Biomedical subjects

Robert G Griffin

Publications and source records attributed to Robert G Griffin.

At least 19 recordsLinked to original sources

PubMed2006

A field-sweep/field-lock system for superconducting magnets--Application to high-field EPR.

We describe a field-lock/field-sweep system for the use in superconducting magnets. The system is based on a commercially available field mapping unit and a custom designed broad-band 1H NMR probe. The NMR signal of a small water sample is used in a feedback loop to set and control the magnetic field to high accuracy. The current instrumental configuration allows field sweeps of +/-0.4 T and a resolution of up to 10(-5) T (0.1 G) and the performance of the system is demonstrated in a high-field electron paramagnetic resonance (EPR) application. The system should also be of utility in other experiments requiring precise and reproducible sweeps of the magnetic field such as DNP, ENDOR or PELDOR.

Electric Conductivity↗

PubMed2006

TOTAPOL: a biradical polarizing agent for dynamic nuclear polarization experiments in aqueous media.

In a previous publication, we described the use of biradicals, in that case two TEMPO molecules tethered by an ethylene glycol chain of variable length, as polarizing agents for microwave driven dynamic nuclear polarization (DNP) experiments. The use of biradicals in place of monomeric paramagnetic centers such as TEMPO yields enhancements that are a factor of approximately 4 larger (epsilon approximately 175 at 5 T and 90 K) and concurrently the concentration of the polarizing agent is a factor of 4 smaller (10 mM electron spins), reducing the residual electron nuclear dipole broadening. In this paper we describe the synthesis and characterization by EPR and DNP/NMR of an improved polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL). Under the same experimental conditions and using 2.5 mm magic angle rotors, this new biradical yields larger enhancements (epsilon approximately 290) at lower concentrations (6 mM electron spins) and has the additional important property that it is compatible with experiments in aqueous media, including salt solutions commonly used in the study of proteins and nucleic acids.

Cyclic N-Oxides↗

PubMed2006

Dynamic nuclear polarization of amyloidogenic peptide nanocrystals: GNNQQNY, a core segment of the yeast prion protein Sup35p.

Dynamic nuclear polarization (DNP) permits a approximately 10(2)-10(3) enhancement of the nuclear spin polarization and therefore increases sensitivity in nuclear magnetic resonance (NMR) experiments. Here, we demonstrate the efficient transfer of DNP-enhanced (1)H polarization from an aqueous, radical-containing solvent matrix into peptide crystals via (1)H-(1)H spin diffusion across the matrix-crystal interface. The samples consist of nanocrystals of the amyloid-forming peptide GNNQQNY(7-13), derived from the yeast prion protein Sup35p, dispersed in a glycerol-water matrix containing a biradical polarizing agent, TOTAPOL. These crystals have an average width of 100-200 nm, and their known crystal structure suggests that the size of the biradical precludes its penetration into the crystal lattice; therefore, intimate contact of the molecules in the nanocrystal core with the polarizing agent is unlikely. This is supported by the observed differences between the time-dependent growth of the enhanced polarization in the solvent versus the nanocrystals. Nevertheless, DNP-enhanced magic-angle spinning (MAS) spectra recorded at 5 T and 90 K exhibit an average signal enhancement epsilon approximately 120. This is slightly lower than the DNP enhancement of the solvent mixture surrounding the crystals (epsilon approximately 160), and we show that it is consistent with spin diffusion across the solvent-matrix interface. In particular, we correlate the expected DNP enhancement to several properties of the sample, such as crystal size, the nuclear T(1), and the average (1)H-(1)H spin diffusion constant. The enhanced (1)H polarization was subsequently transferred to (13)C and (15)N via cross-polarization, and allowed rapid acquisition of two-dimensional (13)C-(13)C correlation data.

Amino Acid Sequence↗

PubMed2006

Description of depolarization effects in double-quantum solid state nuclear magnetic resonance experiments using multipole-multimode Floquet theory.

Using an analytical model based on multipole-multimode Floquet theory (MMFT), we describe the polarization loss (or depolarization) observed in double-quantum (DQ) dipolar recoupling magic angle spinning (MAS) experiments. Specifically, the factors responsible for depolarization are analyzed in terms of higher order corrections to the spin Hamiltonian in addition to the usual phenomenological decay rate constant. From the MMFT model and the effective Hamiltonians, we elucidate the rationale behind the inclusion of a phenomenological damping term in DQ recoupling experiments. As a test of this theoretical approach, the recoupling efficiency of one class of (13)C-(13)C and (13)C-(15)N resonance width dipolar recoupling experiments are investigated at different magnetic field strengths and compared with the more exact numerical simulations. In contrast to existing analytical treatments, the role of higher order corrections is clearly explained in the context of the MMFT approach leading to a better understanding of the underlying spin physics. Furthermore, the analytical model presented herein provides a general framework for describing coherent and incoherent effects in homonuclear and heteronuclear DQ MAS recoupling experiments.

Algorithms↗

PubMed2006

In situ temperature jump high-frequency dynamic nuclear polarization experiments: enhanced sensitivity in liquid-state NMR spectroscopy.

We describe an experiment, in situ temperature jump dynamic nuclear polarization (TJ-DNP), that is demonstrated to enhance sensitivity in liquid-state NMR experiments of low-gamma spins--13C, 15N, etc. The approach consists of polarizing a sample at low temperature using high-frequency (140 GHz) microwaves and a biradical polarizing agent and then melting it rapidly with a pulse of 10.6 microm infrared radiation, followed by observation of the NMR signal in the presence of decoupling. In the absence of polarization losses due to relaxation, the enhancement should be epsilon+ = epsilon(T(obs)/T(mu)(wave)), where epsilon+ is the observed enhancement, epsilon is the enhancement obtained at the temperature where the polarization process occurs, and T(mu)(wave) and T(obs) are the polarization and observation temperatures, respectively. In a single experimental cycle, we observe room-temperature enhancements, epsilon(dagger), of 13C signals in the range 120-400 when using a 140 GHz gyrotron microwave source, T(mu)(wave) = 90 K, and T(obs) = 300 K. In addition, we demonstrate that the experiment can be recycled to perform signal averaging that is customary in contemporary NMR spectroscopy. Presently, the experiment is applicable to samples that can be repeatedly frozen and thawed. TJ-DNP could also serve as the initial polarization step in experiments designed for rapid acquisition of multidimensional spectra.

Carbon Isotopes↗

PubMed2006

Multipole-multimode Floquet theory of rotational resonance width experiments: 13C-13C distance measurements in uniformly labeled solids.

A formal description of zero-quantum (ZQ) NMR processes using multipole-multimode Floquet theory is proposed for studying polarization transfer in magic angle spinning experiments. Specifically, we investigate the factors affecting the accuracy and precision of 13C-13C distance measurements that are based on ZQ-magnetization exchange processes in rotational resonance width experiments. With suitable examples drawn from measurements in N-acetyl-[U-13C,15N]-L-valine-L-leucine, we substantiate our approach and propose methods for improving the accuracy and reliability of such 13C-13C distance measurements in uniformly 13C, 15N-labeled solids. In addition, the theoretical model presented in this article provides a more general framework for describing relaxation phenomena involving multiple decay rate constants in zero-quantum processes.

Carbon Isotopes↗

PubMed2006

Broadband homonuclear correlation spectroscopy at high magnetic fields and MAS frequencies.

We present a new homonuclear recoupling sequence, CMAR, that allows observation of 2D 13C-13C correlation spectra at high magnetic fields and MAS frequencies (10-30 kHz). The main advantages of the sequence are that it provides efficient, broadband dipolar recoupling and concurrently decouples the 1H spins from the 13C's. Thus, no additional 1H decoupling is required during the mixing period, thereby significantly reducing the radio frequency power requirements for the experiment. Thus, CMAR significantly extends the range of applicability of the usual homonuclear recoupling techniques and should be of major interest for structure determinations of biomolecules at high magnetic fields.

Carbon Isotopes↗

PubMed2005

SPINEVOLUTION: a powerful tool for the simulation of solid and liquid state NMR experiments.

Exact numerical simulations of NMR experiments are often required for the development of new techniques and for the extraction of structural and dynamic information from the spectra. Simulations of solid-state magic angle spinning (MAS) experiments can be particularly demanding both computationally and in terms of the programming required to carry them out, even if special simulation software is used. We recently developed a number of approaches that dramatically improve the efficiency and allow a high degree of automation of these computations. In the present paper, we describe SPINEVOLUTION, a highly optimized computer program that implements the new methodology. The algorithms used in the program will be described separately. Although particularly efficient for the simulation of experiments with complex pulse sequences and multi-spin systems in solids, SPINEVOLUTION is a versatile and easy to use tool for the simulation and optimization of virtually any NMR experiment. The performance of SPINEVOLUTION was compared with that of another recently developed NMR simulation package, SIMPSON. Benchmarked on a series of examples, SPINEVOLUTION was consistently found to be orders of magnitude faster. At the time of publication, the program is available gratis for non-commercial use.

Algorithms↗

PubMed2005

Structure of the nitrogen-centered radical formed during inactivation of E. coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine-5'-diphosphate: trapping of the 3'-ketonucleotide.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 has the active site where nucleotide reduction occurs, and R2 contains the diiron tyrosyl radical (Y*) cofactor essential for radical initiation on R1. Mechanism-based inhibitors, such as 2'-azido-2'-deoxyuridine-5'-diphosphate (N(3)UDP), have provided much insight into the reduction mechanism. N(3)UDP is a stoichiometric inactivator that, upon interaction with RNR, results in loss of the Y* in R2 and formation of a nitrogen-centered radical (N*) covalently attached to C225 (R-S-N*-X) in the active site of R1. N(2) is lost prior to N* formation, and after its formation, stoichiometric amounts of 2-methylene-3-furanone, pyrophosphate, and uracil are also generated. On the basis of the hyperfine interactions associated with N*, it was proposed that N* is also covalently attached to the nucleotide through either the oxygen of the 3'-OH (R-S-N*-O-R') or the 3'-C (R-S-N*-C-OH). To distinguish between the proposed structures, the inactivation was carried out with 3'-[(17)O]-N(3)UDP and N* was examined by 9 and 140 GHz EPR spectroscopy. Broadening of the N* signal was detected and the spectrum simulated to obtain the [(17)O] hyperfine tensor. DFT calculations were employed to determine which structures are in best agreement with the simulated hyperfine tensor and our previous ESEEM data. The results are most consistent with the R-S-N*-C-OH structure and provide evidence for the trapping of a 3'-ketonucleotide in the reduction process.

Azides↗

PubMed2005

Multipole-multimode Floquet theory in nuclear magnetic resonance.

In this paper, we present a new analytical approach for describing the spin dynamics of synchronous and asynchronous time-dependent modulations in solid-state nuclear magnetic resonance experiments. The approach, based on multimode Floquet theory, employs the multipole operator basis of Sanctuary for spin description and illustrates the time evolution in the Floquet-Liouville space using the effective Hamiltonians obtained from the contact (or van Vleck) transformation procedure. Since the Hamiltonian and the density operator are expressed in terms of irreducible tensor operators, extensions to higher spin magnitudes (I>12) and multiple spins are quite straightforward and permit analytical treatments for many problems. We outline the general underlying principles involved in this approach with a brief mention of its potential application in other branches of spectroscopy.

Algorithms↗

PubMed2005

Theory of heteronuclear decoupling in solid-state nuclear magnetic resonance using multipole-multimode Floquet theory.

A formal theory for heteronuclear decoupling in solid-state magic angle spinning (MAS) nuclear magnetic resonance experiments is presented as a first application of multipole-multimode Floquet theory. The method permits a straightforward construction of the multispin basis and describes the spin dynamics via effective Floquet Hamiltonians obtained using the van Vleck transformation method in the Floquet-Liouville space. As a test case, we consider a model three-spin system (I2S) under asynchronous time modulations (both MAS and rf irradiation) and derive effective Hamiltonians for describing the spin dynamics in the Floquet-Liouville space during heteronuclear decoupling. Furthermore, we describe and evaluate the origin of cross terms between the various anisotropic interactions and illustrate their exact contributions to the spin dynamics. The theory presented herein should be applicable to the design and understanding of pulse sequences for heteronuclear and homonuclear recoupling and decoupling.

Algorithms↗

PubMed2004

Dynamic nuclear polarization with biradicals.

Dynamic nuclear polarization (DNP) experiments in rotating solids have been performed for the first time using biradicals rather than monomeric paramagnetic centers as polarizing agents. Specifically, two TEMPO radicals were tethered with a poly(ethylene glycol) chain of variable length where the number of glycol units was 2, 3, or 4. NMR experiments show that the signal observed in DNP experiments is approximately inversely proportional to the length of the chain. Thus, the shorter chain with larger electron dipolar couplings yields larger enhancements. The size of the enhancement is a factor of 4 larger than obtained with the identical concentration of monomeric nitroxide radicals achieving a value of approximately 175 for the n = 2 chain.

Cyclic N-Oxides↗

PubMed2004

High-performance selective excitation pulses for solid- and liquid-state NMR spectroscopy.

Computer-optimized selective pulses are routinely used in solution-state NMR spectroscopy. At the same time, their utility and importance for solid-state applications has yet to be fully realized. We suggest a new computational approach that makes the design of soft selective pulses with desired properties relatively straightforward. By applying this technique to the generic selective excitation problem, we have arrived at a family of high performance selective excitation pulses, dubbed E-Family, that allows more flexibility and better performance than analogous pulses previously reported in the literature. The new pulses have been successfully tested in both solid- and solution-state NMR experiments. A theoretical treatment of the effects of chemical shift anisotropy (CSA) on the selective excitation in magic-angle spinning (MAS) experiments in solids is presented. The set of heuristics that comprise our new strategy were incorporated into a general NMR simulation program SPINEVOLUTION.

Anisotropy↗

PubMed2004

Band-selective carbonyl to aliphatic side chain 13C-13C distance measurements in U-13C,15N-labeled solid peptides by magic angle spinning NMR.

We describe three-dimensional magic angle spinning NMR experiments that enable simultaneous band-selective measurement of the multiple distance constraints between carbonyl and side chain carbons in uniformly 13C,15N-labeled peptides. The approaches are designed to circumvent the dipolar truncation and to allow experimental separation of the multiple quantum (MQ) relaxation and dipolar effects. The pulse sequences employ the double quantum (DQ) rotational resonance in the tilted frame (R2TR) to perform selective polarization transfers that reintroduce the 13C'-13Cgamma,delta dipolar interactions. The scheme avoids recoupling of the strongly coupled C'-Calpha and C'-Cbeta spin pairs, therefore minimizing dipolar truncation effects. The experiment is performed in a constant time fashion as a function of the radio frequency irradiation intensity and measures the line shape of the DQ transition. The width and the intensity of this line shape are analyzed in terms of the DQ relaxation and dipolar coupling. The attenuation of the multispin effects in the presence of relaxation enables a two-spin approximation to be employed for the analysis of the experimental data. The systematic error introduced by this approximation is estimated by comparing the results with a three-spin simulation. The contributions of B1-inhomogeneity, CSA orientation effects, and the effects of inhomogeneous line broadening are also estimated. The experiments are demonstrated in model U-13C,15N-labeled peptides, N-acetyl-L-Val-L-Leu and N-formyl-L-Met-L-Leu-L-Phe, where 10 and 6 distances, ranging between 3 and 6 A, were measured, respectively.

Carbon Isotopes↗

PubMed2004

High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy.

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete (13)C and (15)N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 (13)C-(15)N distances between 3 and 6 A were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly (13)C, (15)N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended beta-strand conformation in the amyloid fibrils such that both the main- and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.

Amino Acid Sequence↗

PubMed2004

Solid-phase synthesis and 1H and 13C high-resolution magic angle spinning NMR of 13C-labeled resin-bound saccharides.

We show how high-resolution magic angle-spinning NMR spectroscopy can be used to characterize 13C-labeled saccharides that have been prepared using solid-phase synthesis techniques while they are still bound to a solid-support resin. With the use of 13C-labeled glucose as the starting material, we have successfully synthesized mono-, di- and trisaccharides with uniform 13C labeling of the saccharide rings. Using these materials, we have been able to assign the 13C and 1H spectra and to characterize various impurities on the resin beads.

Algorithms↗

PubMed2003

13C-13C rotational resonance width distance measurements in uniformly 13C-labeled peptides.

The rotational resonance width (R2W) experiment is a constant-time version of the rotational resonance (R2) experiment, in which the magnetization exchange is measured as a function of sample spinning frequency rather than the mixing time. The significant advantage of this experiment over conventional R2 is that both the dipolar coupling and the relaxation parameters can be independently and unambiguously extracted from the magnetization exchange profile. In this paper, we combine R2W with two-dimensional 13C-13C chemical shift correlation spectroscopy and demonstrate the utility of this technique for the site-specific measurement of multiple 13C-13C distances in uniformly labeled solids. The dipolar truncation effects, usually associated with distance measurements in uniformly labeled solids, are considerably attenuated in R2W experiments. Thus, R2W experiments are applicable to uniformly labeled biological systems. To validate this statement, multiple 13C-13C distances (in the range of 3-6 A) were determined in N-acetyl-[U-13C,15N]l-Val-l-Leu with an average precision of +/-0.5 A. Furthermore, the distance constraints extracted using a two-spin model agree well with the X-ray crystallographic data.

Carbon Isotopes↗

PubMed2003

High-frequency dynamic nuclear polarization in MAS spectra of membrane and soluble proteins.

One of the principal promises of solid-state NMR (SSNMR) magic angle spinning (MAS) experiments has been the possibility of determining the structures of molecules in states that are not accessible via X-ray or solution NMR experiments-e.g., membrane or amyloid proteins. However, the low sensitivity of SSNMR often restricts structural studies to small-model compounds and precludes many higher-dimensional solid-state MAS experiments on such systems. To address the sensitivity problem, we have developed experiments that utilize dynamic nuclear polarization (DNP) to enhance sensitivity. In this communication, we report the successful application of MAS DNP to samples of cryoprotected soluble and membrane proteins. In particular, we have observed DNP signal enhancements of up to 50 in 15N MAS spectra of bacteriorhodopsin (bR) and alpha-lytic protease (alpha-LP). The spectra were recorded at approximately 90 K where MAS is experimentally straightforward, and the results suggest that the described protocol will be widely applicable.

Bacteriorhodopsins↗