Abstract
Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR) offers a powerful method for the structural analysis of protein folds. This method can be used to test and build secondary, tertiary, and quaternary structural models as well as measure protein conformational changes in solution. Insertion of two cysteine residues into the protein backbone using molecular biology methods and the subsequent labeling of the cysteine residues with a paramagnetic spin label enables the technique of EPR to be used as a molecular spectroscopic ruler. EPR measures the dipolar interaction between pairs of paramagnetic spin labels to yield internitroxide distances from which quantitative structural information on a protein fold can then be obtained. Interspin dipolar interaction between two spin labels at less than 25 Å are measured using continuous wave (CW) EPR methods. As for any low-resolution distance methods, the positioning of the spin labels and the number of distance constraints to be measured are dependent on the structural question being asked, thus a pattern approach for using distance sets to decipher structure mapping, including protein folds and conformational changes associated with biological activity, is essential. Practical guidelines and hints for the technique of SDSL-EPR are described in this chapter, including methods for spin labeling the protein backbone, CW-EPR data collection at physiological temperatures and two semiquantitative analysis methods to extract interspin distance information from the CW-EPR spectra.
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References
Hubbell, W. L., and Altenbach, C. (1994) Investigation of structure and dynamics in membrane proteins using site-directed spin labeling. Curr Opin Struct Biol. 4, 566–573.
Hubbell, W. L., Gross, A., Langen, R., and Lietzow, M. A. (1998) Recent advances in site-directed spin labeling of proteins. Curr Opin Struct Biol. 8, 649–656.
Hubbell, W. L., McHaourab, H. S., Altenbach, C., and Lietzow, M. A. (1996) Watching proteins move using site-directed spin labeling. Structure. 4, 779–783.
Klug, C. S., Su, W., and Feix, J. B. (1997) Mapping of the residues involved in a proposed beta-strand located in the ferric enterobactin receptor FepA using site-directed spin-labeling. Biochemistry. 36, 13027–13033.
Columbus, L., and Hubbell, W. L. (2002) A new spin on protein dynamics. Trends Biochem Sci. 27, 288–295.
Fajer, P., Brown, L., and Song, L. (2007) Practical Pulsed Dipolar ESR (DEER), in Biological Magnetic Resonance (Hemminga, M., and Berliner, L., Eds.), Vol. 27, Springer, New York, pp. 95–128.
Czogalla, A., Pieciul, A., Jezierski, A., and Sikorski, A. F. (2007) Attaching a spin to a protein - site-directed spin labeling in structural biology. Acta Biochim Pol. 54, 235–244.
Schiemann, O., and Prisner, T. F. (2007) Long-range distance determinations in biomacromolecules by EPR spectroscopy. Q Rev Biophys. 40, 1–53.
Fanucci, G. E., and Cafiso, D. S. (2006) Recent advances and applications of site-directed spin labeling. Curr Opin Struct Biol. 16, 644–653.
Steinhoff, H. J. (2004) Inter- and intra-molecular distances determined by EPR spectroscopy and site-directed spin labeling reveal protein-protein and protein-oligonucleotide interaction. Biol Chem. 385, 913–920.
Borbat, P. P., Costa-Filho, A. J., Earle, K. A., Moscicki, J. K., and Freed, J. H. (2001) Electron spin resonance in studies of membranes and proteins. Science. 291, 266–269.
Hubbell, W. L., Cafiso, D. S., and Altenbach, C. (2000) Identifying conformational changes with site-directed spin labeling. Nat Struct Biol. 7, 735–739.
Hustedt, E. J., and Beth, A. H. (1999) Nitroxide spin–spin interactions: applications to protein structure and dynamics. Annu Rev Biophys Biomol Struct. 28, 129–153.
Perozo, E., Cortes, D. M., and Cuello, L. G. (1999) Structural rearrangements underlying K+−channel activation gating. Science. 285, 73–78.
Cai, Q., Kusnetzow, A. K., Hideg, K., Price, E. A., Haworth, I. S., and Qin, P. Z. (2007) Nanometer distance measurements in RNA using site-directed spin labeling. Biophys J. 93, 2110–2117.
Brown, L. J., Sale, K. L., Hills, R., Rouviere, C., Song, L., Zhang, X., and Fajer, P. G. (2002) Structure of the inhibitory region of troponin by site directed spin labeling electron paramagnetic resonance. Proc Natl Acad Sci U S A. 99, 12765–12770.
Xiao, W., Poirier, M. A., Bennett, M. K., and Shin, Y. K. (2001) The neuronal t-SNARE complex is a parallel four-helix bundle. Nat Struct Biol. 8, 308–311.
Chen, M., Margittai, M., Chen, J., and Langen, R. (2007) Investigation of alpha-synuclein fibril structure by site-directed spin labeling. J Biol Chem. 282, 24970–24979.
Hanson, S. M., Francis, D. J., Vishnivetskiy, S. A., Klug, C. S., and Gurevich, V. V. (2006) Visual arrestin binding to microtubules involves a distinct conformational change. J Biol Chem. 281, 9765–9772.
Radzwill, N., Gerwert, K., and Steinhoff, H. J. (2001) Time-resolved detection of transient movement of helices F and G in doubly spin-labeled bacteriorhodopsin. Biophys J. 80, 2856–2866.
Xiao, W., Brown, L. S., Needleman, R., Lanyi, J. K., and Shin, Y. K. (2000) Light-induced rotation of a transmembrane alpha-helix in bacteriorhodopsin. J Mol Biol. 304, 715–721.
Hustedt, E. J., Stein, R. A., Sethaphong, L., Brandon, S., Zhou, Z., and Desensi, S. C. (2006) Dipolar coupling between nitroxide spin labels: the development and application of a tether-in-a-cone model. Biophys J. 90, 340–356.
Sale, K., Song, L., Liu, Y. S., Perozo, E., and Fajer, P. (2005) Explicit Treatment of Spin Labels in Modeling of Distance Constraints from Dipolar EPR and DEER. J Am Chem Soc. 127, 9334–9335.
Walker, J. M. (2002) SDS Polyacrylamide Gel Electrophoresis of Proteins, in The Protein Protocols Handbook (Walker, J. M., Ed.), Humana, Totowa, NJ, pp. 61–67.
Altenbach, C., Cai, K., Klein-Seetharaman, J., Khorana, H. G., and Hubbell, W. L. (2001) Structure and function in rhodopsin: mapping light-dependent changes in distance between residue 65 in helix TM1 and residues in the sequence 306–319 at the cytoplasmic end of helix TM7 and in helix H8. Biochemistry. 40, 15483–15492.
Langen, R., Isas, J. M., Luecke, H., Haigler, H. T., and Hubbell, W. L. (1998) Membrane-mediated assembly of annexins studied by site-directed spin labeling. J Biol Chem. 273, 22453–22457.
Langen, R., Oh, K. J., Cascio, D., and Hubbell, W. L. (2000) Crystal structures of spin labeled T4 lysozyme mutants: implications for the interpretation of EPR spectra in terms of structure. Biochemistry. 39, 8396–8405.
McHaourab, H. S., Lietzow, M. A., Hideg, K., and Hubbell, W. L. (1996) Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. Biochemistry. 35, 7692–7704.
Perozo, E., Cortes, D. M., and Cuello, L. G. (1998) Three-dimensional architecture and gating mechanism of a K+ channel studied by EPR spectroscopy. Nat Struct Biol. 5, 459–469.
Aitken, A., and Learmonth, M. P. (2002) Protein Determination by UV Absorption, in The Protein Protocols Handbook (Walker, J. M., Ed.), Humana, Totowa, NJ, pp. 3–6.
Waterborg, J. H. (2002) The Lowry Method for Protein Quantitation, in The Protein Protocols Handbook (Walker, J. M., Ed.), Humana, Totowa, NJ, pp. 7–9.
Walker, J. M. (2002) The Bicinchoninic Acid (BCA) Assay for Protein Quantitation, in The Protein Protocols Handbook (Walker, J. M., Ed.), Humana, Totowa, NJ, pp. 11–14.
Kruger, N. J. (2002) The Bradford Method for Protein Quantitation, in The Protein Protocols Handbook (Walker, J. M., Ed.), Humana, Totowa, NJ, pp. 15–21.
Hustedt, E. J., Smirnov, A. I., Laub, C. F., Cobb, C. E., and Beth, A. H. (1997) Molecular distances from dipolar coupled spin-labels: the global analysis of multifrequency continuous wave electron paramagnetic resonance data. Biophys J. 72, 1861–1877.
Steinhoff, H. J., Radzwill, N., Thevis, W., Lenz, V., Brandenburg, D., Antson, A., Dodson, G., and Wollmer, A. (1997) Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure. Biophys J. 73, 3287–3298.
Rabenstein, M. D., and Shin, Y. K. (1995) Determination of the distance between two spin labels attached to a macromolecule. Proc Natl Acad Sci U S A. 92, 8239–8243.
Zhang, F., Chen, Y., Kweon, D. H., Kim, C. S., and Shin, Y. K. (2002) The four-helix bundle of the neuronal target membrane SNARE complex is neither disordered in the middle nor uncoiled at the C-terminal region. J Biol Chem. 277, 24294–24298.
Altenbach, C., Oh, K. J., Trabanino, R. J., Hideg, K., and Hubbell, W. L. (2001) Estimation of inter-residue distances in spin labeled proteins at physiological temperatures: experimental strategies and practical limitations. Biochemistry. 40, 15471–15482.
McHaourab, H. S., Oh, K. J., Fang, C. J., and Hubbell, W. L. (1997) Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling. Biochemistry. 36, 307–316.
Xiao, W., and Shin, Y.-K. (2000) EPR Spectroscopic Ruler: The Deconvolution Method and its Applications, in Biological Magnetic Resonance: Distance Measurements in Biological Systems by EPR (Berliner, L. J., Eaton, S. S., and Eaton, G. R., Eds.), Vol. 19, Kluwer Academic/Plenum Publishers, New York, pp. 249–276.
Perozo, E., Cortes, D. M., Sompornpisut, P., Kloda, A., and Martinac, B. (2002) Open channel structure of MscL and the gating mechanism of mechanosensitive channels. Nature. 418, 942–948.
Persson, M., Harbridge, J. R., Hammarstrom, P., Mitri, R., Martensson, L. G., Carlsson, U., Eaton, G. R., and Eaton, S. S. (2001) Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II. Biophys J. 80, 2886–2897.
Riddles, P. W., Blakeley, R. L., and Zerner, B. (1983) Reassessment of Ellman’s reagent. Methods Enzymol. 91, 49–60.
McHaourab, H. S., Berengian, A. R., and Koteiche, H. A. (1997) Site-directed spin-labeling study of the structure and subunit interactions along a conserved sequence in the alpha-crystallin domain of heat-shock protein 27. Evidence of a conserved subunit interface. Biochemistry. 36, 14627–14634.
Fajer, P. G. (2005) Site directed spin labelling and pulsed dipolar electron paramagnetic resonance (double electron-electron resonance) of force activation in muscle. J Phys Condens Matter. 17, S1459–S1469.
Acknowledgments
This work was supported by the Australian Research Council (ARC). LB is a recipient of an ARC APD fellowship and JC is a recipient of a Macquarie University Research Areas and Centres of Excellence Award (RAACE). The authors thank Mr Michael Howell for his editorial assistance.
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Cooke, J.A., Brown, L.J. (2011). Distance Measurements by Continuous Wave EPR Spectroscopy to Monitor Protein Folding. In: Hill, A., Barnham, K., Bottomley, S., Cappai, R. (eds) Protein Folding, Misfolding, and Disease. Methods in Molecular Biology, vol 752. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-223-0_6
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