VOLUME 26 NUMBER 4 APRIL 1993 Registered in US.Patent and Trademark Office;Copyright 1993 by the American Chemical Society Methodological Advances in Protein NMR AD BAX*AND STEPHANGRZESIEK* Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 Receiued August 13, I992 Since the first experimental observation of nuclear peptides. Nearly a decade ago, protein structure magnetic resonance (NMR) in bulk matter more than determination was added to the realm of applications 45 years ago,lI2 its history has been punctuated by a by the introduction of new systematic procedures for series of revolutionary advances that have greatly spectral analysis, primarily developed by Wiithrich and expanded its horizons. Indeed, methodological and co-~orkers.~During the 19809, development of new instrumental developments witnessed over the past two experimental pulse schemes continued to increase the decades have turned NMR into the most diverse power and applicability of 2D NMR to structural spectroscopic tool currently available. Applications characterization of biopolymers. The most important vary from exploration of natural resources3and medical development was undoubtedly the addition of a third imaging to determination of the three-dimensional frequency dimension to the NMR spectra.14J5 The structure of biologically important macromolecules.Pg concept of 3D NMR is so similar to 2D NMR that no The present Account focuses primarily on the meth- new formalism for the description of such experiments odological advances in this latter application, partic- is required. The main problem that had to be solved ularly as they relate to the study of proteins in solution. for the development of such techniques was a way to After Ernst and Anderson developed Fourier trans- record and process the enormous data matrices asso- form NMR" and demonstrated its use, the introduction ciated with such experiments. The second problem, as of a second frequency dimension in NMR spectroscopy will be discussed later, was that sensitivity of the 3D by Jeener in 197112 provided a critical trigger to the experiments frequently is much lower than for anal- development of this field. An enormous variety of ogous 2D experiments unless the third dimension experimental schemes,all based on the two-dimensional corresponds to the chemical shift of a 13C or 15Nnucleus (2D) concept and largely developed by the group of and isotopic enrichment is used.16J7 The advances in Ernst,l3 expanded the applicability of NMR to the genetic engineering techniques that have occurred in characterization of quite complex molecules, including the last decade enable many proteins to be overproduced natural products, sugars, synthetic polymers, and (1)Bloch, F.;Hansen, W. W.; Packard, M. Phys. Rev. 1946,69,127. Ad Bax was born in the Netherlands. In 1981, he received hls Ph.D In (2)Purcell, E. M.; Torrey, H. C.; Pound, R. V. Phys. Reu. 1946, 69, applied physics from the Deltt University of Technology after conducting most 37-38. of his graduate researchwith Ray Freeman In the Physical Chemlstry Laboratwy (3)Jackson, J. A. Log Analyst 1984, 16-30. at Oxford Unhrersily, working on the development of two-dimensional NMR (4)Wiithrich, K. NMR of Proteins and Nucleic Acids; Wiley: New methods. Following a postdoctoral appointment at Colorado State University, York, 1986. working in solkl-state NMR, he Joined the National Insthutes of Health. where (5)Clore, G. M.; Gronenborn, A. M. Crit. Rev. Biochem. Mol. Biol. he presently is Chief of the Section on Biophysical NMR Spectroscopy. His 1989,24,479-564. work focuses on the development of improved NMR methods for the (6)Kessler, H.;Gehrke, M.; Griesinger, C. Angew. Chem., Int. Ed. characterlzation of the structure and dynamics of biological macromolecules. Engl. 1988,27,490-536. Stephan OIzesiek was born In West Germany and received his Ph.D in (7)Bax, A. Annu. Reo. Biochem. 1989,58, 223-256. physics from the Free Universily of Berlin in 1988 for optical studles of proton (8) Wiithrich, K. Acc. Chem. Res. 1989,22,36-44. release in bacteriomodopsin. After a venture in a small software company, (9)Kaptein, R.; Boelens, R.; Scheek, R.; van Gunsteren, W. F. he receiveda fellowship from the Roche Research Foundation for NMR studies Biochemistry 1988,27,5389-5395. of pharmaceutically relevant blomolecules. Part of his fellowship he spent at (10)Clore, G. M.; Gronenborn, A. M. Science, 1991,252, 1390-1399. the NIH, where he presently is employed as a Vishlng Associate in the Section (11)Ernst, R. R. Adv. Map. Reson. 1966,2,1-137. on Biophysical NMR Spectroscopy. (12)Jeener, J. Ampere Summer School,BaskoPolje, Yugoslavia, 1971. This article not subject to U.S. Copyright. Published 1993 by the American Chemical Society 132 Acc. Chem. Res., Vol. 26, No. 4, 1993 Bax and Grzesiek 90 90 and labeled in microorganisms with the NMR observ- n n n able stable isotopes. With isotopic enrichment, sen- sitivity of many of the heteronuclear 3D experiments is sufficiently high to add yet another frequency dimension to the NMR spectrum, dispersing resonance frequencies in four orthogonal dimensions.1a20 In this Account, we will discuss the advantages and problems associated with extending the dimensionality of the NMR spectrum and will attempt to provide an answer to the question, How many dimensions do we really need? I. Principles of Multidimensional NMR Although the principles of 2D NMR have been reviewed many times, we will briefly reiterate some of these in order to clarify the basis of 3D and 4D NMR. Most of the useful nD NMR experiments are of the t2 so-called ucorrelated"type, in which the chemical shift 15N I DECCWLE I DECOUPLE of a nucleus is correlated with the chemical shifts of Figure 1. Examples of timing diagrams of 2D and 3D NMR other nuclei based on an interaction between them. pulsesequences. (a)2D NOESY experiment. (b) 20'H-detected For example, in the important 2D NOESY experiment, 1H-15N HSQC correlation experiment. (c) 3D pulse scheme for protons are correlated on the basis of the dipole-dipole 3D 15N-separated NOESY-HSQC experiment, obtained by coupling between their magnetic moments, giving rise concatenating schemes a and b. Radiofrequency pulses are marked by vertical bars and have typical durations of tens of to magnetization transfer via the nuclear Overhauser microseconds. Signal is acquired during the time tz (schemes a effect (NOE). The pulse scheme used for the NOESY and b) and tS(c); each scheme is repeated many times while the experiment is sketched in Figure la. In this scheme, duration of tl (and tz, for scheme c) is systematically incremented three RF pulses are applied to the proton spins, and from 0 to -30 ms. the scheme is repeated many times for systematically incremented durations of the time tl. The signals molecular tumbling rate and therefore increases ap- detected during the time tz are modulated by the proximately linearly with the size of the protein. For frequencies present during the time tl, and a two- larger proteins 'H-lH Jcouplings are frequently smaller dimensional Fourier transformation of the acquired than the line width, making the COSY experiment data matrix results in the 2D NMR spectrum. ineffective. Other J correlation techniques, such as In the NOESY spectrum, correlations between a the one depicted in Figure lb, can correlate the resonance of proton A and proton B will be observed frequency of a proton with that of its directly attached if A and B are sufficiently close in space (less than -5 heteroatom (13C or I5N). The heteronuclear one-bond A). However, before the distance information in the couplings, ~JCH(125-160 Hz) and 'JNH(-92 Hz), are NOESY spectrum can be fully interpreted, it is nec- much larger than 3JHH, and frequently as much as 50- essary to assign each of the resonances in the lH NMR 90% of the magnetization can be transferred from spectrum to ita site in the chemical structure. To protons to their directly coupled heteronuclei.16 Con- accomplish this it is also necessary to record so-called sequently, such 2D heteronuclear shift correlation J-correlated experiments, in which magnetization is techniques are highly sensitive and can often be carried transferred between chemically bonded nuclei via the out even without isotopic enrichment. J-coupling mechanism. The oldest 2D NMR pulse The concept of 2D NMR is easily extended to higher scheme, in which magnetization is transferred from one dimensionality. For example, the two pulse schemes proton to another via 'H-lH J coupling, is known as of Figure la,b can be concatenated in a manner depicted the COSY experiment and is probably the most popular in Figure IC,yielding a 3D experiment. The signals, experiment in the NMR analysis of small molecules. acquired during the time t3, are now obtained for many This experiment requires that JHHbe not much smaller different tl and tz durations. As was the case in the 2D than the 'H resonance line width. This line width is NOESY experiment, the data are modulated in the tl approximately proportional to the inverse of the dimension by the frequencies of other nearby protons; however, in the t~dimension the modulation frequency (13)Ernst, R. R.;Bodenhausen, G.; Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions; Clarendon Press: is that of the 15N nucleus that is directly attached to Oxford, 1987. the observed proton. Consequently, a 3D Fourier (14)Vuister, G. W.; Boelens, R.; Kaptein, R. J. Magn. Reson. 1988,80, transformation (with respect to the time variables tl, 176-185. (15)Oschkinat,H.; Griesinger,C.;Kraulis, P. J.;Ssrensen, 0. W.;Ernst, tz, and t3) yields a 3D frequency domain NMR spectrum.