Abstract
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for the study of the structure, dynamics, and folding of proteins in solution. It is particularly powerful when applied to dynamic or flexible systems, such as partially folded molten-globule states of proteins, which are not usually amenable to X-ray crystallography. This chapter describes NMR methods suitable for the characterization of molten-globule states. These include pulsed-field-gradient NMR techniques for the measurement of the hydrodynamic radius, bulk and site-specific hydrogen-deuterium exchange experiments for the identification of regions of secondary structure, and 15N-edited NMR experiments carried out in increasing concentrations of denaturants, which allow the stability of different regions of the molten globule to be probed. Examples of the application of these methods to the study of the low-pH molten globule of human α-lactalbumin are presented.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Arai, M. and Kuwajima, K. (2000) Role of the molten globule state in protein folding. Adv. Protein Chem. 53, 209–282.
Ptitsyn, O. B. (1995) Molten globule and protein folding. Adv. Protein Chem. 47, 83–229.
Ohgushi, M. and Wada, A. (1983) Molten-globule state—a compact form of globular proteins with mobile sidechains. FEBS Lett. 164, 21–24.
Dolgikh, D. A., Abaturov, L. V., Brazhnikov, E. V., Lebedev, I. O., Chirgadze, I. N., and Ptitsyn, O. B. (1983) Acid form of carbonic anhydrase—molten globule with a secondary structure. Dokl. Akad. Nauk., SSSR 272, 1481–1484.
Arai, M. and Kuwajima, K. (1996) Rapid formation of a molten globule intermediate in refolding of α-lactalbumin. Fold. Des. 1, 275–287.
Forge, V., Wijesinha, R. T., Balbach, J., Brew, K., Robinson, C.V., Redfield, C., et al. (1999) Rapid collapse and slow structural reorganisation during the refolding of bovine α-lactalbumin. J. Mol. Biol. 288, 673–688.
Arai, M., Ito, K., Inobe, T., Nakao, M., Maki, K., Kamagata, K., et al. (2002) Fast compaction of α-lactalbumin during folding studied by stopped-flow X-ray scattering. J. Mol. Biol. 321, 121–132.
Hughson, F. M., Wright, P. E., and Baldwin, R. L. (1990) Structural characterization of a partly folded apomyoglobin intermediate. Science 249, 1544–1548.
Jennings, P. A. and Wright, P. E. (1993) Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science 262, 892–896.
Acharya, K. R., Ren, J. S., Stuart, D. I., Phillips, D. C., and Fenna, R. E. (1991) Crystal structure of human α-lactalbumin at 1.7 Å resolution. J. Mol. Biol. 221, 571–581.
Dolgikh, D. A., Abaturov, L. V., Bolotina, I. A., Brazhnikov, E. V., Bychkova, V. E., Bushuev, V. N., et al. (1985) Compact state of a protein molecule with pronounced small-scale mobility: bovine α-lactalbumin. Eur. Biophys. J. 13, 109–121.
Redfield, C., Schulman, B. A., Milhollen, M. A., Kim, P. S., and Dobson, C. M. (1999) α-lactalbumin forms a compact molten globule in the absence of disulfide bonds. Nat. Struct. Biol. 6, 948–952.
Baum, J., Dobson, C. M., Evans, P. A., and Hanley, C. (1989) Characterization of a partly folded protein by NMR methods: studies on the molten globule state of guinea pig α-lactalbumin. Biochemistry 28, 7–13.
Dolgikh, D. A., Gilmanshin, R. I., Brazhnikov, E. V., Bychkova, V. E., Semisotnov, G. V., Venyaminov, S.Yu., et al. (1981) α-lactalbumin: compact state with fluctuating tertiary structure? FEBS Lett. 136, 311–315.
Wu, L. C., Peng, Z.-Y., and Kim, P. S. (1995) Bipartite structure of the α-lactalbumin molten globule. Nat. Struct. Biol. 2, 281–286.
Wu, L. C. and Kim, P. S. (1998) A specific hydrophobic core in the α-lactalbumin molten globule. J. Mol. Biol. 280, 175–182.
Schulman, B. A., Kim, P. S., Dobson, C. M., and Redfield, C. (1997) A residuespecific NMR view of the non-cooperative unfolding of a molten globule. Nat. Struct. Biol. 4, 630–634.
Semisotnov, G. V., Rodionova, N. A., Razgulyaev, O. J., Uversky, V. N., Gripas, A. F., and Gilmanshin, R. I. (1991) Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31, 119–128.
Berne, B. J. and Pecora, R. (1976) Dynamic Light Scattering with Applications to Chemistry, Biology and Physics. Wiley, New York.
Lattman, E. E. (1994) Small-angle X-ray scattering studies of protein-folding. Curr. Opin. Struct. Biol. 4, 87–92.
Stejskal, E. O. and Tanner, J. E. (1965) Spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient. J. Chem. Phys. 42, 288–292.
Gibbs, S. J. and Johnson, C. S., Jr. (1991) A PFG NMR experiment for accurate diffusion and flow studies in the presence of eddy currents. J. Magn. Reson. 93, 395–402.
Jones, J. A., Wilkins, D. K., Smith, L. J., and Dobson, C. M. (1997) Characterisation of protein unfolding by NMR diffusion measurements. J. Biomol. NMR 10, 199–203.
Wilkins, D. K., Grimshaw, S. B., Receveur, V., Dobson, C. M., Jones, J. A., and Smith, L. J. (1999) Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. Biochemistry 38, 16,424–16,431.
Chen, L., Wu, D. H., and Johnson, C. S., Jr. (1995) Determination of the binding isotherm and size of the bovine serum albumin-sodium dodecyl-sulfate complex by diffusion-ordered 2D NMR. J. Phys. Chem. 99, 828–834.
Englander, S. W. and Kallenbach, N. R. (1984) Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q. Rev. Biophys. 16, 521–655.
Woodward, C. K., Simon, I., and Tuchsen, E. (1982) Hydrogen exchange and the dynamic structure of proteins. Mol. Cell. Biochem. 48, 135–160.
Bai, Y., Milne, J. S., Mayne, L., and Englander, S. W. (1993) Primary structure effects on peptide group hydrogen exchange. Proteins 17, 75–86.
Wijesinha-Bettoni, R., Dobson, C. M., and Redfield, C. (2001) Comparison of the denaturant-induced unfolding of the bovine and human α-lactalbumin molten globules. J. Mol. Biol. 312, 261–273.
Jeng, M.-F., Englander, S. W., Elove, G. A., Wand, A. J., and Roder, H. (1990) Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR. Biochemistry 29, 10,433–10,437.
Schulman, B. A., Redfield, C., Peng, Z.-Y., Dobson, C. M., and Kim, P. S. (1995) Different subdomains are most protected from hydrogen exchange in the molten globule and native states of human α-lactalbumin. J. Mol. Biol. 253, 651–657.
Kay, L. E., Keifer, P., and Saarinen, T. (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J. Am. Chem. Soc. 114, 10,663–10,665.
Wang, Y. and Shortle, D. (1995) The equilibrium folding pathway of staphylococcal nuclease: indentification of the most stable chain-chain interactions by NMR and CD spectroscopy. Biochemistry 34, 15,895–15,905.
Wang, Y. and Shortle, D. (1996) A dynamic bundle of four adjacent hydrophobic segments in the denatured state of staphylococcal nuclease. Protein Sci. 5, 1898–1906.
Braun, D., Wider, G., and Wüthrich, K. (1994) Sequence-corrected 15 N “random coil” chemical shifts. J. Am. Chem. Soc. 116, 8466–8469.
Marion, D., Driscoll, P. C., Kay, L. E., Wingfield, P. T., Bax, A., Gronenborn, A. M., et al. (1989) Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn multiple quantum coherence and nuclear Overhauser multiple quantum coherence spectroscopy: application to interleukin 1 β. Biochemistry 28, 6150–6156.
Fiebig, K. M., Schwalbe, H., Buck, M., Smith, L. J., and Dobson, C. M. (1996) Toward a description of the conformation of denatured states of proteins: comparison of a random coil model with NMR measurements. J. Phys. Chem. 100, 2661–2666.
Frenkiel, T., Bauer, C., Carr, M. D., Birdsall, B., and Feeney, J. (1990) HMQCNOESY-HMQC: a three-dimensional NMR experiment which allows detection of nuclear Overhauser effects between protons with overlapping signals. J. Magn. Reson. 90, 420–425.
Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids. Wiley, New York.
Ramboarina, S. and Redfield, C. (2003) Structural characterisation of the human α-lactalbumin molten globule at high temperature. J. Mol. Biol. 330, 1177–1188.
Eliezar, D., Jennings, P. A., Dyson, H. J., and Wright, P. E. (1997) Populating the equilibrium molten globule state of apomyoglobin under suitable conditions for structural characterization by NMR. FEBS Lett. 417, 92–96.
Eliezer, D., Yao, J., Dyson, J. H., and Wright, P. E. (1998) Structural and dynamic characterization of partially folded states of apomyoglobin and implications for protein folding. Nat. Struct. Biol. 5, 148–155.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Redfield, C. (2004). NMR Studies of Partially Folded Molten-Globule States. In: Downing, A.K. (eds) Protein NMR Techniques. Methods in Molecular Biology™, vol 278. Humana Press. https://doi.org/10.1385/1-59259-809-9:233
Download citation
DOI: https://doi.org/10.1385/1-59259-809-9:233
Publisher Name: Humana Press
Print ISBN: 978-1-58829-246-9
Online ISBN: 978-1-59259-809-0
eBook Packages: Springer Protocols