @default.
• W1994855870 hasConceptScore W1994855870C35651441 @default.
• W1994855870 hasConceptScore W1994855870C512185932 @default.
• W1994855870 hasConceptScore W1994855870C54355233 @default.
• W1994855870 hasConceptScore W1994855870C55493867 @default.
• W1994855870 hasConceptScore W1994855870C86803240 @default.
• W1994855870 hasIssue "34" @default.
• W1994855870 hasLocation W19948558701 @default.
• W1994855870 hasLocation W19948558702 @default.
• W1994855870 hasLocation W19948558703 @default.
• W1994855870 hasLocation W19948558704 @default.
• W1994855870 hasOpenAccess W1994855870 @default.
• W1994855870 hasPrimaryLocation W19948558701 @default.
• W1994855870 hasRelatedWork W1485381313 @default.
• W1994855870 hasRelatedWork W1607176723 @default.
• W1994855870 hasRelatedWork W2001963330 @default.
• W1994855870 hasRelatedWork W2016777141 @default.
• W1994855870 hasRelatedWork W2032964163 @default.

• next