Examination of elongation factor Tu for aluminum fluoride binding sites using fluorescence and ¹⁹F‐NMR methodologies

Abstract

This art)tic reports on a companion of the tl,ller~clion tf AP' and F with two GTP.hindmg proteins, clonga;ion factor T-(~F-To} ~nd the humtune scnsllWC regulatory protein ((3 protein) O.z The racthodolo$1¢x tbotcn to elucidate i~ssihle interactions hot.ten protein and alumplum fluoride were Iluor¢~:en~e ~r~clro~:opy and nuclear raagn¢tl¢ r<,~onan¢¢ ()"F.NM R) Bolh prottm~ have tryptoph~n residues near their nucleolide bindlnl| ~lte~, the pu~orted s)t¢ of aluminum fluoride inter~¢lloll It hal bccn assumed for O prnt¢ins (including GQ~) theft ~tluminum fluoride.in the pre~en¢© of Mg ~', rarefies the ntagnes~um coordinated >.phosphate group for the ODP.fou~ of the protein .nd shifts the. protein's confom~a-t~on toward the active GTP.rorm Indeed, ch~nl~es m intrmdc tluoresc©nce of G,.= effected hy aluramura fluoride .re observed The pre~nce of al~m)num fluoride did not ~ffcct the intrinsic fiuore~ccn~c, s~ctra or hfctlmc~, of EF.Tu,GDP. t'~F-NM R w~s then u~d to directly test for bound F-Fluonde alone o, ~)t the prts¢o~.¢ of either protein IW, e = ~lngle ~"F.NMR peak at -10 ppm, ch~ra¢tcnstl¢ of free F" With th~ add,finn ofaluramum 1o 1h¢ protein ,rid F sample~ a ~ccond I'~k, shifted upfi¢ld from the first to -29 ppm. was observed for Go= GDP This (econd pc.k, which has been uss,gncd to protein.bound F , was not ob,~ervcd for EF.Tu GDP These ob~rval|on~ show that Ih~ interaction of AI '~'' and F =. in the pr¢,,cn¢c of' MB q', may ~ qmte d)ffcrent Ixt~ccn the hom'mne.sensmv¢ G prulems, which bind ulumlnum Ihlonde, and the GTPbinding proteins as a whole, which include EF.Tu Cam must therefore b= exercised when structural data oq the elongahon factor, sl~cd~cally on the nucleot~dc s~te, are used to )nierpret data or compose models intended to descnb, e the horrnone.sens=twc regulatory G proteins MATERIALS AND METHODSGuanosme 5 '-dJphosphate (GDP), phenylmct hanesulfonyl floor,de (PMSF), and tns(hydroxymethyl)am,nomethane (Tns) were purchased from Sigma Biocheraicals [8.mH]GDP was obtained through Amersl~am, and dJthtothreltol (DTT) was from Pierce Cheralcal Co AICI~ v, as from Malhnkrodt MgClz and NaF were purchased from J T Baker Chemical Co EF-Tu ~as punned from Escherrchta cob MRE600 paste (Gram Processing, lnc ) by the method of Leberman et al [6] GDP-bmdm8 actw~ty of the EF-Tu was determined using the filter assay as described by Mdler and Welssbach [7], and the total EF.Tu GDP concentrauon v, as determlned using an extmcuon coefficient at 280 nm of 29 200 M -)cra-) [8] Preparatmns were rout,nelY stored at -80"C m 50 mM Tns pH 7 6, I0 ram M8C12, I ram NAN,, 0 $ mM DTT, I0 zM GDP, and I0 ~M PMSF All measurements were performed at 20=C The EF.I'u NMR samples were put into buffer containing 80% DzO Pubhshed by Elsewer Science Pubhshers B V (Biomedical Dwtslon)225

NMR study of the phosphate-binding loops of Thermus thermophilus elongation factor Tu

“…No change is observed in the HMQC spectrum, and so aluminum fluoride is probably not a good transitionstate analogue for EFTu (data not shown). The same conclusion was also reached by Hazlett et al (1991) 12 11 1H (PPM) figure 6: Selective 15N difference decoupling of the downfield resonances as indicated, from fully 15N-labeled EFTu complexed with Mn2+. Neither downfield glycine resonance (A and C) is significantly perturbed by the Mn2+.…”

Formation of a Transition-State Analog of the Ras GTPase Reaction by Ras·GDP, Tetrafluoroaluminate, and GTPase-Activating Proteins

“…Such fluorescent analogs are sensitive to changes in the local structure of the protein (13,14). Even with 5 pM RasmGDP and excess A1F4-, there was little or no change (<3%) of fluorescence (12,15). It has been proposed that the role of GAP might be to stabilize or induce the formation of a transition state in the GTPase reaction (16), and that GAP supplies the helical domain or similar structure that exists in G a proteins (4) and is necessary for GTP hydrolysis (17).…”

Potentiation of N‐Methyl‐d‐Aspartate‐Stimulated Dopamine Release from Rat Brain Slices by Aluminum Fluoride and Carbachol

“…Sternweis and Gilman (1982) first reported that fluoride in the presence of aluminum could stimulate the guanine nucleotide-dependent activation of adenylate cyclase in partially purified cyc-S49 cell membranes. AlF4-has been proposed to be the active species required for this activation and is believed to mimic the y-PO4 ion of GTP and bind to GDP-bound G proteins, thus leading to dissociation of the active cy subunit (Bigay et al, 1987;Hazlett et al, 1991). AlF4-has been used extensively to assess whether G proteins are involved in a variety of cellular processes.…”