JONATHAN BOYD, NICK SOFFE AND lAIN CAMPOELI. WAYS & MEANS~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

NMR at very high fields

Structure 15 April 1994, 2:253-255

IntroductionIn the last decade, there have been exciting develop-ments in techniques for the determination of macro-molecular structure. Crystallography, the traditionalmethod, is undergoing yet more technical improve-ments [1] but nuclear magnetic resonance (NMR) [2]and electron microscopy [3] can now also give atomicresolution structural information. Each of these threestructural methods collects data from specimens invery different conditions and each has its own set oftechnical problems and limitations, but they yield com-plementary information and, taken together, the threeare now giving a remarkable amount of new structuraldetail.

In NMR, the sample conditions are the least demand-ing: an aqueous solution of about 1 mM concentration.No crystals, ordered arrays or heavy-atom derivativesare required and the sample is not exposed to dam-aging radiation. The NMR sample is placed in a mag-netic field and induced radiofrequency signals are de-tected. The stronger the field the higher the resonantradiofrequency. The most effective nucleus to observeis the proton (1H). Proton NMR signals observed ina macromolecule must first be assigned to particularatoms or groups; then a large number of experimen-tal restraints, mainly through-space distances, betweenatom pairs less than 0.5 nm apart, are used to computethe structure [2]. There is, however, a problem: NMRresonances broaden with increasing molecular weight,causing a limit in the size of molecule that can be stud-ied. Some of the problems can be alleviated by theincorporation of NMR-active isotopes such as 13C and15 N since these nuclei can be used to disperse the spec-tra and to filter out unwanted resonances. Generally,however, complete structure determinations by solu-tion state NMR will probably only be carried out onproteins containing less than about 300 residues [4].In spite of this size limit, NMR is proving very valuablein determining the solution structures of small proteins,and folded domains dissected from larger proteins.

Advances in NMR technology have come from variouscumulative instrumental improvements. These includestronger and more uniform magnetic fields, multi-dimensional heteronuclear experiments [5] and im-proved computational methods for processing and cal-culation. The availability of recombinant DNA expres-sion methods that allow production of the requiredamount of isotopically-labelled protein (tens of mil-ligrams) is another major factor in the rapid recent

increase in the number of structures determined byNMR.

Since the first introduction of commercial NMR in-struments in the early 1950s there has been a steadyincrease in the available field strength (Fig. 1). Ironcore magnets are limited to around 2.2Tesla and, inthe 1960s, there was a change to magnets based ona solenoid of suitable conductor, rendered 'super-conducting' at liquid helium temperatures (4.2K atatmospheric pressure). As the field strengths increase,there are formidable technical problems for the mag-net manufacturer, created by the need to keep the wiresuperconducting and stable; modem magnets are madefrom various alloys including niobium/tin and nio-bium/titanium. In addition to absolute field strength,magnets for NMR require an extraordinarily uniformmagnetic field, approaching 1 part in 109, over thesample volume of about 0.5 ml. The improvement ofNMR magnet homogeneity has been another area ofconsiderable advance in the last decade. In spite of theneed to develop new wire and other technology, 1993saw the successful operation of more than one mag-net, custom-built for NMR, operating at field strengthsof 17.6Tesla. At this field 1H nuclei resonate at a radio-frequency of 750 MHz.

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Fig. 1. A plot of magnetic field strength against year of intro-duction. The blue colour indicates iron-based magnets while redindicates superconducting magnets. The numbers correspond tothe frequency, in MHz, at which 1H nuclei resonate at the differ-ent field values.

These new '750 MHz' magnets include one which weuse, manufactured by Oxford Instruments, UK, funded

( Current Biology Ltd ISSN 0969-2126 253

WAYS & MNSJONATHAN BOYD, NICK SOFFE AND AIN CAMPBELL

254 Structure 1994, Vol 2 No 4

by an SERC LINK Protein Engineering initiative withZeneca as the other industrial partner. Like most NMRmagnets this one is cooled to 4.2 K, to make the wiresuperconducting. An additional requirement is to closethe circuit in the solenoid thus producing a permanentcurrent and a very stable field. We have assembled andconstructed our spectrometer at the University of Ox-ford. One of the more interesting aspects of this ex-ercise has been the demanding requirements of theradio frequency 'probe' that induces and detects ra-diofrequency signals from the sample. To avoid pertur-bation of the excellent field uniformity of our magnet,the probes have to be made from materials which ef-fectively have a bulk magnetic susceptibility of close tozero, a matter of some practical difficulty.

What are the advantages of high fields?Higher field strengths potentially offer both improvedspectral resolution and signal-to-noise (S:N) ratio. Sincethe dispersion of resonances increases with field, im-proved resolution results if the linewidths of the res-onances stay the same. Experiments, so far, on ournew spectrometer indicate that there is a significantimprovement in the resolving power of 1H based ex-periments. Fig. 2 shows a small portion of a two di-mensional 15N-lH experiment, carried out on the cy-tokine, granulocyte colony-stimulating factor (G-CSF).This is a rather insoluble helical protein of 19.5 kDa andpresents quite a demanding test for any NMR spectrom-eter [6]. It can be readily seen that the 750 MHz instru-ment has clear resolution advantages for this sample.

Improved S:N ratio is also theoretically expected withincreasing field, because the energy of the NMR signals

is higher and the population difference between the nu-clear energy levels is greater. In fact, over the last twodecades, instrument S:N ratio has increased by consid-erably more than the theoretical expectation becauseof other improvements, such as magnetic field uni-formity and radiofrequency probe performance - thebest instruments of today are over 50 times more sensi-tive than the best of 20 years ago. Nowadays, however,these other instrumental factors are roughly equivalentfor well designed spectrometers operating at the dif-ferent available field strengths. We have observed anapproximately linear increase in S:N ratio with field, onour 500 MHz, 600MHz and 750MHz spectrometers,for single resonances in single-scan experiments onnon-ionic aqueous solutions. In general, NMR exper-iments involve collection of multiple scans with theS:N ratio improving as the square root of the num-ber of scans. This would suggest that the same S:Nratio could be obtained on a 750 MHz spectrometeras on a 500MHz instrument in about half the time.The real situation is, however, somewhat more com-plex and it is better to consider sensitivity, which is theS:N ratio achieved per unit time. This depends on theway the signal is digitized, the type of experiment andthe relaxation properties of the observed resonance.We have noted that, in G-CSF, the proton longitudinalrelaxation times of backbone amide protons increaseby about 40% and the linewidths of 15N resonancesincrease by about 15% on going from 500MHz to750 MHz. While such field dependencies could be anadvantage for analyzing protein dynamics [7] it couldalso decrease the sensitivity attainable at the higherfield. We are still carrying out a full evaluation of thevarious factors, such as relaxation, that affect sensitiv-ity on our 750 MHz instrument; some experiments on

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NMR at very high fields Boyd, Soffe and Campbell 255

proteins indicate that sensitivity may not improve verymuch on going from 500 MHz to 750 MHz. There is,however, little doubt that the information content ofthese NMR spectra (which depends both on sensitivityand resolution) is significantly higher at 750MHz.

What are the disadvantages?One obvious disadvantage is cost. Commercial 750MHzinstruments are currently being quoted at pricesaround £2 000 000 while a 500 MHz spectrometer costsaround 500 000. A special room with a high ceiling(4.5 m) and, possibly, some stray field protection isalso required. These financial implications mean thata 750 MHz instrument is perhaps more likely to formpart of a national facility. As mentioned above, anotherpossible disadvantage is the field-dependence of someNMR relaxation properties; protein 1H resonances donot seem to broaden much with increasing field butthe time taken for thermal equilibrium to be re-estab-lished after a radiofrequency pulse (longitudinal relax-ation) increases. A third potential disadvantage is asso-ciated with the fields applied in addition to the mainstatic magnetic field. Modem heteronuclear NMR ex-periments on macromolecules are very sophisticatedand numerous radiofrequency pulses [5,8] and fieldgradients [9] are used to select appropriate NMR sig-nals. The demands on these various additional fieldsbecome greater with increasing static field strength.The radiofrequency fields, for example, can becomeinadequate to cover the entire spectrum of interestuniformly and the increased strength required to coverthe spectral range can cause significant heating of thesample.

In spite of the potential problems, we believe thatthe advantages of very high fields are significant. It isclear that 1994 will see the installation and operationof several 750MHz NMR spectrometers world-wide.Even higher field strengths will soon become techni-cally feasible and magnet manufacturers expect to be

able to produce magnetic fields equivalent to protonfrequencies of 900 MHz within the next five years. Italso seems likely that other aspects of NMR spectrom-eter technology, such as the development of new so-phisticated heteronuclear pulse sequences [5] will con-tinue to advance. These technical innovations togetherwith the 'brute force' application of very high fields willcontinue. It thus seems likely that the astonishing abilityof modem NMR to explore the structure and dynamicproperties of proteins in solution will carry on beingextended. This will be seen by many to justify the highfinancial cost of the new very high field instruments.

References1. Branden, C.-I. (1994). The new generation of synchrotron ma-

chines. Structure 2, 5-6.2. Wuthrich, K. (1989). Protein structure determination in solution

by nuclear magnetic resonance spectroscopy. Science 243, 45-50.3. De Rosier, DJ. (1993). Tum-of-the-century electron microscopy

Curr. BioL 3, 690-692.4. Wagner, G. (1993). Prospects for NMR of large proteins J

Biomolec. NMR 3, 375-385.5. Bax, A (1991). Experimental NMR techniques for studies of

biopolymers. Curr. Opin. Struct. Biol 1, 1030-1035.6. Werner, J.M., et al, & Campbell, I.D. (1994). Secondary structure

and backbone dynamics of human granulocyte colony-stimulatingfactor. Biochemistry, in press.

7. Kay, LE., Torchia, D.A. & Bax, A (1989). Backbone dynamicsof proteins as studied by 15N inverse detected heteronuclearNMR spectroscopy: an application to staphylococcal nuclease.Biochemistry 28, 8972-8979.

8. Grzesiek, S. & Bax, A. (1993). Amino-acid type determinationin the sequential assignment procedure of uniformly 13C/15N-enriched proteins. J Biomolec. NMR 3, 185-204.

9. Hurd, R.E. (1990). Gradient enhanced spectroscopy. J. MagnReson. 87, 422-428.

Jonathan Boyd, Nick Soffe and lain Campbell, Depart-ment of Biochemistry and Oxford Centre for MolecularScience, University of Oxford, South Parks Road, OxfordOXI 3QU, UK.