Abstract
The development of the biological NMR field over the last decades have been very rapid and widespread. This has included changes in hardware, software, techniques and applications. In terms of macromolecular applications, it is now possible to determine the complete three dimensional structure of a macromolecule in solution, and to characterize its dynamics both in terms of internal motions and the kinetics of conformational changes and interactions with ligands or other macromolecules. The macromolecules studied include mostly proteins, DNA and their complexes, with increasing applications to other macromolecules such as complex carbohydrates and RNA. For the rest of this article we will refer to proteins only for the sake of simplicity.
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References
Ames, J.B., Tanaka, T., Styer, L., and Ikura, M., 1994, Secondary structure of myristoylated recoverin determined by three-dimensional heteronuclear NMR: implications for the calcium-myristoyl switch, Biochemistry 33:10743.
Anglister, J., Grzesiek, S., Ren, H., Klee, C.B., and Bax, A, 1993, Isotope-edited multidimensional NMR of calcineurin B in the presence of the non-deuterated detergent CHAPS,J. Biomol. NMR 3:121.
Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W., and Bax, A., 1992, Backbone dynamics of calmodulinstudied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: The central helix is flexible, Biochemistry 31:5269.
Buck, M., Radford, S JE., and Dobson, C.M., 1993, A partially folded state of hen egg white lysozyme in trifluoroethanol: Structural characterization and implications for protein folding, Biochemistry 32:669.
Craik, DJ., and Higgins, K.A., 1991, Comparison of 1H NMR chemical shifts of bovine and human insulin’s, Peptide Research 4:177.
De Dios, A.C., Pearson, J.G., and Oldfield, E., 1993, Secondary and tertiary structural effects on protein NMR chemical shifts: an ab initio approach, Science 260:1491.
Gagné, S.M., 1994, Calcium-induced structural changes in the regulatory domain of troponin-C by multidimensional NMR spectroscopy, M Sc. Thesis.
Gagné, S.M., Tsuda, S., Li, M.X., Chandra, M., Smillie, L.B., and Sykes, BD., 1994, Quantification of the calcium-induced secondary structural changes in the regulatory domain of troponin-C, Protein Science 3:1961.
Grzesiek, S., and Bax, A, 1993, Measurement of amide proton exchange rates and NOEs with water in 13C/15N-enriched calcineurin B, J. Biomol. NMR 3:627.
Higgins, K.A., Craik, DJ., and Hall, J.G., 1990, 1H NMR studies of monomelic bovine insulin, Biochem. Int. 22:627.
Hobohm, U., Scharf, M., Schneider, R, and Sander, C., 1992, Selection of a representative set of structures from the Brookhaven Protein Data Bank, Protein Science 1:409.
Hua, Q., and Weiss, MA, 1991a, Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: Sequential resonance assignment and implications for protein dynamics and receptor recognition, Biochemistry 30:5505.
Hua, Q., and Weiss, M.A., 1991b, Two-dimensional NMR studies of Des-(B26 - B30)-insulin: sequence- specific resonance assignments and effects of solvent composition, Biochim. Biophys. Acta 1078:101.
Hyberts, S.G., Goldberg, M.S., Havel, T.F., and Wagner, G., 1992, The solution structure of eglin c based on measurements of many NOEs and coupling constants and its comparison with X-ray structures, Protein Science 1:736.
Kline, A.D., and Justice, R.M., 1990, Complete sequence-specific 1H NMR assignments for human insulin, Biochemistry 29:2906.
Lau, S.Y.M, Taneja, A.K, and Hodges, R.S, 1984a, Effects of High-performance liquid chromatographic solvents and hydrophobic matrices on the secondary and quaternary structure of a model protein. Reversed-phase and size-exclusion high-performance liquid chromatography, J. Chrom. 317:129.
Lau, S.Y.M., Taneja, A.K., and Hodges, R.S., 1984b, Synthesis of a Model protein of defined secondary and quaternary structure, J. Biol. Chem. 259:13253.
Morris, AM., MacArthur, M.W., Hutchinson, E.G., and Thornton, J.M., 1992, Proteins 12:345.
Pardi, A., Billeter, M., and Wüthrich, K., 1984, Calibration of the angular dependence of the amide proton- Cα coupling constant JHNα in a globular protein, J. Mol. Biol. 180:741.
Slupsky, C.M., Reinach, F.C. and Sykes, B.D., 1995b, Solution secondary structure of calcium-saturated troponin-C monomer determined by multi-dimensional heteronuclear NMR spectroscopy,Protein Science submitted.
Slupsky, CM Reinach, F.C., Kay, C.M. and Sykes, B.D., 1995a, The calcium-induced dimerization of troponin-C: the mode of interaction and the use of trifluoroethanol as a denaturant of quaternary structure, Biochemistry submitted.
Spera, S., and Bax, A, 1991, Empirical correlation between protein backbone conformation and Cα and Cß 13c nuclear magnetic resonance chemical shifts,J. Am. Chem. Soc. 113:5490.
Weiss, M.A., EliasonJL and States, DJ., 1984, Dynamic filtering by two-dimensional 1H NMR with application to phage 1 repressor, Proc. Natl. Acad. Sci. USA 81:6019.
Wishart, D.S., and Sykes, B.D., 1994a, Chemical shifts as a tool for structure determination Methods Enzymol 239:363.
Wishart, D.S., and Sykes, B.D., 1994b, The 13C chemical shift index: a simple method for the identification of protein secondary structure using 13C chemical shifts, J. Biomol. NMR 4:171.
Wishart, D.S., Sykes, B D., and Richards, F.M., 1991a, Simple techniques for the quantification of protein secondary structure by 1H NMR spectroscopy, FEBS Lett. 293:72.
Wishart, D.S., Sykes, B.D., and Richards, F.M., 1991b, Relationship between nuclear magnetic resonance chemical shift and protein secondary structure, J. Mol. Biol. 222:311.
Wishart, D.S., Sykes, B J., and Richards, F.M., 1992, The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy, Biochemistry 31:1647.
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Peter Wright - TFE of course induces helix but it does something else, it weakens hydrophobic interactions and so most proteins in TFE expand dramatically with a large radius of gyration. Can you be sure at the concentrations that you are dealing with, in troponin C, that you are not getting a significant expansion and disordering in the interior. Brian Sykes - That’s obviously a very important question. But it really doesn’t look like we are at this stage. You can see at this stage of the structural calculation that we are gathering quite good and quite complete data for the structure in TFE. And we don’t see any significantly expanded structure in TFE or anything like that. We do not see any evidence of changes. It would be good to know which residue is responsible for the aggregation or just exactly where is it aggregating. At the time we left we were running a sample where we mixed the 12C-protein and the 13C-protein and tried to look for intermolecular NOE’s. But I guess the answer is, we certainly appreciate your concern but we’ve seen no sort of expansion; we have not gotten anywhere that far in terms of TFE concentration.
Bill Gmeiner - I have a question on the ultracentrifugation techniques and how one goes about doing those tests for dimerization. Do you take the same concentrations that you are working at in the NMR and look for the migration in the ultracentrifuge? Are there any artifactual results that can result from that technique?
Sykes - If you have an older centrifuge, it’s easier, and we do. What you do is load the cell at about 1 mg/ml concentration and then it’s an equilibrium ultracentrifugation so when the cell has reached equilibrium, it’s about a tenth of a mg/ml in one end of the cell and about 2 or 3 mg/ml in the other end of the cell. So it works in a fairly narrowly defined range. All of this corresponds to a range of interest to people like yourself, and all of us, namely 0.1 to 0.5 mM. And then the plot is basically a plot of how the molecule spreads out in the cell and that’s proportional to molecular weight. The problems are just a little more subtle. If you looked at the plot I had at the bottom, it was a beautiful plot that went from monomer to dimer but it doesn’t fit really well to a classic monomer-dimer equilibrium and that’s because there are non-idealities that occur at either end of the cell. Obviously, at the high, centripetal force end you get build up of the protein. I think it gives you a nice result even though there are non idealities that can occur that makes the exact fitting and the exact extrapolation of our constant very difficult. But I think it’s a really good way to see if things are monomer or dimer through the range of interest.
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Sykes, B.D., Slupsky, C.M., Wishart, D.S., Sönnichsen, F.D., Gagné, S.M. (1996). On the Use of NMR in Complex Biological Systems: NMR Studies of Calcium Sensitive Interactions amongst Muscle Proteins. In: Rao, B.D.N., Kemple, M.D. (eds) NMR as a Structural Tool for Macromolecules. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-0387-9_21
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