Nick Soffe

Comparison of techniques for 1H-detected heteronuclear 1H15N Spectroscopy☆

Abstract It has previously been demonstrated that 1 H-detected heteronuclear 1 H 15 N correlation techniques increase the number of proteins that can be usefully studied by NMR. All previous studies of this type have utilized 1 H 15 N heteronuclear multiple-quantum coherence to encode 15 N chemical shift. In this paper we show that 15 N single-quantum coherence has several significant advantages including substantially better 15 N resolution (and a reduction in the transverse relaxation rate of coherence evolving during the t 1 evolution period). Experiments based upon the two techniques are demonstrated and compared using a sample of the c subunit of the Escherichia coli F 1 -F 0 ATP-synthase.

1H- and 13C-n.m.r. assignments and conformational analysis of some monosaccharide and oligosaccharide substrate-analogues of lysozyme

The 1H- and 13C-n.m.r. spectra of solutions of GlcNAc, beta-GlcNAc-(1----4)-GlcNAc, and beta-GlcNAc-(1----4)-beta-GlcNAc-(1----4)-GlcNAc in D2O at 50 degrees are interpreted in terms of the conformations, using a combination of 1D- and 2D-n.m.r. spectroscopy and spectra simulation techniques. Two preferred orientations of the hydroxymethyl group were found for each of these saccharides. The conformations have been compared with those found from X-ray crystallographic data and conformational energy calculations.

NMR at very high fields

One obvious disadvantage is cost. Commercial 750 MHz instruments are currently being quoted at prices around £2 000 000 while a 500 MHz spectrometer costs around £500 000. A special room with a high ceiling (4.5 m) and, possibly, some stray field protection is also required. These financial implications mean that a 750 MHz instrument is perhaps more likely to form part of a national facility. As mentioned above, another possible disadvantage is the field-dependence of some NMR relaxation properties; protein 1 H resonances do not seem to broaden much with increasing field but the time taken for thermal equilibrium to be re-established after a radiofrequency pulse (longitudinal relaxation) increases. A third potential disadvantage is associated with the fields applied in addition to the main static magnetic field. Modern heteronuclear NMR experiments on macromolecules are very sophisticated and numerous radiofrequency pulses [[5]xExperimental NMR techniques for studies of biopolymers. Bax, A. Curr. Opin. Struct. Biol. 1991; 1: 1030–1035Crossref | Scopus (8)See all References, [8]xAmino-acid type determination in the sequential assignment procedure of uniformly 13 C / 15 N-enriched proteins. Grzesiek, S. and Bax, A. J. Biomolec. NMR. 1993; 3: 185–204PubMedSee all References] and field gradients [9xGradient enhanced spectroscopy. Hurd, R.E. J. Magn. Reson. 1990; 87: 422–428See all References][9] are used to select appropriate NMR signals. The demands on these various additional fields become greater with increasing static field strength. The radiofrequency fields, for example, can become inadequate to cover the entire spectrum of interest uniformly and the increased strength required to cover the spectral range can cause significant heating of the sample.In spite of the potential problems, we believe that the advantages of very high fields are significant. It is clear that 1994 will see the installation and operation of several 750 MHz NMR spectrometers world-wide. Even higher field strengths will soon become technically feasible and magnet manufacturers expect to be able to produce magnetic fields equivalent to proton frequencies of 900 MHz within the next five years. It also seems likely that other aspects of NMR spectrometer technology, such as the development of new sophisticated heteronuclear pulse sequences [5xExperimental NMR techniques for studies of biopolymers. Bax, A. Curr. Opin. Struct. Biol. 1991; 1: 1030–1035Crossref | Scopus (8)See all References][5] will continue to advance. These technical innovations together with the ‘brute force’ application of very high fields will continue. It thus seems likely that the astonishing ability of modern NMR to explore the structure and dynamic properties of proteins in solution will carry on being extended. This will be seen by many to justify the high financial cost of the new very high field instruments.

The application of a simple analog phase-shifting device in multiple-quantum NMR

The indirect observation of multiple-quantum coherences offers a useful aid to the established techniques of one-dimensional double-resonance, two-dimensional correlated (COSY), and relayed coherence transfer spectroscopy for the identification of spin systems in large molecules (1-4). Two-dimensional doubleand zeroquantum spectroscopy have been used to obtain chemical-shift correlations in both 13C and ‘H NMR spectra (5-11). It has also been shown that spectral simplification can be achieved using multiple-quantum filtration in conjunction with one or twodimensional experiments (12-16). The order-selective excitation method for the separation of multiple-quantum coherences of order N requires a linear combination of 2N experiments where the excitation pulses are phase shifted in increments of r/N radians (I 7). Recently we have shown that chemical-shift correlations and spectral simplification in the aromatic and methyl regions of protein spectra can be achieved through the use of two-dimensional double-quantum spectroscopy (9, 10). For the selection of doublequantum coherences no modifications to the spectrometer are required when the transmitter offset is placed at the downfield edge of the spectrum because only x/2 phase shifts of the excitation pulses are required. Extension of two-dimensional multiple-quantum spectroscopy to higher order coherences can be achieved with hardware modifications to obtain the required phase shifts. An alternative approach involves the use of composite z pulses (8) but on our spectrometer this has been found to give less satisfactory results because of the incomplete suppression of unwanted coherences. Several digital devices which will produce the necessary radiofrequency phase shifts have been described recently (19-22). Here we describe an analog device which can be used to generate the desired phase shifts. The unit has the advantages of simplicity, low cost, and the ease of interfacing to standard NMR computers (in our case a Nicolet Instruments 1180). The device described relies on a simple pulsedelay technique to provide a continuously adjustable phase shift under the control of a d/a converter. The main disadvantages are relatively slow switching speed and the need for careful calibration to enable accurate phase shifts to be obtained. In practice we find that the unit switches between 0 and 180” in 5 PS. We have not found this to be a disadvantage for the experiments described below. The reproduc-

Specific operator manipulations of heteronuclear scalar-coupled spin systems using phase-modulated selective waveforms

In this Communication we show an alternative and distinctive approach for the uniform selective excitation of scalar-coupled systems with large coupling constants that uses shaped waveforms combined with phase modulation