Dynamic nuclear polarization at 9T using a novel 250GHz gyrotron microwave source.

In this communication, we report enhancements of nuclear spin polarization by dynamic nuclear polarization (DNP) in static and spinning solids at a magnetic field strength of 9T (250 GHz for g=2 electrons, 380 MHz for 1H). In these experiments, 1H enhancements of up to 170+/-50 have been observed in 1-13C-glycine dispersed in a 60:40 glycerol/water matrix at temperatures of 20K; in addition, we have observed significant enhancements in 15N spectra of unoriented pf1-bacteriophage. Finally, enhancements of approximately 17 have been obtained in two-dimensional 13C-13C chemical shift correlation spectra of the amino acid U-13C, 15N-proline during magic angle spinning (MAS), demonstrating the stability of the DNP experiment for sustained acquisition and for quantitative experiments incorporating dipolar recoupling. In all cases, we have exploited the thermal mixing DNP mechanism with the nitroxide radical 4-amino-TEMPO as the paramagnetic dopant. These are the highest frequency DNP experiments performed to date and indicate that significant signal enhancements can be realized using the thermal mixing mechanism even at elevated magnetic fields. In large measure, this is due to the high microwave power output of the 250 GHz gyrotron oscillator used in these experiments.

High-frequency dynamic nuclear polarization in the nuclear rotating frame.

A proton dynamic nuclear polarization (DNP) NMR signal enhancement (epsilon) close to thermal equilibrium, epsilon = 0.89, has been obtained at high field (B(0) = 5 T, nu(epr) = 139.5 GHz) using 15 mM trityl radical in a 40:60 water/glycerol frozen solution at 11 K. The electron-nuclear polarization transfer is performed in the nuclear rotating frame with microwave irradiation during a nuclear spin-lock pulse. The growth of the signal enhancement is governed by the rotating frame nuclear spin-lattice relaxation time (T(1rho)), which is four orders of magnitude shorter than the nuclear spin-lattice relaxation time (T(1n)). Due to the rapid polarization transfer in the nuclear rotating frame the experiment can be recycled at a rate of 1/T(1rho) and is not limited by the much slower lab frame nuclear spin-lattice relaxation rate (1/T(1n)). The increased repetition rate allowed in the nuclear rotating frame provides an effective enhancement per unit time(1/2) of epsilon(t) = 197. The nuclear rotating frame-DNP experiment does not require high microwave power; significant signal enhancements were obtained with a low-power (20 mW) Gunn diode microwave source and no microwave resonant structure. The symmetric trityl radical used as the polarization source is water-soluble and has a narrow EPR linewidth of 10 G at 139.5 GHz making it an ideal polarization source for high-field DNP/NMR studies of biological systems.

Structural changes induced in p21Ras upon GAP-334 complexation as probed by ESEEM spectroscopy and molecular-dynamics simulation.

BACKGROUND: The means by which the protein GAP accelerates GTP hydrolysis, and thereby downregulates growth signaling by p21Ras, is of considerable interest, particularly inasmuch as p21 mutants are implicated in a number of human cancers. A GAP "arginine finger," identified by X-ray crystallography, has been suggested as playing the principal role in the GTP hydrolysis. Mutagenesis studies, however, have shown that the arginine can only partially account for the 10(5)-fold increase in the GAP-accelerated GTPase rate of p21. RESULTS: We report electron spin-echo envelope modulation (ESEEM) studies of GAP-334 complexed with GMPPNP bound p21 in frozen solution, together with molecular-dynamics simulations. Our results indicate that, in solution, the association of GAP-334 with GTP bound p21 induces a conformational change near the metal ion active site of p21. This change significantly reduces the distances from the amide groups of p21 glycine residues 60 and 13 to the divalent metal ion. CONCLUSIONS: The movement of glycine residues 60 and 13 upon the binding of GAP-334 in solution provides a physical basis to interpret prior mutagenesis studies, which indicated that Gly-60 and Gly-13 of p21 play important roles in the GAP-dependent GTPase reaction. Gly-60 and Gly-13 may play direct catalytic roles and stabilize the attacking water molecule and beta,gamma-bridging oxygen, respectively, in p21. The amide proton of Gly-60 could also play an indirect role in catalysis by supplying a crucial hydrogen bonding interaction that stabilizes loop L4 and therefore the position of other important catalytic residues.

Electron spin resonance of TOAC labeled peptides: folding transitions and high frequency spectroscopy.

The unnatural, conformationally constrained nitroxide amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) stabilizes helical structure and provides a means for studying rigidly spin labeled peptides by electron spin resonance (ESR). Two new directions in TOAC research are described. The first investigates intermediates formed during alpha-helix unfolding. Double TOAC labeled alpha-helical peptides were unfolded at low temperature in aqueous solution with increasing concentrations of guanidine hydrochloride. Comparison of ESR spectra from two doubly labeled peptides suggests that 3(10)-helix emerges as an intermediate. The second research direction involves the use of high frequency ESR (140 GHz) at low temperature to assess dipolar couplings and, hence, distances between TOAC pairs in a series of 3(10)-helical peptides. Preliminary simulations suggest that high frequency ESR is able to extract correct distances between 6 and 11 A. In addition, the spectra appear to be very sensitive to the relative orientation of the TOAC labels.

Pulsed electron-nuclear double resonance (ENDOR) at 140 GHz.

We describe a spectrometer for pulsed ENDOR at 140 GHz, which is based on microwave IMPATT diode amplifiers and a probe consisting of a TE011 cavity with a high-quality resonance circuit for variable radiofrequency irradiation. For pulsed EPR we obtain an absolute sensitivity of 3x10(9) spins/Gauss at 20 K. The performance of the spectrometer is demonstrated with pulsed ENDOR spectra of a standard bis-diphenylene-phenyl-allyl (BDPA) doped into polystyrene and of the tyrosyl radical from E. coli ribonucleotide reductase (RNR). The EPR spectrum of the RNR tyrosyl radical displays substantial g-anisotropy at 5 T and is used to demonstrate orientation-selective Davies-ENDOR.

The effects of cryoprotection on the structure and activity of p21 ras: implications for electron spin-echo envelope modulation spectroscopy.

Electron spin-echo envelope modulation (ESEEM) spectroscopy is widely used to investigate the active sites of biological molecules in frozen solutions. Various cryoprotection techniques, particularly the addition of co-solvents, are commonly employed in the preparation of such samples. In conjunction with ESEEM studies of Mn(II) guanosine nucleotide complexes of p21 ras, we have investigated the effects of cryoprotection on the spectroscopy, the structure, and the activity of this protein. Echo decay times, which typically govern ESEEM spectral resolution, were found to vary linearly with the concentration of glycerol or methyl alpha-D-glucopyranoside (MG), with both additives equally effective on a per-mole basis. The effect of glycerol and MG on the ESEEM amplitudes of various protein nucleiwas studied in ras p21.Mn(II). 5'guanylylimido-diphosphate(p21.Mn(II)-GMPPNP) complexes: these additives did not alter the distances of these nuclei from the Mn(II) ion. In particular, in p21 incorporating [2H-3]Thr, the Mn(II)-[2H-3]Thr35 distance was found to be unaffected by the concentration of cryoprotectant or the rate of freezing. The proximity of the cryoprotectants to the Mn(II) ion was probed by 2H ESEEM in solutions made with d5-glycerol and d7-methyl alpha-D-glucopyranoside (d7-MG). In p21.Mn(II)GMPPNP, the large deuterium modulations from the d5-glycerol exhibit saturation behavior with increasing d5-glycerol concentration, implying that glycerol, a widely used cryoprotectant, replaces the aquo ligands of the Mn(II) ion. The interaction between the Mn(II) ion of p21 and MG, however, is less intimate: the deuterium ESEEM amplitudes are much smaller for samples prepared with d7-MG than with d5-glycerol. Several polyhydroxylic compounds were found to have essentially no effect on the ability of the guanosine 5'-triphosphate (GTP) hydrolysis activating protein, GAP334, to catalyze hydrolysis of p21. guanosine 5'-triphosphate. This observation implies that the introduction of cryoprotectant does not significantly perturb the structure of p21 and gives insight into the mechanism of the GTPase reaction.

The frozen solution structure of p21 ras determined by ESEEM spectroscopy reveals weak coordination of Thr35 to the active site metal ion.

BACKGROUND: The G protein p21 ras is a molecular switch in the signal transduction pathway for cellular growth and differentiation. Hydrolysis of tightly bound GTP alters the conformation of p21, terminating the signal. The coordination of the p21 residue Thr35 to Mg2+ in its active site, which has been observed in the crystal structure of p21 in complex with a GTP-analog GMPPNP but not with GDP, has been proposed to drive the conformational change accompanying nucleotide substitution and may have a role in the GTP hydrolysis reaction itself. However, previous electron spin-echo envelope modulation (ESEEM) studies of selectively 2H beta-threonine and 15N-threonine labeled p21.Mn2+ GMPPNP suggest that Thr35 only weakly coordinates the metal ion in the growth-active GTP-bound state of p21. RESULTS: A 13C beta-Thr35 to Mn2+ distance of 4.3 +/- 0.2 A and a 15N epsilon-Lys16 to Mn2+ distance of 5.3 +/- 0.2 A were determined from ESEEM spectra of the selectively 13C beta-Thr and 15N epsilon-Lys labeled p21.Mn2+ GMPPNP frozen solution structure. The 13C beta-Thr35 to Mn2+ distance is greater than that (3.16 A) observed in the crystal structure. In contrast, the 15N epsilon-Lys16 to Mn2+ distance is in good agreement with the 5.1 A crystal structure distance. CONCLUSIONS: The 13C beta of Thr35 is more distant from the active site Mn2+ in the frozen solution structure than in the crystal structure of p21.Mg2+ GMPPNP, indicating that Thr35 only weakly coordinates the metal ion in frozen solution. Thr35 coordination of the metal ion is therefore unlikely to drive the conformational change between GTP- and GDP-bound states of p21. Thr35 may be essential for GTPase-activating protein (GAP)-stimulated GTP hydrolysis and/or signal transduction for other reasons.

High frequency (139.5 GHz) electron paramagnetic resonance spectroscopy of the GTP form of p21 ras with selective 17O labeling of threonine.

Electron paramagnetic resonance spectroscopy at 139.5 GHz has been used to study p21 ras complexed with Mn(II) and guanosine 5'-(beta, gamma-imidotriphosphate), an analog of GTP. The p21 sample studied was selectively labeled with [17O gamma]threonine to a final enrichment of 30%. A Mn(II)-17O hyperfine interaction was observed, but the value of the coupling constant, 0.11 +/- 0.04 mT, is the smallest such value yet reported. Ab initio calculations indicate that this value is consistent with direct coordination of the threonine hydroxyl group and provide an estimate for the Mn(II)-17O bond length of 2.7 A. The measured hyperfine coupling constant and associated bond length starkly contrast with typical values for Mn(II)-17O coordination complexes, namely, approximately 0.25 mT and approximately 2.2 A, respectively. This contrast underscores the peculiar weakness of this Mn(II)-O interaction in p21 and persuasively argues that the nucleotide-induced conformational change, which is known to encompass the region of p21 involving Thr35, is not driven by Mn(II) coordination of the Thr35 hydroxyl group.

Characterization of the active site of p21 ras by electron spin-echo envelope modulation spectroscopy with selective labeling: comparisons between GDP and GTP forms.

Selectively labeled samples of human H- or N-ras p21 ligated to MnIIGDP or MnIIGMPPNP were studied by electron spin-echo envelope modulation spectroscopy in order to define the protein environment around the divalent metal. We incorporated [4-13C]-labeled Asx into p21.MnIIGDP and found that the distance from the carboxyl 13C of Asp57 to MnII is approximately 4.1 A. Our result is consistent with indirect coordination of this residue to the metal. From a [2-2H]Thr-labeled sample, we estimate that the distance from the MnII ion to the 2H of Thr35 is at least 5.8 A. Thus, the only protein or nucleotide ligands to the metal appear to be Ser17 and the beta-phosphate of GDP, as previously reported [Larsen, R. G., Halkides, C. J., Redfield, A. G., & Singel, D. J. (1992b) J. Am. Chem. Soc. 114, 9608-9611]. In the 5'-guanylylimido diphosphate (GMPPNP) form of p21, Thr35 has been reported by X-ray crystallography to be a ligand of the metal via its hydroxyl group, and this residue appears to play a key role in the biologically important conformational change upon nucleotide substitution [Pai, E. F., Krengel, U., Petsko, G., Goody, R. S., Kabsch, W., & Wittinghofer, A. (1990) EMBO J. 9, 2351-2359]. The ESEEM spectrum of p21.MnIIGMPPNP labeled with [2-2H]Thr yields a MnII-2H distance of 4.9 A, a distance inconsistent with strong coordination. A sample of p21 in which the Thr residues were fully labeled with 13C and 15N yielded a value of 5.0 A for the distance from MnII to the amide nitrogen of Thr35, while the 13C signal is much smaller than expected if Thr35 were coordinated. A [15N]serine/glycine-labeled sample gives a distance to the amide 15N of Ser17 of 3.9 A, consistent with the X-ray structure; a [4-13C]-labeled Asx sample of p21 gives a distance of approximately 4 A between MnII and the label of Asp57, again implying indirect coordination. Both of these values are very similar to those found for the GDP form of the protein. The results for Thr35, however, reveal a structural difference between the GDP and GTP forms in the region of Thr35. In addition, the position of this residue is found to be different from the crystal structure and in a manner suggesting that the metal ligation of Thr35 does not drive the conformational change that accompanies nucleotide substitution.