Modern NMR in Undergraduate Education ... - ACS Publications

Chapter 1

Modern NMR in Undergraduate Education: Introduction

Downloaded by KINGSTON UNIV on February 8, 2015 | http://pubs.acs.org Publication Date: August 16, 2007 | doi: 10.1021/bk-2007-0969.ch001

Gerhard Wagner Elkan R. Blout Professor, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115

The rapid increase of knowledge has made most scientists specialists of a small area of expertise, and a comprehensive knowledge of all sciences is impossible today. In contrast, some of the greatest thinkers of past centuries, such as Newton, Leibnitz or Goethe had a near universal knowledge and engaged in a wide spectrum of intellectual activities, often ranging from mathematics, physics, chemistry, astronomy, biology or geology to philosophy and politics. Indeed, Gottfried Wilhelm Leibnitz (1646-1716), the inventor of differentials, was called a "Walking Encyclopedia" by his employer George Luis of Hanover who later became King George I of England. In an earlier unsuccessful political mission as an envoy of the Elector of Mainz he tried to convince the king Louis XIV of France to give up attacking the Alsace and pursue the conquest of Egypt instead. Scientists are hardly politically active any more to the better or worse, and fortunately we have now easy access to encyclopedic knowledge through the internet. However, most scientists miss the joy to be active in a wide spectrum of sciences. Rare exceptions are those of us who are engaged in nuclear magnetic resonance spectroscopy, which draws its powerfroma wide range of fields in science and mathematics. It is one of the beauties of NMR that it provides an intellectual playing field entertaining physicists, chemists, mathematicians, electrical and mechanical engineers, biochemists, biologists and physicians. A single individual may be active in one particular of these areas or cover a wide spectrum. Using NMR skillfully requires basic physical understanding. Those who like it can dig into quantum mechanics, density matrix theory, spin dynamics, average Hamiltonian

© 2007 American Chemical Society

In Modern NMR Spectroscopy in Education; Rovnyak, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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2 theory, or advanced data processing. On the other hand, a biologist may prefer to solve a beautiful protein structure without needing to understand the physical basis of the method in much detail, and the results obtained can be of high significance for elucidating biological processes or designing drugs for fighting human disease. I did not know much about the potential of NMR and the dramatic development of this technology was hardly foreseeable when I became interested in this method. I was a physics undergraduate student at the Technical University of Munich when I became attracted by resonance phenomena. However, when I considered pursuing NMR spectroscopy for a Ph.D. project I was asked: "Why do you want to do NMR? Everything has been done in NMR already." By then indeed, NMR was established as an analytical tool to characterize and confirm the chemical structures of small molecules. Much was known about the features that define an NMR spectrum, such as chemical shifts and scalar couplings [1], spin decoupling, the nuclear Overhauser effect, and relaxation phenomena in general [2]. Also most basic features of solid-state NMR spectra were known [3]. Thus, my supervisors discouraged me to enter this field. However, I had seen some H NMR spectra of the protein hemoglobin and was startled by the complexity and plethora of signals found in the spectra. I thought that there must be a lot of wonderful information hidden in these signals. I imagined it could be exciting to go treasure hunting, and I decided to pursue a career in NMR spectroscopy. The development of NMR in the 1970s and 1980s was to a large extent driven by spectroscopy with proteins and to some degree with nucleic acids. Protein NMR spectra were complex and challenging, and demanded new technologies. It was obvious that there was a lot of information in protein NMR spectra, in particular when magnets of higherfieldsbecame available, resulting in a better dispersion of the resonances. However, it was entirely unclear what the numerous spectral features would eventually reveal. There was hope that structural information could be obtainedfromanalyzing chemical shifts since the basic principles responsible for the variation of resonance positions were known, such as ring-current shifts, effects of electronegativity and hydrogen bonding. However, this turned out to be inadequate for deriving solid structural information. Scalar coupling constants were recognized early on as a valuable source of structural information and much effort went into deriving quantitative relations with dihedral angles.[4] Indeed such information was found sufficient for determining structures of small cyclic peptides [5] but was inadequate for obtaining structural information for proteins. The single most important obstacle preventing access to the treasures of protein NMR spectra was the lack of technologies to assign the NMR resonances to individual nuclei of a protein. The most powerful assignment technology available early on was spin decoupling, which could identify pairs or !



3 spins connected by scalar coupling. However, locating these spin pairs in a protein was impossible and additional methods had to be explored. Initially, assignments were only attempted for proteins with known crystal structures. Chemical modifications were introduced to create binding sites for paramagnetic lanthanides, and the effects on protein resonances were used to obtain early assignments of a few resonances [6]. The breakthrough with protein assignments came with the proper use of the nuclear Overhauser effect (NOE) in combination with spin decoupling. It had been known for a long time that saturation of individual spins caused changes of resonance intensities of nearby spins. Originally, in these experiments a particular resonance was irradiated for several seconds, and many resonance were found to experience intensity changes due to direct NOEs and multi-step spin diffusion. Thus, scientists were afraid that NOEs would only be marginally useful for measuring intra-protein distances accurately enough to have an impact for assignments and structure elucidation [7, 8]. However, this fear was proven unsubstantiated when it was shown that NOE experiments with short irradiation times could be quite selective and yield quantitative distance information [9]. Using this approach combined with spin decoupling allowed thefirstsequencespecific assignment of a large part of a small protein in simple ID NMR experiments [10]. However the real break-through came only with the development of 2D and multi-dimensional NMR, which was developed by physical chemists [11, 12]. An important aspect of the development of NMR was the possibility to play with spins. The increasing sophistication of NMR spectrometers allowed physically oriented scientists to manipulate ensembles of nuclear spins and follow the response on the computer screen. Thus, NMR was essentially "handson quantum mechanics". As a consequence of such efforts, two-dimensional NMR was invented, which was extended to multi-dimensional NMR experiments. Initially, 2D NMR methods were all homonuclear and allowed solving protein structures of up to 10 or 15 kDa. Crucial for a further development was the ability to label proteins and nucleic acids with the stable isotopes N and C. This and more elaborate labeling methods, together with increasingly sophisticated experiments made possible determination of protein and nucleic acid structures in the molecular weight range up to 50 kDa and beyond. Despite many spectacular success stories of NMR spectroscopy one has to think where this technique can provide unique and most valuable contributions. NMR can solve protein structures but what is the impact when we have X-ray crystallography as a structural method that is probably faster and less limited by molecular size? Clearly there are many proteins that do not crystallize and often their structures can be solved with NMR. Getting a structure is clearly faster than not getting it at all. However, both NMR and crystallography are quite complementary. As an example of mutual benefit, we were recently approached by a local 15

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4 crystallographer asking whether we could have a look at a protein that is found at the tip of a rotavirus spike. They had tried to crystallize the protein for a year without success. Placed in the magnet it exhibited excellent NMR spectra, and the structure could be solved. Inspecting the molecular model revealed that there wereflexibletails that might have prevented crystallization. More importantly, the structure had similarity to a fold found in sialic acid-binding proteins. Realizing this, a simple sialic acid was added, and the flexible tails were trimmed. This version of the protein immediately crystallized, and the costructure could quickly be solved with X-ray methods.[13] Probably the most valuable and unique power of NMR lies in detecting and characterizing protein interactions. In contrast to crystallography, where crystal contacts may be erroneously interpreted as binding interfaces, NMR only sees true and physiological contacts.[14] Even very weak interactions with equilibrium dissociation constants up to 10 mM can be seen as small chemical shift changes. NMR is the most reliable and only technique to directly identify and characterize such weak interactions. This is usually pursued by observing changes in 2D *Η- Ν or H- C correlated spectra upon titration with a putative ligand. NMR is a "litmus test" for proving protein interactions that may have been suggested based on other biochemical techniques, and often such claimed interactions turn out to be not existent. Often protein interactions are weak and transient to allow for rearrangement of components during the assembly and alteration of mega-complexes that may accomplish different functions during a well-defined time course of events. Examples are the assembly of the pre-initiation complex of eukaryotic translation initiation where numerous factors join and leave the 40S subunit to ready the ribosome for protein synthesis. Many of these factors or their domains are small enough to be analyzable by NMR and multiple interfaces can be identified with a variety of NMR mapping experiments.[15] This can reveal topologies of factor association in dynamic assemblies of mega complexes. Another powerful aspect of NMR is its ability to characterize weak interactions with small chemicals. Small-molecule libraries have become increasingly available to academic laboratories and can be exploited for probing cellular processes. Inhibitors of protein-protein interactions can be found with simple high-throughput screening methods, such as using fluorescencepolarization. [16, 17] However, the inhibitors that can be found are typically rather weak, in the low μΜ range. Thus, the complexes with target proteins may not be suitable for crystal-structure analysis but can be characterized with NMR methods. Designing potent inhibitors of protein interactions could become a powerful approach for developing novel molecules of therapeutic value. The information about protein complexes with small molecules obtainable with NMR is suitable for a rational improvement of the affinities of these inhibitors. This is afieldfor fruitful collaborations between structural biologists, chemists and medicinal chemists and has the potential of contributing new means forfightinghuman disease. 15

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5 A rather new and promising application of NMR is in the field of metabolomics, which tries to characterize the state of whole cells, organs or even whole living beings based on the entirety of all metabolites, the metabolome.[18] While this is most efficiently pursued with mass spectroscopy, NMR is complementary and can provide quantitative measurements of metabolite levels from ID and 2D spectra, and it can be employed to identify the chemical structures of so far unknown metabolites. Usually two classes of samples are compared, such as from sick and healthy individuals, or from animals with and without a drug treatment, orfromcells with and without a mutant protein. Considering the large number of metabolites found in body fluids or cell extracts, data are usually analyzed with multi-variate statistical tools, such as principle component analysis (PCA) or partial least squares discriminant analysis (PLSDA). This reveals the molecules for which concentrations are most different between the two groups of samples. Mass spectroscopy and NMR can then identify these molecules, which might be biomarkers of a disease. This approach has the potential of elucidating disease pathways and even identifying new drug targets. To obtain valid results it is important to use ultimate care in sample preparation and statistical analysis. NMR has made dramatic and unexpected advances in a large range of areas. For quite a while its uses in chemistry and structural biology were separate. But recently the two fields are crossing over with the rising interest in chemical biology and metabolomics. Technical advances have been astounding, with the development of ultra high field magnets that are now available at 22.3 (950 MHz) and will soon be at 23.488 Ts (1 GHz) or above. However, most projects can be carried out at lower fields, such as at 400, 500 and 600 MHz spectrometers. Cryogenically-cooled probes have boosted the sensitivity of spectrometers up to five fold, and the sophistication of pulse sequences, data acquisition and processing is booming. NMR is afieldwith a wide spectrum of applications and certainly will continue to come up with unexpected and fascinating new achievements.

References 1. Gutowsky, H.S., McCall, D.W., and Slichter, CP., Nuclear Magnetic Resonance Multiplets in Liquids. The Journal of Chemical Physics, 1953. 21(2): p. 279-292. 2. Solomon, I., Relaxation processes in a system of two spins. Phys. Rev., 1955. 99: p. 559-565. 3. Abragam, Α., The Principles of Nuclear Magnetism. The International series of Monographs on Physics, ed. W.C. Marshall and D.H. Wilkinson. 1961, London: Oxford University Press. 4. Bystrov, V.F., Spin-spin coupling and the conformational states of peptide systems. Progr. NMR Spectrosc., 1976. 10: p. 41-81.


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Meraldi, J.P., Schwyzer, R., Tun-Kyi, Α., and Wuthrich, K., Conformational studies of cyclic pentapeptides by proton magnetic resonance spectroscopy. Helv Chim Acta, 1972. 55(6): p. 1962-1973. Marinetti, T.D., Snyder, G.H., and Sykes, B.D., Nitrotyrosine chelation of nuclear magnetic resonance shift probes in proteins: application to bovine pancreatic trypsin inhibitor. Biochemistry, 1977. 16(4): p. 647-653. Kalk, A. and Berendsen, H.J.C., Proton magnetic relaxation and spin diffusion in proteins. J. Magn. Resonance, 1976. 24: p. 346-366. Sykes, B.D., Hull, W.E., and Snyder, G.H., Experimental evidence for the role of cross-relaxation in proton nuclear magnetic resonance spin lattice relaxation time measurements in proteins. Biophys J, 1978. 21(2): p. 137146. Wagner, G. and Wüthrich, K., Truncated driven nuclear overhauser effect (TOE). A new technique for studies of selective H- H Overhauser effects in the presence of spin diffusion. J. Magn. Reson., 1979. 33: p. 675-680. Dubs, Α., Wagner, G., and Wuthrich, K., Individual assignments of amide proton resonances in the proton NMR spectrum of the basic pancreatic trypsin inhibitor. Biochim Biophys Acta, 1979. 577(1): p. 177-194. Jeener, J. in Ampére Interational Summer School. 1971. Basko Polje, Yugoslavia. Aue, W.P., Bartholdi, E., and Ernst, R.R., Two-dimensional Spectroscopy. Application to NMR. J. Chem. Phys., 1976. 64: p. 2229-2246. Dormitzer, P.R., Sun, Z.Y., Wagner, G., and Harrison, S.C., The rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site. Embo J, 2002. 21(5): p. 885-897. Ferentz, Α., Opperman, T., Walker, G., and Wagner, G., Dimerization of the UmuD' protein in solution and its implications for regulation of SOS mutagenesis. Nature Structural Biology, 1997. 4(12): p. 979-983. Marintchev, A. and Wagner, G., Translation initiation: structures, mechanisms and evolution. Q Rev Biophys, 2004. 37(3-4): p. 197-284. Lugovskoy, Α.Α., Degterev, A.I., Fahmy, A.F., Zhou, P., Gross, J.D., Yuan, J., and Wagner, G., A novel approach for characterizing protein ligand complexes: molecular basis for specificity of small-molecule Bcl-2 inhibitors. J Am Chem Soc, 2002. 124(7): p. 1234-1240. Moerke, N.J., Aktas, H., Chen, H., Cantel, S., Reibarkh, M., Fahmy, Α., Gross, J.D., Degterev, Α., Yuan, J., Chorev, M., Halperin, J.A., and Wagner, G., Small Molecule Inhibition of the Interaction Between the Translation Initiation Factors eIF4E and eIF4G. Cell, 2007: p. in press. Nicholson, J.K., Connelly, J., Lindon, J.C., and Holmes, E., Metabonomics: a platform for studying drug toxicity and gene function. Nat Rev Drug Discov, 2002. 1(2): p. 153-161. 1

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Modern NMR in Undergraduate Education ... - ACS Publications

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