Abstract
As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure.
Similar content being viewed by others
Abbreviations
- NMR:
-
Nuclear magnetic resonance
- SDSL:
-
Site-directed spin-labelling
- ESR:
-
Electron spin resonance
- EPR:
-
Electron paramagnetic resonance
- GHOST:
-
Condensation algorithm that filters and groups the solutions found in optimization runs
- NTAIL:
-
C-terminal domain of nucleoprotein of the measles virus
References
Alexander N, Al-Mestarihi A, Bortolus M, McHaourab H, Meiler J (2008) De novo high-resolution protein structure determination from sparse spin-labeling EPR data. Structure 16:181–195
Arora A, Tamm LK (2001) Biophysical approaches to membrane protein structure determination. Curr Opin Struct Biol 11:540–547
Athanasiadis A, Anderluh G, Macek P, Turk D (2001) Crystal structure of the soluble form of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina. Structure 9:341–346
Bax A (1989) Two-dimensional NMR and protein structure. Annu Rev Biochem 58:223–256
Belle V, Fournel A, Woudstra M, Ranaldi S, Prieri F, Thome V, Currault J, Verger R, Guigliarelli B, Carriere F (2007) Probing the opening of the pancreatic lipase lid using site-directed spin labeling and EPR spectroscopy. Biochem 46:2205–2214
Belle V, Rouger S, Costanzo S, Liquiere E, Štrancar J, Guigliarelli B, Fournel A, Longhi S (2008) Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy. Proteins 73:973–988
Bourhis J-M, Canard B, Longhi S (2007) Predicting protein disorder and induced folding: from theoretical principles to practical applications. Curr Protein Pept Sci 8:135–149
Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M (1983) CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J Comput Chem 4:187–217
Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–921
Castellani F, van Rossum B, Diehl A, Schubert M, Rehbein K, Oschkinat H (2002) Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy. Nature 420:98–102
Dominguez C, Boelens R, Bonvin AMJJ (2003) HADDOCK: a protein–protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125:1731–1737
Dunbrack JRL (2002) Rotamer libraries in the 21st century. Curr Opin Struct Biol 12:431–440
Dunbrack JRL, Cohen FE (1997) Bayesian statistical analysis of protein side-chain rotamer preferences. Protein Sci 6:1661–1681
Dunbrack JRL, Karplus M (1993) Backbone-dependent rotamer library for proteins application to side-chain prediction. J Mol Biol 230:543–574
Dunker AK, Lawson JD, Brown CJ, Williams RM, Romero P, Oh JS, Oldfield CJ, Campen AM, Ratliff CM, Hipps KW, Ausio J, Nissen MS, Reeves R, Kang C, Kissinger CR, Bailey RW, Griswold MD, Chiu W, Garner EC, Obradovic Z (2001) Intrinsically disordered protein. J Mol Graph Model 19:26–59
Dunker AK, Cortese MS, Romero P, Iakoucheva LM, Uversky VN (2005) Flexible nets: the roles of intrinsic disorder in protein interaction networks. FEBS J 272:5129–5148
Dunker AK, Oldfield C, Meng J, Romero P, Yang J, Chen J, Vacic V, Obradovic Z, Uversky V (2008) The unfoldomics decade: an update on intrinsically disordered proteins. BMC Genomics 9:S1
Dyson HJ, Wright PE (2005) Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6:197–208
Eiben AE, Smith JE (2003) Introduction to evolutionary computing. Springer, New York
Engh RA, Huber R (1991) Accurate bond and angle parameters for X-ray protein structure refinement. Acta Crystallogr Sect A 47:392–400
Fajer M, Fajer PG, Sale KL (2007) Molecular modeling of spin labels. In: Hemminga MA, Berliner L (eds) ESR spectroscopy in membrane biophysics. Springer, New York, pp 253–259
Fanucci GE, Cafiso DS (2006) Recent advances and applications of site-directed spin labeling. Curr Opin Struct Biol 16:644–653
Fasman GD (1996) Circular dichroism and the conformational analysis of biomolecules. Plenum Press, New York
Ferron F, Longhi S, Canard B, Karlin D (2006) A practical overview of protein disorder prediction methods. Proteins 65:1–14
Filipič B, Štrancar J (2001) Tuning EPR spectral parameters with a genetic algorithm. Appl Soft Comput 1:83–90
Fink AL (2005) Natively unfolded proteins. Curr Opin Struct Biol 15:35–41
Fleishman SJ, Unger VM, Ben-Tal N (2006) Transmembrane protein structures without X-rays. Trends Biochem Sci 31:106–113
Fogel DB, Bäck T, Michalewicz Z (2000) Evolutionary computation. Philadelphia, Institute of Physics Publishing, Bristol
Grigoryan G, Ochoa A, Keating AE (2007) Computing van der Waals energies in the context of the rotamer approximation. Proteins 68:863–878
Hemminga MA (2007) Introduction and future of site-directed spin labeling of membrane proteins. In: Hemminga MA, Berliner L (eds) ESR Spectroscopy in Membrane Biophysics. pp, Springer, pp 1–16
Henderson R (2004) Realizing the potential of electron cryo-microscopy. Q Rev Biophys 37:3–13
Hinds MG, Zhang W, Anderluh G, Hansen PE, Norton RS (2002) Solution structure of the eukaryotic pore-forming cytolysin equinatoxin II: implications for pore formation. J Mol Biol 315:1219–1229
Ho BK, Thomas A, Brasseur R (2003) Revisiting the Ramachandran plot: hard-sphere repulsion, electrostatics, and H-bonding in the α-helix. Protein Sci 12:2508–2522
Huang H, Cafiso DS (2008) Conformation and membrane position of the region linking the two C2 domains in synaptotagmin 1 by site-directed spin labeling. Biochem 47:12380–12388
Hubbell WL, Gross A, Langen R, Lietzow MA (1998) Recent advances in site-directed spin labeling of proteins. Curr Opin Struct Biol 8:649–656
Jao CC, Hegde BG, Chen J, Haworth IS, Langen R (2008) Structure of membrane-bound α-synuclein from site-directed spin labeling and computational refinement. Proc Natl Acad Sci USA 105:19666–19671
Johansson AC, Lindahl E (2006) Amino-acid solvation structure in transmembrane helices from molecular dynamics simulations. Biophys J 91:4450–4463
Kavalenka AA, Filipič B, Hemminga MA, Štrancar J (2005) Speeding up a genetic algorithm for EPR-based spin label characterization of biosystem complexity. J Chem Inf Mod 45:1628–1635
Kavalenka A, Hemminga MA, Štrancar J (2009a) Optimization of membrane protein structure based on SDSL-ESR constraints and conformational space modeling. Biophys J (submitted)
Kavalenka A, Urbančič I, Belle V, Rouger S, Costanzo S, Kure S, Fournel A, Longhi S, Guigliarelli B, Štrancar J (2009b) Conformational analysis of the partially disordered measles virus NTAIL–XD complex explored by SDSL EPR spectroscopy. Biophys J (submitted)
Kelly SM, Price NC (2000) The use of circular dichroism in the investigation of protein structure and function. Curr Protein Pept Sci 1:349–384
Kirkpatrick S, Gelatt CD Jr, Vecchi MP (1983) Optimization by simulated annealing. Science 220:671–680
Lacapère JJ, Pebay-Peyroula E, Neumann JM, Etchebest C (2007) Determining membrane protein structures: still a challenge! Trends Biochem Sci 32:259–270. Epub 2007 May 3. Review.
Lehninger AL, Nelson DL, Cox MM (2005) Lehninger principles of biochemistry, 4th edn edn. W.H. Freeman, New York
Li Q, Fung LW-M (2009) Structural and dynamic study of the tetramerization region of non-erythroid α-spectrin: a frayed helix revealed by site-directed spin labeling electron paramagnetic resonance. Biochem 48:206–215
Lovell SC, Word JM, Richardson JS, Richardson DC (2000) The penultimate rotamer library. Proteins 40:389–408
MacCallum JL, Bennett WFD, Tieleman DP (2008) Distribution of amino acids in a lipid bilayer from computer simulations. Biophys J 94:3393–3404
MacKerell AD, Bashford D, Bellott M, Dunbrack RL, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S, Joseph-McCarthy D, Kuchnir L, Kuczera K, Lau FTK, Mattos C, Michnick S, Ngo T, Nguyen DT, Prodhom B, Reiher WE, Roux B, Schlenkrich M, Smith JC, Stote R, Straub J, Watanabe M, Wiorkiewicz-Kuczera J, Yin D, Karplus M (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102:3586–3616
Malovrh P, Viero G, Serra MD, Podlesek Z, Lakey JH, Macek P, Menestrina G, Anderluh G (2003) A novel mechanism of pore formation: membrane penetration by the N-terminal amphipathic region of equinatoxin. J Biol Chem 278:22678–22685
Marsh D (1981) Electron Spin Resonance: Spin Labels. In: Grell E (ed) Membrane Spectroscopy. Springer-Verlag, Berlin New York, pp 51–142
Marsh D (2008) Protein modulation of lipids, and vice versa, in membranes. Biochim Biophys Acta 1778:1545–1575
Metropolis N, Rosenbluth AW, Rosenbluth MN, Teller AH, Teller E (1953) Equation of state calculations by fast computing machines. J Chem Phys 21:1087–1092
Muller DJ, Engel A (2008) Strategies to prepare and characterize native membrane proteins and protein membranes by AFM. Curr Opin Colloid Interface Sci 13:338–350
Nordio PL (1976) General magnetic resonance theory. In: Berliner LJ (ed) Spin labeling: theory and applications. Academic Press, New York, pp 5–51
Pebay-Peyroula E (2008) Biophysical analysis of membrane proteins: investigating structure and function. Wiley-VCH, Weinheim
Petoukhov MV, Svergun DI (2005) Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophys J 89:1237–1250
Pistolesi S, Ferro E, Santucci A, Basosi R, Trabalzini L, Pogni R (2006) Molecular motion of spin labeled side chains in the C-terminal domain of RGL2 protein: A SDSL-EPR and MD study. Biophys Chem 123:49–57
Ramya Bhargavi G, Sheik SS, Velmurugan D, Sekar K (2003) Side-chain conformation angles of amino acids: effect of temperature factor cut-off. J Struct Biol 143:181–184
Receveur-Bréchot V, Bourhis JM, Uversky VN, Canard B, Longhi S (2006) Assessing protein disorder and induced folding. Proteins 62:24–45
Schindler H, Seelig J (1973) EPR spectra of spin labels in lipid bilayers. J Chem Phys 59:1841–1850
Shetty RP, De Bakker PI, DePristo MA, Blundell TL (2003) Advantages of fine-grained side chain conformer libraries. Protein Eng 16:963–969
Steinhoff H-J, Savitsky A, Wegener C, Pfeiffer M, Plato M, Mцbius K (2000) High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin. Biochim Biophys Acta 1457:253–262
Stopar D, Štrancar J, Spruijt RB, Hemminga MA (2005) Exploring the local conformational space of a membrane protein by site-directed spin labeling. J Chem Inf Mod 45:1621–1627
Stopar D, Štrancar J, Spruijt RB, Hemminga MA (2006) Motional restrictions of membrane proteins: a site-directed spin labeling study. Biophys J 91:3341–3348
Štrancar J (2007) Advanced ESR spectroscopy in membrane biophysics. In: Hemminga MA, Berliner L (eds) ESR spectroscopy in membrane biophysics. Springer, New York, pp 49–93
Štrancar J, Sentjurc M, Schara M (2000) Fast and accurate characterization of biological membranes by EPR spectral simulations of nitroxides. J Magn Reson 142:254–265
Štrancar J, Koklic T, Arsov Z, Filipic B, Stopar D, Hemminga MA (2005) Spin label EPR-based characterization of biosystem complexity. J Chem Inf Mod 45:394–406
Štrancar J, Kavalenka A, Ziherl P, Stopar D, Hemminga MA (2009) Analysis of side chain rotational restrictions of membrane-embedded proteins by spin-label ESR spectroscopy. J Magn Reson 197:245–248
Svergun DI, Koch MHJ (2003) Small-angle scattering studies of biological macromolecules in solution. Rep Prog Phys 66:1735–1782
Timsit Y, Allemand F, Chiaruttini C, Springer M (2006) Coexistence of two protein folding states in the crystal structure of ribosomal protein L20. EMBO Rep 7:1013–1018
Tombolato F, Ferrarini A, Freed JH (2006) Dynamics of the nitroxide side chain in spin-labeled proteins. J Phys Chem B 110:26248–26259
Tompa P (2002) Intrinsically unstructured proteins. Trends Biochem Sci 27:527–533
Torres J, Stevens TJ, Samsó M (2003) Membrane proteins: the ‘Wild West' of structural biology. Trends Biochem Sci 28:137–144. Review. Erratum in: Trends Biochem Sci 2003 28:174
Uversky VN (2002) Natively unfolded proteins: a point where biology waits for physics. Protein Sci 11:739–756
Vasquez M (1996) Modeling side-chain conformation. Curr Opin Struct Biol 6:217–221
Vermeer LS, de Groot BL, Reat V, Milon A, Czaplicki J (2007) Acyl chain order parameter profiles in phospholipid bilayers: computation from molecular dynamics simulations and comparison with 2H NMR experiments. Eur Biophys J 36:919–931
Watts A, Straus SK, Grage SL, Kamihira M, Lam YH, Zhao X (2004) Membrane protein structure determination using solid-state NMR. Methods Mol Biol 278:403–473
White S (2009) Membrane proteins of known 3D structure. http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
Wiener MC, White SH (1992) Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of X-ray and neutron diffraction data III. Complete structure. Biophys J 61:434–447
Winkler FK, D’Arcy A, Hunziker W (1990) Structure of human pancreatic lipase. Nature 343:771–774
Word JM, Lovell SC, LaBean TH, Taylor HC, Zalis ME, Presley BK, Richardson JS, Richardson DC (1999) Visualizing and quantifying molecular goodness-of-fit: small-probe contact dots with explicit hydrogen atoms. J Mol Biol 285:1711–1733
Wright PE, Dyson HJ (2009) Linking folding and binding. Curr Opin Struct Biol 19:31–38
Wüthrich K (1986) NMR of proteins and nucleic acids. Wiley, New York
Xiang Z, Honig B (2001) Extending the accuracy limits of prediction for side-chain conformations. J Mol Biol 311:421–430
Acknowledgments
We wish to thank David Stopar and Primoz Ziherl (University of Ljubljana) for many valuable discussions at the very beginning of development of the methodology, Valerie Belle and Bruno Guigliarelli (BIP CNRS Marseille) and Sonia Longhi (AFMB CNRS Marseille) for helping us to develop the algorithm for cleaning the motional pattern profiles on the NTAIL problem. We are also grateful to Jan Premru for important contribution in analysis of the rotamer libraries. This work was carried out with the financial support of the Slovenian Research Agency (program “Experimental biophysics of complex systems”, P1-0060) and COST P15 action support (through a Short Term Scientific Meeting grant).
Author information
Authors and Affiliations
Corresponding author
Additional information
The more you see: Spectroscopy in molecular biophysics.
Rights and permissions
About this article
Cite this article
Štrancar, J., Kavalenka, A., Urbančič, I. et al. SDSL-ESR-based protein structure characterization. Eur Biophys J 39, 499–511 (2010). https://doi.org/10.1007/s00249-009-0510-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-009-0510-5