In the Classroom
NMR Spectroscopy and Its Value: A Primer Sudha Veeraraghavan Department of Biochemistry and Molecular Biology, University of Texas–Houston Medical School and the Graduate School of Biomedical Sciences, Houston, TX 77030;
[email protected] Since the first published proton magnetic resonance spectrum taken in solution (1), NMR spectroscopy has become the staple of chemists to characterize small molecules and in quality control. Undergraduate students in colleges with sufficient resources may even have the opportunity to analyze chemical compounds in upper-level analytical chemistry or organic chemistry courses. However, among first-year graduate students in biomedical sciences at this university, only about 15% of students in each of my classes (by show of hands) has been exposed to NMR spectroscopy or know of its value. Thus a core course in the graduate biomedical science curriculum exposes first-year graduate students to a variety of spectroscopic, biophysical, and structural biological approaches to investigate biologically interesting problems. In this context, I teach a primer on NMR spectroscopy. It includes two intense hours of introduction to NMR and covers terminology and basics as well as commercial, biochemical, pharmaceutical, and biophysical applications. The aims of these lectures are to familiarize the novice with the premise of NMR spectroscopy, provide a brief history, illustrate its strengths and weaknesses, and ease students into reading NMR-based research articles. While students who have never encountered NMR spectroscopy learn that it is one of the major modern methods, students who might have used NMR spectroscopy in undergraduate laboratories also benefit from learning about various applications. Over the last four years, I have modified this approach as described below to include a greater degree of student participation in terms of initiating them into reading NMR-based original research articles (not only reviews). Introduction to NMR To make the lectures memorable, I start with use of NMR in testing the integrity of expensive wines (2) and of aloe vera used in cosmetics and health care products (3). Following this, I briefly discuss magnets, magnetic field strengths, and nuclei as magnets. I then introduce concepts in NMR (List 1) including Bo, NMR-active nuclei, gyromagnetic ratio, the B1 field, Larmor frequency, continuous wave versus pulsed-field NMR, relaxation, free-induction decay, Fourier transformation, frequency domain spectra or lines, chemical shifts and units (Hz, ppm), line-widths, and coupling constants. Many of these topics are described in detail in several NMR books (4–10). Students are also referred to Chapters 1, 3, 4, and 5 of the online NMR textbook by Hornak (11) as it provides animations and the ability to move between related topics readily. Explanation of pulsed NMR is particularly enjoyable as described by Derome (4) or in the introduction to Ernst’s Nobel Prize lecture by Forsen (12). As introduction to the practical side, I also discuss components in the NMR spectrometer, including cut-away views to identify various key components, provide a hands-on view of an old probe to visualize the Helmholtz or Saddle coil, and introduce the concept of modern higher sensitivity cryo or cooled probes.
Furthermore, the narrations about accidental NMR quenches and their consequences, and description of our in-house NMR spectrometer that remained unquenched and bubbling while under water after tropical storm Allison (2001) elicits a variety of questions and promotes discussions about how an NMR spectrometer is built and how it works. NMR is a relatively insensitive technique, frequently requiring samples at high micromolar to millimolar concentrations. This is because the net change in the population of spins between different energy levels is small compared to total number of spins in each of these state (13). To put it in perspective, compare sensitivity of NMR to that of other methods such as fluorescence spectroscopy or spectropolarimetry (circular dichroism) that require low micromolar concentrations, or that of electrophoretic gel mobility shift assay that uses radioactivity, which allows detection of femtomolar concentrations of DNA. Students also become aware that NMR is a non-destructive spectroscopic method as opposed to, say, exposure to irradiation with X-rays, which may render samples non-reusable. By the end of the first lecture, students have learned that: (i) NMR is rooted in Bloch equations (students are not required to memorize or derive these) and signal size depends on Bo field strength (ii) Magnetization relaxes with time to generate free induction decay (FID) that can be transformed into a frequency-domain signal by Fourier transformation (7) (iii) Fine structure in NMR lines is due to coupling constants ( J) (iv) Relaxation of magnetization can be through lattice (solvent, T1) or other spins in the same or neighboring molecules (T2) (8)
List 1. NMR Terms
Nuclear spin quantum number (or simply, spin) Precession Resonance Field strength (Bo, B1) Continuous-wave (scan frequency) Pulsed NMR Free-induction decay (FID) Fourier transform (FT) One- and multi-dimensional NMR (1D, 2D, 3D, …) Homo- and hetero-nuclear (proton only vs multiple nuclei) Chemical shift (δ; in ppm of Bo field vs Hz) Coupling constant (J) Line-width (width at half-maximal height, in Hz) Relaxation time (T1, T2) Nuclear Overhauser effect (NOE) Residual dipolar coupling (RDC)
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 85 No. 4 April 2008 • Journal of Chemical Education
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In the Classroom Table 1. A Brief History of NMR Date
Individual(s)
Contribution
1930s, 1940s
O. Stern (34), I. I. Rabi (35)
Discovery of nuclear magnetic resonance in molecular beams
1946
F. Bloch, E. Purcell (36)
First NMR signal detected (Nobel Prize: 1952)
1949
N. F. Ramsey (Rabi’s student)
First successful chemical shift theory
W. D. Knight
Chemical shift discovered (in solids)
1949
E. L. Hahn, R. R. Ernst
Discovery of spin echos
1951
J. T. Arnold
Chemical shift discovered (in liquids)
1963
A. Saupe, G. Englert
NMR in partially oriented media, residual dipolar coupling
1966
R. R. Ernst (37), Anderson
Fourier transform in NMR (Nobel Prize: 1991)
1971
J. Janeer
2D NMR experiments proposed
1973
Mansfield
Space selection with field gradients proposed
1970s
N. Bloembergen
Non-linear optics and spectroscopy sets stage for MRI
1973
P. C. Lauterbur, P. Mansfield (38)
Application of MR scanning, pictorial map (Nobel Prize: 2003)
1971–1977
R. V. Damadian
Whole body imaging, NMR tissue relaxation
1980s
K. Wuthrich (39)
NMR spectra used to solve macromolecular structure (Nobel Prize: 2002)
Various groups
Modern multidimensional NMR methods to study structure and dynamics of macromolecules; Membrane protein NMR; Signal selection using pulsed-field gradients
Various groups
LC–NMR; Protein fold using residual dipolar coupling; Reduced-dimensionality NMR; Selective excitation using pulsed NMR
1990s-now
Although Jeneer envisioned the possibility of two-dimensional NMR in 1971 (14), multi-dimensional NMR was only realized in the 1980s (15). For courses in which a more detailed discussion of multi-dimensional NMR is possible, I recommend Jelinski’s article (16) on the topic as an opener. The first lecture ends with examples describing the use of relaxation measurements to examine water content in wood pulp (17) and concrete (18) and NMR spectroscopy in water quality management as examples of NMR utility to industry and the general public. I also present Table 1 and point out the long history of NMR spectroscopy and its recent advances in the biomolecular arena as segue to the next lecture. NMR in Biological Research There are numerous applications of NMR (List 2) and it is important to choose the applications most suited to the type of students being addressed. I choose to spend a greater quantity of time discussing just a few applications in biological sciences. This serves two purposes: (i) provide relevant information without being overwhelming and (ii) keeps students interested, which allows active participation. The second lecture is divided into three parts and demonstrates the value of concepts introduced in the first lecture. Macromolecular NMR Here the focus is on the study of proteins and peptides although it could just as easily be about DNA, RNA, or other materials. First, students are introduced briefly to sample requirements, such as molecular weight limits, solvents, aggregation status, and protein purity and stability (19). Students also learn about the four most common variables that can be used to improve resolution or to reduce or alter resonance overlaps, that is, sample and salt concentrations, temperature, and pH. 538
List 2. Some NMR Applications Studies on small molecules Analysis: organic synthesis, quality control (chemistry; pharmacology; food, cosmetics and health care industries; engineering) Studies of macromolecules Reaction mechanism: Is the reactive species charged? Is it protonated? (enzymology) Thermodynamics and kinetics: protein folding or unfolding, stability (biophysics/physical biochemistry) Structural biology Protein secondary structure: α-helices, β-strands, turns, random coil Three-dimensional structure(s): DNA, RNA, protein (structural biology) Quaternary structure: residual dipolar coupling (biology) Dynamics: single vs multiple conformations, conformational selection and activity (enzymology), local dynamics (ring flip, loops, DNA/RNA base stacking, H-bonding) (various) Molecular interactions Ligand binding sites and affinities (Kd) (biochemistry, chemistry, pharmacology) Metal binding (various) Medicine, drug discovery, research (diagnosis, disease prevention, public health) Drug discovery Magnetic resonance imaging (MRI) In situ studies (studies with cells or tissues) In vivo studies (whole organism or tissues within) Metabolomics (pharmacology, biochemistry, physiology; complements mRNA and protein profiles)
Students learn to assess information content of NMR spectra, that is, foldedness of proteins (dispersed resonances), small versus large size (for example, aggregations and large complexes have greater line-widths as opposed to peptides or small mono-
Journal of Chemical Education • Vol. 85 No. 4 April 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Classroom
meric proteins), and regions of resonances in NMR spectra of biomolecules (ref 7: Chapters 2 and 3 ) including random coil chemical shifts of amino acids (20). Proton NMR spectroscopy is also an excellent universal assay for studying many biochemical reactions or ligand binding activities, for example, (i) pH titration to measure the protonation states of histidine side chains during enzymatic reactions, (ii) solvent accessible surface area, and (iii) substrate-induced conformational changes (ref 21: Chapter 3). Three-Dimensional Structures by NMR NMR is a well-established method for determining detailed, atomic resolution, three-dimensional structures (22, 23). There are several excellent reviews and books that describe modern NMR methods used to determine the three-dimensional structures of peptides and proteins (24, 25). Given the limited time for the lecture, rather than dwell on the intricacies of the methods, I provide a simplistic overview of structure determination by NMR: (a) Resonance overlaps in one-dimensional 1H-NMR make it difficult to assign all peaks to their corresponding nuclei. Therefore, it is necessary to spread the peaks into additional dimensions. This is possible using uniformly, partially, or selectively 15N-, 13C-, or 2H-labeled samples and multidimensional heteronuclear experiments. (b) Heteronuclear correlation data (for example, 1H,15N-HSQC) provide a unique fingerprint for each protein since the resonance of each proton in the molecule is influenced by its overall structure, tumbling times, and the microenvironment surrounding a given nucleus. (c) Triple-resonance strategies are frequently used in sequencespecific resonance assignments. Furthermore, protein secondary structure can be determined just from chemical shift data (26). (d) Transfer of these assignments to “through-space” contacts observed in nuclear Overhauser effect spectra (NOESY). (e) Orientational information for bond vectors from residual dipolar couplings. (f ) Structure calculations using NOE-derived internuclear distances, dihedral angles, chemical shifts, and/or residual dipolar couplings. Any one of a large number of original articles that use NMR to solve protein or peptide structure can be used to illustrate these concepts. I have used our own recent work describing the first three-dimensional structure of the DNA-binding domain of transcription enhancer factors (27). In doing so, I point out that analysis of the electrostatic charge surface provided us with clues about the DNA-recognition helix, which we then confirmed using mutational analysis. It helps students realize how new models can be generated through such structural analyses, starting with models freely available at the Protein Data Bank (28), which is a repository for three-dimensional structures of proteins and peptides. It is also worth noting that recent advances in NMR, such as segmental labeling, TROSY, enhanced-sensitivity pulse programs, and improved probe design, permit investigations of even very large molecules (~700 kD) (25, 29, 30). In expanded NMR courses, it will be important to also include various aspects of protein dynamics; a recent report indicates that pre-existing dynamics in proteins may be critical to its function (31). NMR in Drug Discovery and Design Traditionally, NMR has been used to identify targets by screening for compounds that bind proteins of interest. In the recent years, structure-based drug design has come of age. When
the structure of a protein is known and its sequence-specific assignments available, it is possible to map the binding sites of various compounds on the protein surface. Such information is used to design new targets or drugs. It may also reduce the amount of random screening of naturally occurring compounds. I use the Jahnke and Widmer article (32) for this section as it covers a range of relevant topics with good examples. A high-throughput method developed at Abbott Laboratories under Stephen Fesik is the SAR (structure–activity relationship)-by-NMR approach (31). In this method, thousands of compounds are screened in a semi-automated manner and then the compounds that are able to bind the protein are identified, their binding sites mapped, and chemistries of different compounds binding at adjacent sites on the protein combined to make better drug targets. Another approach is the SHAPES strategy developed at Vertex Pharmaceuticals by Jonathan Moore and colleagues (33) that uses a library of compounds with different shapes, and elaboration of the shapes is used to design better binders or inhibitors. Reporter screening, which establishes relative binding affinities, and transferred-NOE experiments for mapping binding sites of weakly associated complexes (with millimolar dissociation constants) are also covered. The usefulness of combining multiple approaches in structure-based drug design and discovery is portrayed by Jahnke and Widmer via examples of ligands that bind phosphotyrosine phosphatase-1B and papillomaviral E2 proteins. The latter is especially interesting given the now controversial introduction of immunization against human papillomaviruses in Texas. Reinforcement of Concepts and the Beginnings of NMR Self-Education At the end of the two lectures, students receive a homework problem set with instructions to work independently. Here is a typical set of questions: (i) compute resonance frequencies of proton, carbon, and nitrogen at multiple Bo field strengths (requires knowledge of gyromagnetic ratios and mathematical relationship between Bo and gyromagnetic ratio). (ii) If 300 MHz and 800 MHz NMR spectrometers were available, what magnetic field strengths would be better for higher resolution spectra and why? (iii) If a regular triple-axis gradient probe and a higher sensitivity cryogenic probe were available, which should one choose to collect two-dimensional 1H,15N-HSQC spectrum of a sample at low concentration of 0.1 mM? (iv) Visit the Protein Data Bank and list how many three-dimensional structures have been determined using NMR-spectroscopy compared to all known three-dimensional structures. (v) Read an original article (not a review article) that used NMR spectroscopy to investigate protein structure–function or drug discovery that was published within the last 4 months. Some examples of journals are Journal of Biological Chemistry, Biochemistry, Proceedings of the National Academy of Sciences, or Protein Science. How was NMR used in the investigation? Summarize the results and the role of NMR spectroscopy with regard to the authors’ findings, in your own words. Email a portable document file (PDF) of the article along with the answers to the instructor. With regard to the last question, I find that about 90% of the students choose a completely different article from those chosen by their classmates. Perhaps it is an indication that each student is drawn to a different article based on their own research interests and level of understanding of the subject matter.
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 85 No. 4 April 2008 • Journal of Chemical Education
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In the Classroom
Concluding Remarks It is possible to introduce basic concepts and utility of NMR spectroscopy to a complete novice within a short span of just 2.5 contact hours. More importantly, the lectures and associated homework can eliminate unwarranted fear of the high-end technology. I emphasize to my students that although many of them may never actually use an NMR spectrometer, it is important they realize its potential and be familiar enough to converse with other researchers and possibly to establish fruitful collaborations in their future research careers. Literature Cited 1. Arnold, J. T.; Dharmatti, S. S.; Packard, M. E. J. Chem. Phys. 1951, 19, 507. 2. Weekley, A. J.; Bruins, P.; Sisto, M.; Augustine, M. P. J. Magn. Reson. 2003, 161, 91–98. 3. Diehl, S.; Teichmuller, E. E. Agro. Food Industry Hi-Tech 1998, 9, 14–16. 4. Derome, A. E. Modern NMR Techniques for Chemistry Research, 1st ed.; Pergamon Press: Oxford, 1988. 5. Friebolin, H. Basic One- and Two-Dimensional NMR Spectroscopy, 3rd ed.; Wiley-VCH: New York, 1998. 6. Wuthrich, K. NMR in Biological Research: Peptides and Proteins; North-Holland Publishing Company: Amsterdam, 1976. 7. Wuthrich, K. NMR of Proteins and Nucleic Acids, 1st ed.; John Wiley & Sons, Inc.: New York, 1986. 8. Claridge, T. D. W. High-Resolution NMR Techniques in Organic Chemistry, 1st ed.; Elsevier: Oxford, 2004. 9. Sanders, J. K. M.; Hunter, B. K. Modern NMR Spectroscopy: A Guide for Chemists; Oxford University Press: New York, 1993. 10. Macomber, R. S. NMR Spectroscopy. Essential Theory and Practice, 1st ed.; Harcourt Brace Javanovich: San Diego, 1988. 11. Hornak, J. P. The Basics of NMR [Online]; 1997–1999. http:// www.cis.rit.edu/htbooks/nmr/bnmr.htm (accessed Jan 2008). 12. Ernst, M. K.; Dunn, L. L.; Rice, N. R. Mol. Cell. Biol. 1995, 15, 872–882. 13. James, T. L. In Biophysics Textbook Online. 1998. http://www. biophysics.org/education/james.pdf (accessed Jan 2008). 14. Jeneer, J. In Ampere International Summer School; Basko Polje: Yugoslavia, 1971. 15. Wagner, G. Magnetic Resonance in Chemistry 2003, 41, S3–S15. 16. Jelinski, L. W. Chem. Eng. News 1984, 62 (Nov 5), 26–47. 17. Barale, P. J.; Fong, C. G.; Green, M. A.; Luft, P. A.; McInturff, A. D.; Reimer, J. A.; Yahnke, M. IEEE Transactions on Applied Superconductivity 2002, 12, 975–978. 18. Kohl, F.; Wolter, B.; Knapp, F. In Non-Destructive Testing in Civil Engineering; Wiggenhauser, H., Ed.; Fraunhofer Publica: Berlin, 2003. 19. Primrose, W. U. Sample Preparation; IRL Press: Oxford, 1993. 20. Schwarzinger, S.; Kroon, G. J.; Foss, T. R.; Wright, P. E.; Dyson, H. J. J. Biomol. NMR 2000, 18, 43–48. http://www.bmrb.wisc.
edu/ref_info/pentapeptide.tbl (accessed Jan 2008).
21. Hausser, K. H.; Kalbitzer, H. R. NMR in Medicine and Biology; Springer-Verlag: Berlin, 1991.
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22. MacArthur, M. W.; Driscoll, P. C.; Thornton, J. M. Trends Biotechnol 1994, 12, 149–153. 23. Machius, M. Curr. Opin. Nephrol. Hypertens. 2003, 12, 431–438. 24. Cavanagh, J.; Fairbrother, W. J.; Palmer, A. G., III; Skelton, N. J. Protein NMR Spectroscopy. Principles and Practice, 1st ed.; Academic Press: San Diego, 1996. 25. Biological Magnetic Resonance; Rama Krishna, N., Berliner, L. J., Eds.; Kluwer Academic/Plenum Publishers: New York, 2003; Vol. 20. 26. Use of Chemical Shifts in Macromolecular Structure Determination; Wishart, D. S., Case, D. A., Eds.; Academic Press: San Diego, 2001; Vol. 338. 27. Anbanandam, A.; Albarado, D. C.; Nguyen, C. T.; Halder, G.; Gao, X.; Veeraraghavan, S. Proc. Natl. Acad. Sci. USA 2006, 103, 17225–17230. 28. Protein Data Bank Home Page. http//:www.rcsb.org (accessed
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29. Wider, G. Biotechniques 2000, 29, 1278–1282, 1284–1290, 1292 (passim). 30. Tugarinov, V.; Sprangers, R.; Kay, L. E. J. Am. Chem. Soc. 2007, 129, 1743–1750. 31. Eisenmesser, E. Z.; Millet, O.; Labeikovsky, W.; Korzhnev, D. M.; Wolf-Watz, M.; Bosco, D. A.; Skalicky, J. J.; Kay, L. E.; Kern, D. Nature 2005, 438, 117–121. 32. Jahnke, W.; Widmer, H. Cell. Mol. Life. Sci. 2004, 61, 580– 599. 33. Fejzo, J.; Lepre, C. A.; Peng, J. W.; Bemis, G. W.; Ajay; Murcko, M. A.; Moore, J. M. Chem. Biol. 1999, 6, 755–769. 34. The Nobel Prize in Physics 1943: Otto Stern. http://nobelprize. org/nobel_prizes/physics/laureates/1943/index.html (accessed Jan 2008). 35. The Nobel Prize in Physics 1944: Isidor Isaac Rabi. http://nobelprize.org/nobel_prizes/physics/laureates/1944/index.html (accessed Jan 2008). 36. The Nobel Prize in Physics 1952: Felix Bloch and Edward Mills Purcell. http://nobelprize.org/nobel_prizes/physics/laureates/1952/ index.html (accessed Jan 2008). 37. The Nobel Prize in Chemistry 1991: Richard R. Ernst. http:// nobelprize.org/nobel_prizes/chemistry/laureates/1991/index.html (accessed Jan 2008). 38. The Nobel Prize in Physiology or Medicine 2003: Paul C. Lauterbur and Sir Peter Mansfield. http://nobelprize.org/nobel_prizes/ medicine/laureates/2003/ (accessed Jan 2008). 39. The Nobel Prize in Chemistry 2002: Kurt Wüthrich. http:// nobelprize.org/nobel_prizes/chemistry/laureates/2002/index.html (accessed Jan 2008).
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