Home LiteratureArticle Details
PMID: 20157634 Published · ppublish English Journal Article

Resolving Conformational and Rotameric Exchange in Spin-Labeled Proteins Using Saturation Recovery EPR.

Applied magnetic resonance ·Vol. 37 ·No. 1-4 ·2010-01-01 ·Pages 363

Bridges MD, Hideg K, Hubbell WL

Abstract

The function of many proteins involves equilibria between conformational substates, and to elucidate mechanisms of function it is essential to have experimental tools to detect the presence of conformational substates and to determine the time scale of exchange between them. Site-directed spin labeling (SDSL) has the potential to serve this purpose. In proteins containing a nitroxide side chain (R1), multicomponent electron paramagnetic resonance (EPR) spectra can arise either from equilibria involving different conformational substates or rotamers of R1. To employ SDSL to uniquely identify conformational equilibria, it is thus essential to distinguish between these origins of multicomponent spectra. Here we show that this is possible based on the time scale for exchange of the nitroxide between distinct environments that give rise to multicomponent EPR spectra; rotamer exchange for R1 lies in the ≈0.1-1 μs range, while conformational exchange is at least an order of magnitude slower. The time scales of exchange events are determined by saturation recovery EPR, and in favorable cases, the exchange rate constants between substates with lifetimes of approximately 1-70 μs can be estimated by the approach.

Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Bridges Michael D
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-7008, USA.
Hideg Kálmán
Hubbell Wayne L
References (65)
65 references, click to expand
  1. Natively unfolded proteins.
    Curr Opin Struct Biol. 2005 Feb;15(1):35-41 PMID: 15718131
  2. Ligand binding alters the backbone mobility of intestinal fatty acid-binding protein as monitored by 15N NMR relaxation and 1H exchange.
    Biochemistry. 1997 Feb 25;36(8):2278-90 PMID: 9047330
  3. Backbone dynamics of an oncogenic mutant of Cdc42Hs shows increased flexibility at the nucleotide-binding site.
    Biochemistry. 2004 Aug 10;43(31):9968-77 PMID: 15287724
  4. Structural origin of weakly ordered nitroxide motion in spin-labeled proteins.
    Protein Sci. 2009 May;18(5):893-908 PMID: 19384990
  5. Molecular motion of spin labeled side chains in alpha-helices: analysis by variation of side chain structure.
    Biochemistry. 2001 Apr 3;40(13):3828-46 PMID: 11300763
  6. The energy landscapes and motions of proteins.
    Science. 1991 Dec 13;254(5038):1598-603 PMID: 1749933
  7. Impact of electron-electron spin interaction on electron spin relaxation of nitroxide diradicals and tetraradical in glassy solvents between 10 and 300 k.
    J Phys Chem B. 2008 Mar 13;112(10):2818-28 PMID: 18284225
  8. Dynamics of the transition between open and closed conformations in a calmodulin C-terminal domain mutant.
    Structure. 2001 Mar 7;9(3):185-95 PMID: 11286885
  9. Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect.
    Science. 1992 Jan 10;255(5041):178-83 PMID: 1553543
  10. Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy.
    Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14171-6 PMID: 12381795
  11. Pervasive conformational fluctuations on microsecond time scales in a fibronectin type III domain.
    Nat Struct Biol. 1998 Jan;5(1):55-9 PMID: 9437430
  12. Oxygen transport parameter in membranes as deduced by saturation recovery measurements of spin-lattice relaxation times of spin labels.
    Proc Natl Acad Sci U S A. 1982 Mar;79(6):1854-8 PMID: 6952236
  13. Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy.
    Chem Rev. 2006 May;106(5):1700-19 PMID: 16683750
  14. Implications of using approximate Bloch-McConnell equations in NMR analyses of chemically exchanging systems: application to the electron self-exchange of plastocyanin.
    J Magn Reson. 2003 Aug;163(2):215-27 PMID: 12914837
  15. Inherent flexibility in a potent inhibitor of blood coagulation, recombinant nematode anticoagulant protein c2.
    Eur J Biochem. 1999 Oct;265(2):539-48 PMID: 10504384
  16. Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA.
    Biochemistry. 2004 Jun 15;43(23):7273-87 PMID: 15182173
  17. Mechanism of coupled folding and binding of an intrinsically disordered protein.
    Nature. 2007 Jun 21;447(7147):1021-5 PMID: 17522630
  18. Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale.
    Biochemistry. 1973 Oct 9;12(21):4171-9 PMID: 4200894
  19. Solution of the nitroxide spin-label spectral overlap problem using pulse electron spin resonance.
    Biophys J. 1988 Apr;53(4):525-31 PMID: 2838099
  20. Relating side-chain mobility in proteins to rotameric transitions: insights from molecular dynamics simulations and NMR.
    J Biomol NMR. 2005 Jun;32(2):151-62 PMID: 16034666
  21. Dynamic personalities of proteins.
    Nature. 2007 Dec 13;450(7172):964-72 PMID: 18075575
  22. Disulfide bond isomerization in basic pancreatic trypsin inhibitor: multisite chemical exchange quantified by CPMG relaxation dispersion and chemical shift modeling.
    J Am Chem Soc. 2003 Nov 26;125(47):14324-35 PMID: 14624581
  23. Demonstration of short-lived complexes of cytochrome c with cytochrome bc1 by EPR spectroscopy: implications for the mechanism of interprotein electron transfer.
    J Biol Chem. 2008 Sep 5;283(36):24826-36 PMID: 18617515
  24. Antibody multispecificity mediated by conformational diversity.
    Science. 2003 Feb 28;299(5611):1362-7 PMID: 12610298
  25. Site-directed spin labeling of proteins. Applications to diphtheria toxin.
    Methods Mol Biol. 2000;145:147-69 PMID: 10820721
  26. Insights into the mobility of methyl-bearing side chains in proteins from (3)J(CC) and (3)J(CN) couplings.
    J Am Chem Soc. 2003 Jul 23;125(29):8959-66 PMID: 12862493
  27. Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR.
    Biophys J. 2005 Sep;89(3):2103-12 PMID: 15994891
  28. Hydrogen exchange.
    Annu Rev Biochem. 1972;41:903-24 PMID: 4563445
  29. Pulse EPR detection of lipid exchange between protein-rich raft and bulk domains in the membrane: methodology development and its application to studies of influenza viral membrane.
    Biophys J. 2001 Feb;80(2):738-48 PMID: 11159441
  30. Explanation of spin-lattice relaxation rates of spin labels obtained with multifrequency saturation recovery EPR.
    J Phys Chem A. 2005 May 12;109(18):4049-61 PMID: 16833727
  31. Exploring subdomain cooperativity in T4 lysozyme II: uncovering the C-terminal subdomain as a hidden intermediate in the kinetic folding pathway.
    Protein Sci. 2007 May;16(5):852-62 PMID: 17400925
  32. Lateral diffusion of lipids in membranes by pulse saturation recovery electron spin resonance.
    Proc Natl Acad Sci U S A. 1987 Feb;84(4):964-8 PMID: 3029766
  33. Thermodynamic fluctuations in protein molecules.
    Proc Natl Acad Sci U S A. 1976 Aug;73(8):2740-1 PMID: 1066687
  34. Function and structure of inherently disordered proteins.
    Curr Opin Struct Biol. 2008 Dec;18(6):756-64 PMID: 18952168
  35. A new spin on protein dynamics.
    Trends Biochem Sci. 2002 Jun;27(6):288-95 PMID: 12069788
  36. mRNA guanylation catalyzed by the S-adenosylmethionine-dependent guanylyltransferase of bamboo mosaic virus.
    J Biol Chem. 2005 Apr 1;280(13):13153-62 PMID: 15677480
  37. Sequence of late molecular events in the activation of rhodopsin.
    Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20290-5 PMID: 18077356
  38. Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics.
    Biochemistry. 1996 Jun 18;35(24):7692-704 PMID: 8672470
  39. Structural determinants of nitroxide motion in spin-labeled proteins: tertiary contact and solvent-inaccessible sites in helix G of T4 lysozyme.
    Protein Sci. 2007 Jun;16(6):1069-86 PMID: 17473014
  40. Backbone dynamics and energetics of a calmodulin domain mutant exchanging between closed and open conformations.
    J Mol Biol. 1999 Jun 11;289(3):603-17 PMID: 10356332
  41. A hierarchy of timescales in protein dynamics is linked to enzyme catalysis.
    Nature. 2007 Dec 6;450(7171):913-6 PMID: 18026087
  42. Intrinsic dynamics of an enzyme underlies catalysis.
    Nature. 2005 Nov 3;438(7064):117-21 PMID: 16267559
  43. Structural determinants of nitroxide motion in spin-labeled proteins: solvent-exposed sites in helix B of T4 lysozyme.
    Protein Sci. 2008 Feb;17(2):228-39 PMID: 18096642
  44. Enhanced protein flexibility caused by a destabilizing amino acid replacement in BPTI.
    J Mol Biol. 1997 May 30;269(1):154-64 PMID: 9193007
  45. Probing molecular motion by NMR.
    Curr Opin Struct Biol. 1997 Oct;7(5):732-7 PMID: 9345634
  46. Dynamics of ligand binding to myoglobin.
    Biochemistry. 1975 Dec 2;14(24):5355-73 PMID: 1191643
  47. Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution.
    Science. 2008 Jun 13;320(5882):1471-5 PMID: 18556554
  48. Fast relaxation processes inn a protein revealed by the decay kinetics of tryptophan fluorescence.
    Biochemistry. 1974 Dec 3;13(25):5170-8 PMID: 4611479
  49. Linking folding and binding.
    Curr Opin Struct Biol. 2009 Feb;19(1):31-8 PMID: 19157855
  50. Equilibrium and pressure-jump relaxation studies of the conformational transitions of P13MTCP1.
    J Mol Biol. 2002 Jul 12;320(3):609-28 PMID: 12096913
  51. Orientation and motion of amphiphilic spin labels in membranes.
    Proc Natl Acad Sci U S A. 1969 Sep;64(1):20-7 PMID: 4312749
  52. A novel pressure-jump apparatus for the microvolume analysis of protein-ligand and protein-protein interactions: its application to nucleotide binding to skeletal-muscle and smooth-muscle myosin subfragment-1.
    Biochem J. 2002 Sep 1;366(Pt 2):643-51 PMID: 12010120
  53. Crystal structures of spin labeled T4 lysozyme mutants: implications for the interpretation of EPR spectra in terms of structure.
    Biochemistry. 2000 Jul 25;39(29):8396-405 PMID: 10913245
  54. Binding induced conformational changes of proteins correlate with their intrinsic fluctuations: a case study of antibodies.
    BMC Struct Biol. 2007 May 17;7:31 PMID: 17509130
  55. Biophysics: proteins hunt and gather.
    Nature. 2007 Jun 21;447(7147):920-1 PMID: 17581572
  56. Millisecond time scale conformational flexibility in a hyperthermophile protein at ambient temperature.
    Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3166-70 PMID: 10716696
  57. Subdomain interactions as a determinant in the folding and stability of T4 lysozyme.
    Protein Sci. 1998 Jan;7(1):96-104 PMID: 9514264
  58. Osmolyte perturbation reveals conformational equilibria in spin-labeled proteins.
    Protein Sci. 2009 Aug;18(8):1637-52 PMID: 19585559
  59. Accessibility and dynamics of nitroxide side chains in T4 lysozyme measured by saturation recovery EPR.
    Biophys J. 2005 Sep;89(3):2059-68 PMID: 15994892
  60. Novel regulation and function of Src tyrosine kinase.
    Cell Mol Life Sci. 2002 Mar;59(3):456-62 PMID: 11964124
  61. Primary structure effects on peptide group hydrogen exchange.
    Biochemistry. 1972 Jan 18;11(2):150-8 PMID: 5061873
  62. New tools provide new insights in NMR studies of protein dynamics.
    Science. 2006 Apr 14;312(5771):224-8 PMID: 16614210
  63. Electron spin-lattice relaxation of nitroxyl radicals in temperature ranges that span glassy solutions to low-viscosity liquids.
    J Magn Reson. 2008 Mar;191(1):66-77 PMID: 18166493
  64. The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains.
    J Biol Chem. 2002 Oct 18;277(42):39401-8 PMID: 12171941
  65. A novel reversible thiol-specific spin label: papain active site labeling and inhibition.
    Anal Biochem. 1982 Jan 15;119(2):450-5 PMID: 6280514
Article Info
Journal
Applied magnetic resonance
Abbr.
Appl Magn Reson
ISSN
0937-9347
DOI
10.1007/s00723-009-0079-2
Published
2010-01-01
Pages
363
Language
English
Region
Austria
NLM ID
9204100
PMCID
PMC2821067
Grants
NEI NIH HHS · R01 EY005216-27 · United States
NEI NIH HHS · R01 EY005216-29 · United States
NEI NIH HHS · R01 EY005216 · United States
NEI NIH HHS · R37 EY005216 · United States
NEI NIH HHS · T32 EY007026 · United States
NIBIB NIH HHS · P41 EB001980 · United States
External Links
PubMed source DOI: 10.1007/s00723-009-0079-2 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2