In the Laboratory
Proton NMR Studies of the Conformation of an Octapeptide
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An NMR Exercise for Biophysical Chemistry Anne M. Rehart and J. T. Gerig* Department of Chemistry, University of California, Santa Barbara, Santa Barbara, CA 93106; *
[email protected] Nuclear magnetic resonance (NMR) spectroscopy has become an indispensable tool for finding the three-dimensional structures of peptides, small proteins and other biological molecules (1, 2). In comparison with X-ray or other diffraction techniques, structure definition by NMR methods has the advantage that the molecule of interest is examined in solution and therefore is not subject to the possibly structure-distorting interactions present in the solid state. Further, since crystals are not required for an NMR experiment, structural information may be obtained for biochemical systems that are impossible to crystallize. Given the importance of NMR-based methods for structural biochemistry and biophysics, it is important that students beginning study of these fields develop a basic understanding of the nature and limitations of NMR methods contemporaneously with the instruction they receive in X-ray diffraction and other methods for obtaining structural information about biological systems. However, it seems to us impractical to expect students in beginning courses to carry out an experiment that involves collection of the NMR data that are needed. A rather high degree of experimental sophistication and computer literacy would be required to make such an experiment a successful learning experience, and the investment of student time required to achieve these at the needed level is not realistic. Perhaps most importantly, few departments can afford to make the required instrumentation available to beginning students. Our department desired to provide an introduction to NMR methods for structure elucidation in a new biochemistry laboratory course but recognized these limitations. Instead, an exercise based on genuine experimental data, obtained with a “real” sample, was developed.
The biological activities of angiotensin II and its analogs surely are related to the conformation of these peptides both in solution and as they are bound to their receptor sites (3). The conformation of [Sar1]angiotensin II in dimethylsulfoxide solution has been studied by proton NMR (4, 5). Nuclear Overhauser effects between the protons of several of the side chains of the octapeptide have been observed and they support the conclusion that the peptide predominantly takes up a folded conformation in this solvent, which brings these side chains close to each other. The goal set for students is to find this conformation. A disadvantage to the use of [Sar1]angiotensin is that the octapeptide contains a proline residue. Rotational isomerism at the peptide bond joining this residue to the adjacent His-7, as indicated in the structure below, leads to the presence of two distinct conformations for the peptide. One of these is dominant (∼80%), but the other is present in sufficiently high amounts that resonances and cross peaks from it are intense enough to allow confusion to arise when students are attempting to assign resonances to each amino acid. To simplify the proton spectrum of the peptide, a small amount of D2O was added to the sample examined. Under these conditions the signals from the peptide (NHCO) N–H protons and all other acidic hydrogens of the peptide disappear from the spectrum because these protons have been replaced by deuterons. O H
O
C
H
C
O N
C C
C H H
trans
N
C O
cis
System Studied Beginning biochemistry students typically have been exposed to proton and carbon-13 NMR spectroscopy as part of their organic chemistry course and are familiar with the concepts of chemical shift and spin coupling. An important step in using NMR observations to define the three-dimensional structure of a peptide is assignment of multiplets in the proton spectrum to specific protons of the molecule. This process relies on two-dimensional spectra and can be difficult for beginning students if an amino acid is present in the structure more than once. We thus sought a molecule that (i) was biologically significant, (ii) was composed of a small number of amino acids, none of which appeared more than once in the sequence, (iii) was readily available, and (iv) exhibited a nonrandom conformation. Experimental investigation and consideration of the literature showed that these requirements were reasonably well met by the octapeptide [Sar1]angiotensin II (Sar-Arg-Val-Tyr-Ile-His-Pro-Phe). 892
Experimental Procedure [Sar1]angiotensin II is available from Sigma Chemical Co. and is shipped in 5-mg lots. To prepare the sample for this exercise, 0.5 mL of deuterated DMSO (99.9% D, Aldrich) was added directly to the sample vial received from the supplier and this was followed by 2 drops of deuterium oxide (99.9%, Aldrich). The resulting solution (∼8 mM peptide) was transferred to a clean, dry 5-mm NMR tube. The tube was stoppered with a polyethylene cap, which was then wrapped in Parafilm. For retention and re-use of the sample over a long period of time it is suggested that the sample tube be sealed permanently, since DMSO is hygroscopic. A one-dimensional proton spectrum of a sample of [Sar1]angiotensin II prepared in the way described is shown in Figure 1. When the sample temperature is 25o the fine structure of many of the multiplets is reasonably well resolved. At a sample temperature of 1° the signals are appreciably
Journal of Chemical Education • Vol. 77 No. 7 July 2000 • JChemEd.chem.wisc.edu
In the Laboratory
Background Material
9
8
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ppm
[Sar1]angio-
Figure 1. One-dimensional proton NMR spectrum of tensin II in d6-DMSO with a trace of D2O added, 25°, 500 MHz. Signals from water, the residual protons of the solvent, and acetate were suppressed as indicated in the text.
broader, although their positions are generally little altered by the reduction in temperature. The lower temperature favors the dominant conformation of the octapeptide, reduces the rate of rotation at the His6–Pro7 peptide bond, and improves the detectability of proton–proton Overhauser effects. The viscosity of DMSO is increased from about 2.0 cP to about 4.2 cP by this change of temperature (7) and presumably the line broadening observed is due to viscosity effects. The experimental data collected for this exercise are available in several formats from our World Wide Web site (http:// www2.chem.ucsb.edu/~nmr_edu/jchemed/welcome.htm). Plots of processed data displayed with a variety of expansions are provided in PDF format. Plots in the PDF format can be visualized or printed using a version of the Adobe Acrobat Reader that is available for all computing platforms without charge (http://www.adobe.com). Raw spectral data in the Varian Vnmr format is also provided. These files may be processed locally if access to Vnmr is available. The software packages for instruments produced by other manufacturers typically contain programs that can be used to convert Vnmr data files into a usable format. Data in Vnmr format can be read by a number of third-party software packages for processing and analysis of multi-dimension NMR data. Finally, we provide processed data sets that can be used with ANSIG and SPARKY, 2D NMR spectral analysis programs that are available without charge over the Internet (8). Several versions of an exercise in three-dimensional structure determination by NMR are possible with the materials we have assembled. At the most basic level the NOESY spectra along with the signal assignment list we have provided can be used to determine structural constraints. At a somewhat more advanced level the COSY and TOCSY spectra can be used to obtain the assignments, which would then be employed in an analysis of the NOESY data. The raw experimental data can be used in more advanced exercises, which could include experience with the processing and display of 2D NMR data or the use of commercial and freeware programs to assist in the assignment of the spectrum and in the quantitation of the NOESY cross peaks.
Although discussions of 2D NMR experiments appear in current organic spectroscopy and structure analysis texts (9, 10), students undertaking our exercise in 3D structure determination will likely need introduction to or review of several concepts related to 2D NMR. Cheatham has provided introductory descriptions of COSY and NOESY (11). There are advanced explanations of these available (12, 13). The notion of using NMR data to indicate possible conformations of small molecules has been the basis for a number of experiments described in this Journal (14–16 ). Jones has provided a useful introduction to current research techniques in protein folding (17 ) and Lundberg has described the wide variety of software available for education and research in NMR spectroscopy (18). We make available a 16-page handout for students at our Web site and on JCE Online.W The handout provides introductions to the concepts of two-dimensional spectroscopy and the particular 2D experiments (COSY, TOCSY, NOESY) that are used to obtain structural information for Sar1-angiotensin. A discussion of structure determination by constraint satisfaction and suggested procedures for finding the conformation of [Sar1]angiotensin II are also provided in this handout. Discussion Three sharp lines appear at 1.85, 2.50, and ∼3.6 ppm in the proton NMR spectrum of samples prepared as indicated above. These arise from acetate (present in the commercial peptide), the residual protons in the DMSO-d6 solvent, and water (HOD), respectively. Although these signals were suppressed in our experiments, some artifacts appear at these shifts in the 2D spectra. Their presence is not a significant stumbling block to successful completion of this exercise; these and the signals from the minor conformer remind students that all experiments produce artifacts and complications that must be recognized and taken into consideration when analyzing the data produced by those experiments. However, plots of 2D spectra with these artifactual features removed are supplied as part of the materials available at the WWW site for instructors who prefer them. It is pedagogically desirable to compare the best structures obtained by all students in a class when they have completed the exercise. Since a large number of NOEs are not observed, the three-dimensional structure of the peptide is not strongly constrained and a variety of structures satisfying the available data will be possible (19). Our experience with the exercise we have described has been relatively limited. We find so far that an introductory lecture during which copies of the various spectra are distributed and the basic concepts indicated above are discussed is sufficient to get students started with the exercise. After a week or so a second lecture or discussion is held to answer questions and provide additional guidance. Able students can typically produce a reasonable structure for the peptide with 5–10 hours of effort, depending on their familiarity with the software used.
JChemEd.chem.wisc.edu • Vol. 77 No. 7 July 2000 • Journal of Chemical Education
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In the Laboratory
Acknowledgments Development of this exercise was supported in part by the National Science Foundation (Grant DUE-9850596). NMR instrumentation in the Department has been acquired with the assistance of awards from the NSF. W
Supplemental Material
Supplemental material for this article is available in this issue of JCE Online. Literature Cited 1. Voet, D.; Voet, J. G. Biochemistry; Wiley: New York, 1995. 2. Mathews, C. K.; Van Holde, K. E. Biochemistry; Benjamin/ Cummings: Menlo Park, CA, 1996. 3. Smeby, R R.; Fermandijian, S. In Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins; Weinstein, N., Ed.; Dekker: New York, 1976; pp 117–162. 4. Matsoukas, J. M.; Bigam, G.; Zhou, N.; Moore, G. J. Peptides 1990, 11, 359–366. 5. Matsoukas, J. M.; Hondrelis, J.; Keramida, M.; Mavromoustakos, T.; Makriyannis, A.; Yamdagni, R.; Wu, Q.; Moore, G. J. J. Biol. Chem. 1994, 269, 5303–5312.
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6. Smallcombe, S. H.; Patt, S. L.; Keifer, P. A. J. Magn. Reson. A 1995, 117, 295–303. 7. Daubert, T. E.; Danner, R. P., Physical and Thermodynamic Properties of Pure Chemicals; Hemisphere: New York, 1989. 8. ANSIG is available at http://www-ccmr-nmr.bioc.cam.ac.uk/ public/ANSIG/ansig.html; SPARKY is available at http:// www.cgl.ucsf.edu/home/sparky/ (both accessed Mar 2000). 9. Macomber, R. S. A Complete Introduction to Modern NMR Spectroscopy; Wiley: New York, 1998. 10. Crews, P.; Rodriquez, J.; Jaspars, M. Organic Structure Analysis; Oxford University Press: New York, 1998. 11. Cheatham, S. J. Chem. Educ. 1989, 66, 111–117. 12. Cavanaugh, J.; Fairbrother, W. J.; Palmer, A. G. III; Skelton, N. J. Protein NMR Spectroscopy; Academic: New York, 1966. 13. Protein NMR Techniques; Reid, D. G., Ed.; Humana: Totowa, NJ, 1997. 14. Mills, N. S. J. Chem. Educ. 1996, 73, 1190–1193. 15. Lee, M. J. Chem. Educ. 1996, 73, 184–187. 16. Anderson, E. L.; Li, D.; Owen, N. L. J. Chem. Educ. 1992, 69, 846–849. 17. Jones, C. M. J. Chem. Educ. 1997, 74, 1306–1310. 18. Lundberg, P. J. Chem. Educ. 1997, 74, 1489–1491. 19. The program molmol can be useful for these purposes. The program may be obtained at http://www.mol.biol.ethz.ch/ wuthrich/software/molmol (accessed Mar 2000).
Journal of Chemical Education • Vol. 77 No. 7 July 2000 • JChemEd.chem.wisc.edu
