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A F Miller

Publications and source records attributed to A F Miller.

At least 19 recordsLinked to original sources
PubMed2001

Host-guest study of left-handed polyproline II helix formation.

The importance of the left-handed polyproline II (PPII) helical conformation has recently become apparent. This conformation generally is involved in two important functions: protein-protein interactions and structural integrity. PPII helices play vital roles in a variety of processes including signal transduction, transcription, and cell motility. Proline-rich regions of sequence are often assumed to adopt this structure. Remarkably, little is known about the physical determinants of this secondary structure type. In this study, we have explored the formation of PPII helices by a short poly(proline) peptide. In addition, the results from experiments used to determine the propensities for apolar residues, plus glycine, asparagine, and glutamine, to adopt this structure in a poly(proline)-based host peptide are reported here. Proline possesses the highest intrinsic propensity, with glutamine, alanine, and glycine having surprisingly high propensities. beta-Branched residues possess the lowest propensities of the residues examined. It is postulated that propensities possessed by apolar residues are due in part to peptide-solvent interactions, and that the remarkably high propensity possessed by glutamine may be due to a side chain to backbone hydrogen bond. These data are the first step toward a molecular understanding of the formation of this important, and yet little studied, secondary structure.

Circular Dichroism↗
PubMed2001

Novel insights into the basis for Escherichia coli superoxide dismutase's metal ion specificity from Mn-substituted FeSOD and its very high E(m).

Fe and Mn are both entrained to the same chemical reaction in apparently superimposable superoxide dismutase (SOD) proteins. However, neither Fe-substituted MnSOD nor Mn-substituted FeSOD is active. We have proposed that the two SOD proteins must apply very different redox tuning to their respective metal ions and that tuning appropriate for one metal ion results in a reduction potential (E(m)) for the other metal ion that is either too low (Fe) or too high (Mn) [Vance and Miller (1998) J. Am. Chem. Soc. 120, 461-467]. We have demonstrated that this is true for Fe-substituted MnSOD from Escherichia coli and that this metal ion-protein combination retains the ability to reduce but not oxidize superoxide. We now demonstrate that the corollary is also true: Mn-substituted FeSOD [Mn(Fe)SOD] has a very high E(m). Specifically, we have measured the E(m) of E. coli MnSOD to be 290 mV vs NHE. We have generated Mn(Fe)SOD and find that Mn is bound in an environment similar to that of the native (Mn)SOD protein. However, the E(m) is greater than 960 mV vs NHE and much higher than MnSOD's E(m) of 290 mV. We propose that the different tuning stems from different hydrogen bonding between the proteins and a molecule of solvent that is coordinated to the metal ion in both cases. Because a proton is taken up by SOD upon reduction, the protein can exert very strong control over the E(m), by modulating the degree to which coordinated solvent is protonated, in both oxidation states. Thus, coordinated solvent molecules may have widespread significance as "adapters" by which proteins can control the reactivity of bound metal ions.

Binding Sites↗
PubMed2001

Quantitative structure-activity relationships in two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H:nitroreductase.

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (kcat/Km) ranging from 10(4) to 10(7) M(-1) s(-1). In agreement with a previously proposed scheme of two-step four-electron reduction of nitroaromatics by NR (Koder, R. L., and Miller, A.-F. (1998) Biochim. Biophys. Acta 1387, 395-405), 2 mol NADH per mole mononitrocompound were oxidized. An oxidation of excess NADH by polinitrobenzenes, including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O2 consumption. This type of "redox cycling" was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The initial reduction of tetryl and other polinitrophenyl-N-nitramines by E. cloacae NR was analogous to a two-step four-electron reduction mechanism of TNT and other nitroaromatics. The logs kcat/Km of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationship shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.

Aniline Compounds↗
PubMed2001

Retro-nitroreductase, a putative evolutionary precursor to Enterobacter cloacae strain 96-3 nitroreductase.

Enterobacter cloacae strain 96-3 nitroreductase (NR) is a homodimeric flavoenzyme that catalyzes the pyridine nucleotide-dependent four-electron reduction of a variety of nitroaromatic compounds, including the explosives TNT (2,4,6-trinitrotoluene), RDX (1,3,5-trinitro-1,3,5-triazine), tetryl (2,4,6-trinitrophenyl-N-methylnitramine), and pentryl (2,4,6-trinitrophenyl-N-nitroaminoethylnitrate). The enzyme was initially characterized by Bryant et al. from a strain of Enterobacter that had been isolated from a weapons dump in La Jolla, CA. The enzyme displays a catalytic efficiency for nitroreduction at least 10-fold higher than that of several highly homologous bacterial nitroreductases and has long been thought to have evolved to be a more efficient nitroreductase due to the high nitroaromatic compound concentrations in its environment. We report the cloning and biochemical characterization of a nitroreductase gene from a clinical isolate of Enterobacter cloacae, a strain that presumably had not encountered high concentrations of nitroaromatics. The new enzyme, which we term retro-nitroreductase, had an amino acid sequence 96.7% identical to NR, and most differences are relatively conservative. The catalytic efficiency of the new enzyme is twofold less than that of NR for the oxidation of NADH and is not significantly different from the value observed for NR for the reduction of dinitrobenzyl alcohol. We conclude that NR has not significantly evolved to be a more efficient nitroreductase as a result of its environment, and the relatively high catalytic activity of the enzyme is a general property of Enterobacter cloacae nitroreductases.

Amino Acid Sequence↗
PubMed2000

Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase.

We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller. J. Am. Chem. Soc. 120(3) (1998) 461-467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn2+, Fe3+ or Fe2+ are bound, based on EPR and NMR spectroscopy. However, the mutant's Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSOD's activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn2+, Fe3+ and 1H in the presence of Fe2+ distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe2+ SOD and Fe2+ (Mn)SOD by 15N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe2+ in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower Em in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH- coordinated to the metal ion.

Binding Sites↗
PubMed2000

Bone morphogenetic protein-9. An autocrine/paracrine cytokine in the liver.

Bone morphogenetic proteins (BMPs) occupy important roles during development serving to direct cells through specific differentiation programs. While several BMPs are essential for embryonic viability, their significance in mediating intercellular communication in the context of adult organ systems remains largely unknown. In the adult rat we characterized the tissue- and cell-specific transcription and translation of BMP-9. Utilizing a ribonuclease protection assay, we determined that in the adult animal, BMP-9 expression occurs predominantly in the liver. Furthermore, we determined that the non-parenchymal cells of the liver, i.e. endothelial, Kupffer, and stellate cells, are the major sources of this message. Western analyses corroborate the ribonuclease protection assay results, confirming that LEC and KC contain an abundance of immunoreactive BMP-9. Using [(125)I]BMP-9, a receptor with specific binding affinity for BMP-9 was characterized in primary cultures of hepatic endothelial cells and Kupffer cells. BMP-9 binding to these cell types was observed to be fully reversible and highly specific for this ligand. Additionally, we demonstrate that BMP-9 is specifically internalized upon binding to its receptor. This may represent a novel BMP receptor and is the first to be characterized in primary cultures of mature liver non-parenchymal cells. Our results depict BMP-9 as a potential autocrine/paracrine mediator in the hepatic reticuloendothelial system.

Animals↗
PubMed2000

Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.

We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide dismutase (Fe(II)SOD and Fe(III)SOD) from Escherichia coli. Despite the severe relaxivity of high-spin Fe(III), we have been able to assign resonances to ligand His' delta1 protons near 100 ppm, and beta and alpha protons collectively between 20 and 50 ppm, in Fe(III)SOD. In the reduced state, we have assigned all but 7 ligand protons, in most cases residue-specifically. A pair of previously unreported broad resonances at 25.9 and 22.1 ppm has been conclusively assigned to the beta protons of Asp 156, superseding earlier assignments (Ming et al. (1994) Inorg. Chem., 33, 83-87). We have exploited higher temperatures to resolve previously unobserved ortho-like ligand His proton resonances, and specific isotopic labeling to distinguish between the possibilities of 82 and epsilon1 protons. These are the closest protein protons to Fe(II) and therefore they have the broadest (approximately 4,000 Hz) and most difficult to detect resonances. Our assignments permit interpretation of temperature dependences of chemical shifts, pH dependences and H/D exchange rates in terms of a hydrogen bond network and the Fe(II) electronic state. Interestingly, Fe(II)SOD's axial His ligand chemical shifts are similar to those of the axial His ligand of Rhodopseudomonas palustris cytochrome c' (Bertini et al. (1988) Inorg. Chem., 37, 4814-4821 ) suggesting that Fe(II)SOD's equatorial His2Asp- ligation is able to reproduce some of the electronic, and thus possibly chemical, properties of heme coordination for Fe2+.

Binding Sites↗
PubMed2000

Two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H nitroreductase: description of quantitative structure-activity relationships.

Enterobacter cloacae NAD(P)H:nitroreductase catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (kcat/Km) ranging from 10(4) M(-1) s(-1) to 10(7) M(-1) s(-1), and oxidizing 2 moles NADH per mole mononitrocompound. Oxidation of excess NADH by polynitrobenzenes including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O2 consumption. This type of 'redox cycling' was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The logs kcat/Km of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron- or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationships shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.

Electrons↗
PubMed1998

Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase.

Enterobacter cloacae nitroreductase (NR) is a flavoprotein which catalyzes the pyridine nucleotide-dependent reduction of nitroaromatics. Initial velocity and inhibition studies have been performed which establish unambiguously a ping-pong kinetic mechanism. NADH oxidation proceeds stereospecifically with the transfer of the pro-R hydrogen to the enzyme and the amide moiety of the nicotinamide appears to be the principal mediator of the interaction between NR and NADH. 2,4-Dinitrotoluene is the most efficient oxidizing substrate examined, with a kcat/KM an order of magnitude higher than those of p-nitrobenzoate, FMN, FAD or riboflavin. Dicoumarol is a potent inhibitor competitive vs. NADH with a Ki of 62 nM. Several compounds containing a carboxyl group are also competitive inhibitors vs. NADH. Yonetani-Theorell analysis of dicoumarol and acetate inhibition indicates that their binding is mutually exclusive, which suggests that the two inhibitors bind to the same site on the enzyme. NAD+ does not exhibit product inhibition and in the absence of an electron acceptor, no isotope exchange between NADH and 32P-NAD+ could be detected. NR catalyzes the 4-electron reduction of nitrobenzene to hydroxylaminobenzene with no optically detectable net formation of the putative two-electron intermediate nitrosobenzene.

Acetates↗
PubMed1998

Spectroscopic comparisons of the pH dependencies of Fe-substituted (Mn)superoxide dismutase and Fe-superoxide dismutase.

We have compared the active sites of Escherichia coli Fe-substituted (Mn)superoxide dismutase [Fe-sub-(Mn)SOD] and Fe-SOD to elucidate the basis for the inactivity of Fe-sub-(Mn)SOD, despite its apparent similarity to Fe-SOD. The active site of (reduced) Fe2+-sub-(Mn)SOD is qualitatively similar to that of native Fe2+-SOD, indicating similar active site structures and coordination environments for Fe2+. Its nativelike pK is indicative of nativelike local electrostatics, and consistent with Fe2+-sub-(Mn)SOD's retention of ability to reduce O2*- [Vance and Miller (1998) J. Am. Chem. Soc. 120(3), 461-467]. The active site of (oxidized) Fe3+-sub-(Mn)SOD differs from that of Fe3+-SOD with respect to the EPR signals produced at both neutral and high pH, indicating different coordination environments for Fe3+. Although Fe3+-sub-(Mn)SOD binds the small anions N3- and F-, the KD for N3- is tighter than that of Fe3+-SOD, suggesting that the (Mn)SOD protein favors anion binding more than does the (Fe)SOD protein. The EPR spectral consequences of binding F- are reminiscent of those observed upon binding the first F- to Fe3+-SOD, but the EPR spectrum obtained upon binding N3- is different, consistent with crystallographic observation of a different binding mode for N3- in Thermus thermophilus Mn-SOD than Fe-SOD [Lah, M., et al. (1995) Biochemistry 34, 1646-1660]. We find a pK of 8.5 to be associated with dramatic changes in the EPR spectrum. In addition, we confirm the pK between 6 and 7 that has previously been reported based on changes in the optical signal and N3- binding [Yamakura, F., et al. (1995) Eur. J. Biochem. 227, 700-706]. However, this latter pK appears to be associated with much subtler changes in the EPR spectrum. The non-native pKs observed in Fe3+-sub-(Mn)SOD and the differences in the Fe3+ coordination indicated by the EPR spectra are consistent with Fe3+-sub-(Mn)SOD's inability to oxidize O2*- and suggest that its low E degrees is due to perturbation of the oxidized state.

Binding Sites↗
PubMed1998

Overexpression, isotopic labeling, and spectral characterization of Enterobacter cloacae nitroreductase.

Bacterial nitroreductases have generated much interest recently due to their central roles in both nitroaromatic bioremediation and nitroaromatic toxicity, mutagenicity, and carcinogenicity. Enterobacter cloacae nitroreductase (NR) has been subcloned into the pET overexpression system and purified to homogeneity via a four-step procedure resulting in a final yield of 65.7 mg per liter. Overexpression in minimal media containing 15NH4Cl as the sole source of nitrogen yielded 37.6 mg per liter of homogenous NR containing > 99 atom % 15N. A series of melting curves generated under a variety of solvent conditions established the optimal conditions for NR stability as pH 7.5, low ionic strength phosphate buffer. A two-dimensional 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance spectrum demonstrates this enzyme to be amenable to study by high-resolution multidimensional NMR in combination with amino-acid-specific isotopic labeling. Optical spectra of the purified enzyme suggest that the noncovalently bound flavin mononucleotide cofactor binds in a hydrophobic environment and is in the neutral and anionic protonation states in the oxidized and two-electron reduced oxidation states, respectively. NR exhibits a novel visible region circular dichroism spectrum which has a small distinct negative band at 366 nm and a large positive ellipticity at 454 nm with a shoulder centered at 480 nm.

Base Sequence↗
PubMed1997

Mutation of tyrosine 34 to phenylalanine eliminates the active site pK of reduced iron-containing superoxide dismutase.

We have compared the magnetic resonance properties and pH dependence of wild-type and mutant Fe-containing superoxide dismutase (Fe-SOD) in which the conserved active site tyrosine (Tyr 34) is replaced by phenylalanine. The EPR spectrum of the oxidized state and the NMR spectrum of the paramagnetically shifted resonances of the reduced state indicate that in both states the active site is relatively unperturbed by the mutation. Similarly, the mutant Fe-SOD retains approximately 41% of wild-type catalytic activity on a per Fe basis. However, replacement of Tyr 34 by Phe abolishes both NMR spectroscopic signatures of the active site pK of 8.5 of (reduced) Fe2+-SOD. Neither accessibility to base-catalyzed exchange nor the chemical shifts of active site residues are affected by pH in the range of 6.5-10.5 in Y34F Fe2+-SOD. Thus, the active site pK of 8.5 of Fe2+-SOD most likely corresponds to deprotonation of Tyr 34. The widespread chemical shift changes associated with the pK could reflect Tyr 34's participation in the active site hydrogen bonding network and the network's propagation of the effects of deprotonating Tyr 34 to the Fe2+. Deprotonation of Tyr 34 can also explain the dramatic decrease in active site accessibility to base-catalyzed exchange as the result of electrostatic repulsion between the exchange catalyst OH- and the (Tyr 34)- ion formed at high pH. Similar electrostatic repulsion between (Tyr 34)- and the substrate O2.- is also consistent with the observed increase in KM above pH 9.

Binding Sites↗
PubMed1997

Spectroscopic measurement of a long-predicted active site pK in iron-superoxide dismutase from Escherichia coli.

The accepted mechanism of Fe-containing superoxide dismutase (Fe-SOD) activity and inhibition by anions implies the existence of a group with a pK of 8.6-9.0 in the active site of reduced Fe-SOD [Bull, C. & Fee, J. A. (1985) J. Am. Chem. Soc. 107, 3295-3304]. We have performed pH titrations of reduced Fe-SOD by NMR spectroscopy and observe a pK of 8.5 at 30 degrees C which is the only pK affecting the active site between pH 5.5 and 10.5. Thus, we present the first spectroscopic evidence of the predicted pK. Although the pK is associated with chemical shift changes for almost all of the resonances of the active site, resonance line widths and the T1 of a ligand proton are not significantly affected by the pK, indicating that there is no significant conformational change and only relatively minor effects on the electronic spin properties of Fe2+. The changes in chemical shift are probably caused by changes in hydrogen bonding to a ligand and attendant subtle perturbation of the Fe2+ paramagnetism upon loss of the proton with the pK of 8.5. The pK is also associated with a dramatic restriction of the exchange of at least one ligand proton. Thus, active site accessibility to solvent and OH- decreases by more than 2 orders of magnitude upon loss of the proton with the pK of 8.5. Since OH- is a competitive inhibitor of Fe-SOD, and thus a substrate analog, this dramatic and unusual decrease in accessibility to OH- is consistent with the increase in the K(M) for O2.- that is associated with a pK near 9.

Binding Sites↗
PubMed1997

Measurement of the degree of coupled isotopic enrichment of different positions in an antibiotic peptide by NMR.

An experimental strategy for determining the extent to which multiply isotopically labeled fragments are incorporated intact into relatively complicated compounds of interest is presented. The NMR methods employed are based on isotope-filtered one-dimensional spectra and difference HSQC spectra incorporating a spin echo designed to report on the presence of a second NMR active isotope at a coupled site. They supplement existing methods for determining the extent of isotopic incorporation at individual sites to reveal whether two coupled labeled sites in a precursor are incorporated as an intact unit into products. The methods described also circumvent 1H signal overlap and distinguish between the effects of different nitrogens coupled to individual carbons. The somewhat complicated case of valclavam illustrates the method's utility in measuring the J coupling constants between 13C and nearby sites that are only fractionally labeled with 15N, and measuring the fraction of molecules in which 13C is coupled to 15N, at each of several sites. The 15N of [2-13C, 15N]-labeled glycine is found to be incorporated into all three N positions of valclavam but most heavily into the N11 position. Specifically, 15N and 13C are incorporated into the N11 and C10 positions together as an 15N13C fragment approximately 8% of the time, whereas 15N is incorporated largely independently at the other positions.

Carbon Isotopes↗
PubMed1997

Selective 15N labeling and direct observation by NMR of the active-site glutamine of Fe-containing superoxide dismutase.

The glutamine in position 69 is one of only three conserved active-site amino acid differences between Fe- and Mn-containing superoxide dismutases (SODs). We have refined the conditions for extremely selective labeling of the side chains of glutamine with 15N, and thus obtained dramatically simplified spectra, despite the large size of Fe-SOD. The improved resolution afforded by such highly specific labeling permits the use of direct 15N detection to observe and assign Gln 69, even though its distance to the paramagnetic Fe2+ is only 5 A. Selective glutamine side-chain labeling is inexpensive and has general utility for large (and paramagnet-containing) proteins.

Binding Sites↗
PubMed1995

Solution structure of the MutT enzyme, a nucleoside triphosphate pyrophosphohydrolase.

The MutT enzyme (129 residues) catalyzes the hydrolysis of normal and mutagenic nucleoside triphosphates, such as 8-oxo-dGTP, by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed beta-sheet connected by the loop I-alpha-helix I--loop II motif, by two tight turns, and by loop III, and terminated by loop IV--alpha-helix II [Abeygunawardana et al. (1993) Biochemistry 32, 13071-13080; Weber et al. (1993) Biochemistry 32, 13081-13087). Complete side-chain assignments of 1H and 13C resonances have now been made by 3D C(CO)NH and HCCH-TOCSY experiments. A total of 1461 interproton proximities (11 per residue), obtained by 3D 15N-resolved NOESY-HSQC and 3D 13C-resolved NOESY-HSQC spectra, including 372 long-range NOEs, as well as 65 dihedral angle (phi) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure of MutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR. The structure is globular and compact with the parallel portion of the beta-sheet sandwiched between the two alpha-helices, forming an alpha+beta fold. The essential divalent cation has previously been shown to bind near residues Gly-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to bind near residues Leu-54 and Val-58 by NMR relaxation methods [Frick et al. (1995) Biochemistry 34, 5577-5586].(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1993

An NMR comparison of the changes produced by different guanosine 5'-triphosphate analogs in wild-type and oncogenic mutant p21ras.

We have used nuclear magnetic resonance spectroscopy to compare the conformational changes produced by replacement of bound GDP by the GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanylyl (beta, gamma-imido)diphosphate (GMPPNP) in wild-type p21ras as well as the oncogenic mutant (G12D)p21ras. We have used isotope-edited nuclear magnetic resonance spectroscopy to observe the amide resonances of selectively [15N]glycine and [15N]isoleucine labeled p21ras-nucleotide complexes. We find that eight of the nine resonances that respond strongly to GTP gamma S and GMPPNP binding are the same but that the nature of the effect appears different. With GTP gamma S, seven new resonances replace the eight resonances specifically associated with GDP-p21ras, but in GMPPNP-p21ras only two resonances replace the GDP-specific resonances that are lost. The resonance of Gly 60 is clearly shown to be responsive to replacement of GDP by GMPPNP, in addition to glycines 10, 12, 13, 15, and 75 and isoleucines 36, 21, and one other, that were found to respond to GTP gamma S by Miller et al. [Miller, A.-F., Papastavros, M. Z., & Redfield, A.G. (1992) Biochemistry 31, 10208-10216). The two GMPPNP-specific resonances observed appear in positions similar to GTP gamma S-specific resonances, and the GTP gamma S-specific resonances, although not lost altogether, are weaker than the GDP-specific resonances they replace. Thus, the two GTP analogs have similar effects on the spectrum of p21ras, suggesting that the effects are due to features common to both analogs. We propose that active site resonance intensities are specifically attenuated when GTP analogs are bound because interactions with the gamma-phosphate of GTP analogs couple the flexible loops 2 and 4 to the rigid loop 1 of the active site. The conformational heterogeneity and dynamics of loops 2 and 4 would be constrained by loop 1 but also transmitted to it. Coupled conformational exchange on a common intermediate time scale could explain the simultaneous loss of resonances from all three loops in the active site. In our comparison of wild-type and (G12D) GDP-p21ras, we find that the resonance of Ile 36 is not visible in (G12D)p21ras. In (G12D)p21ras, replacement of GDP by GTP gamma S causes the resonances of glycines 10, 13, 15, 60, and 75 and isoleucine 21 and four others to shift from their GDP-specific positions. GTP gamma S-specific resonances are observed for all but two of these.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗