Abstract
The study of protein folding has been conventionally hampered by the assumption that all single-domain proteins fold by an all-or-none process (two-state folding) that makes it impossible to resolve folding mechanisms experimentally. Here we describe an experimental method for the thermodynamic analysis of protein folding at atomic resolution using nuclear magnetic resonance (NMR). The method is specifically developed for the study of small proteins that fold autonomously into basic supersecondary structure motifs, and that do so in the sub-millisecond timescale (folding archetypes). From the NMR experiments we obtain hundreds of atomic unfolding curves that are subsequently analyzed leading to the determination of the characteristic network of folding interactions. The application of this approach to a comprehensive catalog of elementary folding archetypes holds the promise of becoming the first experimental approach capable of unraveling the basic rules connecting protein structure and folding mechanism.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bryngelson JD, Onuchic JN, Socci ND et al (1995) Funnels, pathways, and the energy landscape of protein-folding—a synthesis. Proteins: Struct Funct Genet 21:167–195
Pande VJ (2008) Computer simulations of protein folding. In: Muñoz V (ed) Protein folding, misfolding and aggregation: classical themes and novel approaches, RSC, Cambridge, pp 161–187
Jackson SE (1998) How do small single-domain proteins fold? Fold Des 3:R81–R91
Kubelka J, Hofrichter J, Eaton WA (2004) The protein folding “speed limit”. Curr Opin Struct Biol 14:76–88
Muñoz V (2007) Conformational dynamics and ensembles in protein folding. Annu Rev Biophys Biomol Struct 36:395–412
Naganathan AN, Doshi U, Fung A et al (2006) Dynamics, energetics, and structure in protein folding. Biochemistry 45:8466–8475
Yang WY, Gruebele M (2003) Folding at the speed limit. Nature 423:193–197
Muñoz V, Sanchez-Ruiz JM (2004) Exploring protein-folding ensembles: a variable-barrier model for the analysis of equilibrium unfolding experiments. Proc Natl Acad Sci USA 101:17646–17651
Naganathan AN, Sanchez-Ruiz JM, Muñoz V (2005) Direct measurement of barrier heights in protein folding. J Am Chem Soc 127:17970–17971
Naganathan AN, Perez-Jimenez R, Sanchez-Ruiz JM et al (2005) Robustness of downhill folding: guidelines for the analysis of equilibrium folding experiments on small proteins. Biochemistry 44:7435–7449
Muñoz V (2002) Thermodynamics and kinetics of downhill protein folding investigated with a simple statistical mechanical model. Int J Quant Chem 90:1522–1528
Garcia-Mira MM, Sadqi M, Fischer N et al (2002) Experimental identification of downhill protein folding. Science 298:2191–2195
Sadqi M, Fushman D, Muñoz V (2006) Atom-by-atom analysis of global downhill protein folding. Nature 442:317–21
Fung A, Li P, Godoy-Ruiz R et al (2008) Expanding the realm of ultrafast protein folding: gpW, a midsize natural single-domain with alpha + beta topology that folds downhill. J Am Chem Soc 130:7489–7495
Marti-Renom MA, Stuart A, Fiser A et al (2000) Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct 29:291–325
De Sancho D, Munoz V (2011) Integrated prediction of protein folding and unfolding rates from only size and structural class. Phys Chem Chem Phys 13: 17030–17043
Bax A, Grzesiek S (1993) Methodological advances in protein NMR. Acc Chem Res 26:131–138
Cavanagh J, Fairbrother WJ III, AGP, Rance M et al (1996) Chemical exchange effects in NMR spectroscopy, in protein NMR spectroscopy: principles and practice, Academic Press, San Diego, p 391–404
Delaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293
Garret DS, Powers R, Gronenborn AM et al (1991) A common sense approach to peak picking in two-, three-, and four-dimensional spectra using computer analysis of contour diagrams. J Magn Reson 95:214–220
Brandes U, Wagner D (2004) In: Juenger M, Mutzel P (ed) Visone—analysis and visualization of social networks, in Graph drawing software, Springer, New York, p 321–340
Schanda P, Brutscher B (2005) Very fast two-dimensional NMR Spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds. J Am Chem Soc 127:8014–8015
Amman C, Meier P, Merbach AE (1982) A simple multinuclear NMR thermometer. J Magn Reson 46:319–321
Naganathan AN, Muñoz V (2008) Determining denaturation midpoints in multiprobe equilibrium protein folding experiments. Biochemistry 47:6752–6761
Sadqi M, Fushman D, Muñoz V (2007) Structural biology—analysis of protein-folding cooperativity—reply. Nature 445:E17–E18
Acknowledgments
This work was supported by the Marie Curie Excellence Award MEXT-CT-2006-042334, and the grants BFU2008-03237, BFU2008-03278 and CONSOLIDER CSD2009-00088 from the Spanish Ministry of Science and Innovation (MICINN).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sborgi, L., Verma, A., Sadqi, M., de Alba, E., Muñoz, V. (2012). Protein Folding at Atomic Resolution: Analysis of Autonomously Folding Supersecondary Structure Motifs by Nuclear Magnetic Resonance. In: Kister, A. (eds) Protein Supersecondary Structures. Methods in Molecular Biology, vol 932. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-065-6_13
Download citation
DOI: https://doi.org/10.1007/978-1-62703-065-6_13
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-064-9
Online ISBN: 978-1-62703-065-6
eBook Packages: Springer Protocols