Rolf Tschudin

Three-Dimensional Triple-Resonance NMR Spectroscopy of Isotopically Enriched Proteins

correlates the amide ‘H and “N shifts with the 13C shift of the carbonyl resonance of the preceding amino acid. A second experiment (HNCA) correlates the intraresidue amide ‘H and 15N shifts with the CLY chemical shift. This experiment often also provides a weak correlation between the amide NH and 15N resonances of one amino acid and the Ca resonance of the preceding amino acid. A third experiment (HCACO) correlates the Ha and GY shifts with the intraresidue carbonyl shift. Finally, a 3D relay experiment, HCA( CO)N, correlates Ha and Cal resonances of one residue with the “N frequency of the succeeding residue. The principles of these experiments are described in terms of the operator formalism. To optimize spectral resolution, special attention is paid to removal of undesired J splittings in the 3D spectra. Technical details regarding the implementation of these triple-resonance experiments on a commercial spectrometer are also provided. The experiments are demonstrated for the protein calmodulin ( 16.7 kDa).

Comparison of different modes of two-dimensional reverse-correlation NMR for the study of proteins

Different two-dimensional NMR schemes for generating ‘H-detected ‘H-“N and ‘H13C correlation spectra are compared. It is shown that the resolution in the dimension that represents the “C or “N chemical shift depends on the type of correlation scheme used. For “N NMR studies of proteins, it is found that experiments that involve “N single-quantum coherence offer improved resolution compared to multiple-quantum correlation experiments, mainly because the ‘H- ‘H dipolar broadening of the multiplequantum coherence is stronger than the heteronuclear dipolar broadening of “N, but also because ofthe presence ofunresolved Jsplittings in the F, dimension ofthe multiplequantum correlation spectra. For 13C, the heteronuclear dipolar interaction is much larger and the ‘H-13C multiple-quantum relaxation is slower than the “C transverse relaxation; however, because of the presence of ‘H- ‘H Jcouplings in the F, dimension of such spectra, in practice the multiplequantum type correlation experiments often offer no gain or even a small loss in resolution, compared to experiments that use transverse 13C magnetization during the evolution period. A modified pulse scheme that increases F, resolution by elimination of scalar relaxation of the second kind is proposed. Experiments for the proteins calmodulin, uniformly enriched with “N, and staphylococcal nuclease, labeled with 13C in the Ca position of all Pro residues are demonstrated.

Measurement of homo- and heteronuclear J couplings from quantitative J correlation

Publisher Summary This chapter discusses a third method, referred to as quantitative “ J correlation.” In this approach, the J coupling is obtained from the fraction of magnetization that is transferred from a nucleus to its J -coupled partner. Quantitative J correlation is a useful and general approach for measuring a large variety of two- and three-bond homo- and heteronuclear J couplings, providing access to the study of a large number of dihedral angles in proteins. The approach is not limited to isotopically enriched proteins and has also been used to measure a large number of heteronuclear 1 H- 13 C J couplings in the cyclic decapeptide gramicidin S. The quantitative J correlation experiments are particularly useful for defining the X∼ angle, as a substantial number of J couplings defined by this angle can now be measured. The large number of J couplings overdetermines χ l if there exist only a single rotametric state. In cases of rotamer averaging, however, this large number of independent J measurements provides a unique opportunity to characterize the motional averaging process.

Three-dimensional NOESY-HMQC spectroscopy of a 13C-labeled protein

The analysis of NMR spectra of med ium-size ( lo-20 kDa) proteins is often difficult because of severe signal overlap. A number of isotope-edited 2D experiments (I7) and, more recently, 3D NMR experiments have been used to alleviate this problem (8-12). Spreading spectral information in three independent frequency dimensions greatly reduces spectral overlap and thereby simplifies the process of analysis. The heteronuclear 3D experiments (I.?, 10-12) are particularly useful in this respect, because the total number of resonances remains unchanged relative to the corresponding homonuclear ‘H 2D spectrum; the chemical shift of the heteronucleus is merely used to disperse the resonances in the regular 2D spectrum along a third axis. All applications of heteronuclear 3D experiments to proteins publ ished to date combine the commonly used homonuclear ‘H experiments NOESY (14,15) and HOHAHA ( 16, 17) with the ‘H-detected heteronuclear mu ltiplequantum correlation (HMQC) experiment ( 18-20). Recently, it has been shown that the HMQC experiment does not provide as high a resolution as some more complex pulse sequences (21). However, the resolution in the dimension of the heteronuclear chemical shift is usually lim ited by digital resolution, and the potentially lower resolution does not play a role. So far, all protein applications of heteronuclear 3D NMR have utilized 15N as the heteronucleus. Here we report the first use of heteronuclear 3D protein NMR using the r3C chemical shift to spread the ‘H resonances. Although at first sight, the change from “N to 13C may appear simple, there were a number of problems that had made it uncertain whether this approach would be successful. F irst, the heteronuclear dipolar l ine-broadening effect of 13C on the resonance of its directly attached proton(s) is severe, typically causing a twofold increase of the regular proton linewidth. This sharply reduces sensitivity in those 3D experiments where one of the magnetization transfer steps relies on ‘H‘H Jcoupling. Our experiments with fully (about 95%) ‘3C-labeled protein also showed a reduction in the proton T, by about 30% relative to the unlabeled protein, and it was not certain a priori whether this strong heteronuclear dipolar relaxation would have a negative influence on the quality of the NOESY spectrum. A second problem arises from the presence of relatively large r Jcc couplings, varying from about 60 Hz for C,-carbonyl and aromatic couplings to about 35 Hz for couplings between aliphatic carbons. Attempting to resolve these splittings in the 3D experiment would increase the number of resonances and thus decrease signal-to-noise. As shown by Markley and co-workers ( 22-24)) using a relatively low level of 13C labeling reduces the * 3Cr3C broaden-

A broadband pulsed radio frequency electron paramagnetic resonance spectrometer for biological applications

A time-domain radio frequency (rf) electron paramagnetic resonance (EPR) spectrometer/imager (EPRI) capable of detecting and imaging free radicals in biological objects is described. The magnetic field was 10 mT which corresponds to a resonance frequency of 300 MHz for paramagnetic species. Short pulses of 20–70 ns from the signal generator, with rise times of less than 4 ns, were generated using high speed gates, which after amplification to 283 Vpp, were deposited into a resonator containing the object of interest. Cylindrical resonators containing parallel loops at uniform spacing were used for imaging experiments. The resonators were maintained at the resonant frequency by tuning and matching capacitors. A parallel resistor and overcoupled circuit was used to achieve Q values in the range 20–30. The transmit and receive arms were isolated using a transmit/receive diplexer. The dead time following the trailing edge of the pulse was about 450 ns. The first stage of the receive arm contained a low noise,...

Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N

SummaryIt is demonstrated that sequential resonance assignment of the backbone 1Hα and 15N resonances of proteins can be obtained without recourse to the backbone amide protons, an approach which should be useful for assignment of regions with rapidly exchanging backbone amide protons and for proteins rich in proline residues. The method relies on the combined use of two 2D experiments, HA(CA)N and HA(CACO)N or their 3D analogs, which correlate 1Hα with the intraresidue 15N and with the 15N resonance of the next residue. The experiments are preferably conducted in D2O, where very high resolution in the 15N dimension can be achieved by using 2H decoupling. The approach is demonstrated for a sample of human ubiquitin, uniformly enriched in 13C and 15N. Complete backbone and 13Cβ/1Hβ resonance assignments are presented.

Comparison of sickle cell hemoglobin gelation kinetics measured by NMR and optical methods

Abstract We describe a technique for monitoring the kinetics of sickle cell hemoglobin gelation by observing the change in the amplitude and linewidth of the water proton magnetic resonance. The resulting kinetic progress curves are very similar to those obtained by optical birefringence and turbidity methods. The curves consist of a delay, followed by a rapidly accelerating signal change which terminates quickly. From a study of the temperature dependence of the delay time, it is shown that all three techniques see the onset of gelation simultaneously. The origin of the change in physical properties upon gelation is briefly discussed in relation to the component steps of the reaction.

Water suppression using a combination of hard and soft pulses

Numerous methods are available for suppression of the Hz0 resonance in the NMR spectra of water-soluble compounds. These methods include presaturation (I, 2), excitation with a soft pulse (3) or a Redfield pulse (4), rapid scan correlation (5), binomial hard-pulse sequences (6-9), and methods that exploit the different relaxation characteristics of HZ0 and solute protons (10-13). In recent years, the binomial hard-pulse sequences and variations thereof (14, 15) have become most widely used. Morris and Smith (16) showed that the binomial sequences can be combined with a single nonselective 90” pulse to provide an excitation spectrum which has nearly maximal excitation over a wide frequency range except for a narrow region around the HZ0 resonance which has near-zero excitation. The phase distortions resulting from this type of excitation are severe which leads to problems when this type of composite pulse is used in phase-sensitive two-dimensional NMR experiments. Levitt and Roberts (I 7) very recently proposed a new hard-pulse sequence which has a much weaker phase dependence across the spectrum while still providing good water suppression. However, the remaining small phase distortions, which present no problem in 1D NMR, can present difficulties in 2D NMR spectra. For example, cross peaks in a NOESY spectrum are typically two orders of magnitude weaker than diagonal peaks and even small phase distortions of the diagonal resonances give rise to serious distortions in the 2D spectrum. A recently proposed echo scheme (18) yields pure absorptive resonances across the entire spectrum but gives poor excitation close to the water resonance. Here we demonstrate the use of a hard/soft pulse combination first suggested by Gupta and Redfield (I 9). In principle, this type of hard/soft pulse combination can solve both the excitation window and the phasing problems mentioned above. However, it should be noted that the hard/soft pulse combinations described below require very stable spectrometer hardware. Conceptually the method is extremely simple. With the carrier placed on the HZ0 resonance, a weak 90?, pulse of duration T (T = 5 ms) rotates the HZ0 magnetization from the z axis to the -y axis (Fig. la), while leaving all resonances more than about 50 Hz removed from the HZ0 resonance along the z axis. A subsequent nonselective 90,” pulse rotates the HZ0 magnetization back to the z axis and rotates the resonances

High-speed digitizer/averager data-acquisition system for fourier transform electron paramagnetic resonance spectroscopy

A high‐speed digitizer/averager data‐acquisition system designed and built as part of a 300‐MHz Fourier transform electron paramagnetic resonance spectrometer is described. There are two key features of the system: (1) the maximum digitizing rate is 300 Msamples/s and (2) a 256‐point free‐induction‐decay signal running summation can be updated in less than 3 μs. At the maximum digitizing rate, the system can sum 65 536 FIDs in 220 ms. The system consists of an analog‐to‐digital converter/adder unit (ADCA) and an IBM compatible personal computer. The ADCA is comprised of a digitizer, high‐speed sample buffers, high‐speed adders/memory, and control hardware. Design techniques, such as parallel processing, utilized to meet the high‐speed performance requirements are described. Trigger and timing signals for the system are derived from the spectrometer. System efficiency, synchronization, and time base stability are demonstrated in the spectrometer at a sampling frequency of 200 MHz. Signal‐to‐noise ratio enh...

Stochastic excitation and Hadamard correlation spectroscopy with bandwidth extension in RF FT-EPR.

The application of correlation spectroscopy employing stochastic excitation and the Hadamard transform to time-domain Fourier transform electron paramagnetic resonance (FT-EPR) spectroscopy in the radiofrequency (RF) band is described. An existing, time-domain FT-EPR spectrometer system with a Larmor frequency (L(f)) of 300 MHz was used to develop this technique by incorporating a pseudo-random pulse sequence generator to output the maximum length binary sequence (MLBS, 10- and 11-bit). Software developed to control the EPR system setup, acquire the signals, and post process the data, is outlined. The software incorporates the Hadamard transform algorithm to perform the required cross-correlation of the acquired signal and the MLBS after stochastic excitation. To accommodate the EPR signals, bandwidth extension was accomplished by sampling at a rate many times faster than the RF pulse repetition rate, and subsequent digital signal processing of the data. The results of these experiments showed that there was a decrease in the total acquisition time, and an improved free induction decay (FID) signal-to-noise (S/N) ratio compared to the conventional coherent averaging approach. These techniques have the potential to reduce the RF pulse power to the levels used in continuous wave (CW) EPR while retaining the advantage of time-domain EPR methods. These methods have the potential to facilitate the progression to in vivo FT-EPR imaging of larger volumes.