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Intrinsically Disordered Protein Analysis pp 405–419Cite as

Deconstructing Time-Resolved Optical Rotatory Dispersion Kinetic Measurements of Cytochrome c Folding: From Molten Globule to the Native State

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 895))

Abstract

The far-UV time-resolved optical rotatory dispersion (TRORD) technique has contributed significantly to our understanding of nanosecond secondary structure kinetics in protein folding and function reactions. For reduced cytochrome c, protein folding kinetics have been probed largely from the unfolded to the native state. Here we provide details about sample preparation and the TRORD apparatus and measurements for studying folding from a partly unfolded state to the native secondary structure conformation of reduced cytochrome c.

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References

  1. Lowry TM (1935) Optical rotatory power. Longmans, Green and Co., London

    Google Scholar 

  2. Keston A, Lospalluto J (1953) Simple ultrasensitive spectropolarimeters. Fed Proc 12:229

    Google Scholar 

  3. Poulsen KG (1960) Evaluation of standard model-D Keston polarimetric attachment for the Beckman Du spectrophotometer. Anal Chem 32:410–413

    Article  CAS  Google Scholar 

  4. Gallop PM (1957) Simplified formula for the operation of the Keston-type polarimeter. Rev Sci Instrum 28:209

    Article  CAS  Google Scholar 

  5. Lewis JW et al (1985) New technique for measuring circular dichroism changes on a nanosecond time scale—application to (carbonmonoxy)myoglobin and (carbonmonoxy)hemoglobin. J Phys Chem 89:289–294

    Article  CAS  Google Scholar 

  6. Milder SJ et al (1990) Assignments of ground-state and excited-state spectra from time-resolved absorption and circular dichroism measurements of the 2E state of (Δ)-Cr(Bpy) 3+3 . Inorg Chem 29:2506–2511

    Article  CAS  Google Scholar 

  7. Chen E et al (2010) Nanosecond time-resolved polarization spectroscopies: tools for probing protein reaction mechanisms. Methods 52(1):3–11

    Article  PubMed  CAS  Google Scholar 

  8. Chen EF et al (2005) Nanosecond laser temperature-jump optical rotatory dispersion: application to early events in protein folding/unfolding. Rev Sci Instrum 76:083120

    Article  Google Scholar 

  9. Chen EF et al (1993) Time-resolved UV circular dichroism of phytochrome A: folding of the N-terminal region. J Am Chem Soc 115:9854–9855

    Article  CAS  Google Scholar 

  10. Chen EF et al (1997) Dynamics of the N-terminal α-helix unfolding in the photoreversion reaction of phytochrome A. Biochemistry 36:4903–4908

    Article  PubMed  CAS  Google Scholar 

  11. Chen EF et al (2007) A LOV story: the signaling state of the Phot1 LOV2 photocycle involves chromophore-triggered protein structure relaxation, as probed by far-UV time-resolved optical rotatory dispersion spectroscopy. Biochemistry 46:4619–4624

    Article  PubMed  CAS  Google Scholar 

  12. Chen EF et al (2003) Dynamics of protein and chromophore structural changes in the photocycle of photoactive yellow protein monitored by time-resolved optical rotatory dispersion. Biochemistry 42:2062–2071

    Article  PubMed  CAS  Google Scholar 

  13. Chen EF, Kliger DS (1996) Time-resolved near UV circular dichroism and absorption studies of carbonmonoxymyoglobin photolysis intermediates. Inorg Chim Acta 242:149–158

    Article  CAS  Google Scholar 

  14. Chen EF et al (1998) Time-resolved circular dichroism studies of protein folding intermediates of cytochrome c. Biochemistry 37:5589–5598

    Article  PubMed  CAS  Google Scholar 

  15. Chen EF et al (1999) Far-UV time-resolved circular dichroism detection of electron-transfer-triggered cytochrome c folding. J Am Chem Soc 121:3811–3817

    Article  CAS  Google Scholar 

  16. Chen EF et al (2003) Earliest events in protein folding: submicrosecond secondary structure formation in reduced cytochrome c. J Phys Chem A 107:8149–8155

    Article  CAS  Google Scholar 

  17. Chen EF et al (2004) The earliest events in protein folding: a structural requirement for ultrafast folding in cytochrome c. J Am Chem Soc 126:11175–11181

    Article  PubMed  CAS  Google Scholar 

  18. Chen EF et al (2003) The kinetics of helix unfolding of an azobenzene cross-linked peptide probed by nanosecond time-resolved optical rotatory dispersion. J Am Chem Soc 125:12443–12449

    Article  PubMed  CAS  Google Scholar 

  19. Chen E et al (2007) Non-native heme-histidine ligation promotes microsecond time scale secondary structure formation in reduced horse heart cytochrome c. Biochemistry 46:12463–12472

    Article  PubMed  CAS  Google Scholar 

  20. Chen E et al (2008) The folding kinetics of the SDS-induced molten globule form of reduced cytochrome c. Biochemistry 47:5450–5459

    Article  PubMed  CAS  Google Scholar 

  21. Moscowitz A (1962) Theoretical aspects of optical activity. 1. Small molecules. Adv Chem Phys 4:67–112

    Article  Google Scholar 

  22. Latypov RF et al (2008) Folding mechanism of reduced cytochrome c: equilibrium and kinetic properties in the presence of carbon monoxide. J Mol Biol 383:437–453

    Article  PubMed  CAS  Google Scholar 

  23. Jones CM et al (1993) Fast events in protein folding initiated by nanosecond laser photolysis. Proc Natl Acad Sci U S A 90:11860–11864

    Article  PubMed  CAS  Google Scholar 

  24. Pascher T et al (1996) Protein folding triggered by electron transfer. Science 271:1558–1560

    Article  PubMed  CAS  Google Scholar 

  25. Mines GA et al (1996) Cytochrome c folding triggered by electron transfer. Chem Biol 3:491–497

    Article  PubMed  CAS  Google Scholar 

  26. Telford JR et al (1998) Protein folding triggered by electron transfer. Acc Chem Res 31:755–763

    Article  CAS  Google Scholar 

  27. Pascher T (2001) Temperature and driving force dependence of the folding rate of reduced horse heart cytochrome c. Biochemistry 40:5812–5820

    Article  PubMed  CAS  Google Scholar 

  28. Hagen SJ et al (2002) Rapid intrachain binding of histidine-26 and histidine-33 to heme in unfolded ferrocytochrome c. Biochemistry 41:1372–1380

    Article  PubMed  CAS  Google Scholar 

  29. Pabit SA et al (2004) Internal friction controls the speed of protein folding from a compact configuration. Biochemistry 43:12532–12538

    Article  PubMed  CAS  Google Scholar 

  30. Colon W, Roder H (1996) Kinetic intermediates in the formation of the cytochrome c molten globule. Nat Struct Biol 3:1019–1025

    Article  PubMed  CAS  Google Scholar 

  31. Sanghera N, Pinheiro TJ (2000) Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles. Protein Sci 9:1194–1202

    Article  PubMed  CAS  Google Scholar 

  32. Dolgikh DA et al (1985) Compact state of a protein molecule with pronounced small-scale mobility: bovine alpha-lactalbumin. Eur Biophys J 13:109–121

    Article  PubMed  CAS  Google Scholar 

  33. Moza B et al (2006) A unique molten globule state occurs during unfolding of cytochrome c by LiClO4 near physiological pH and temperature: structural and thermodynamic characterization. Biochemistry 45:4695–4702

    Article  PubMed  CAS  Google Scholar 

  34. Dolgikh DA et al (1984) “Molten-globule” state accumulates in carbonic anhydrase folding. FEBS Lett 165:88–92

    Article  PubMed  CAS  Google Scholar 

  35. Pinheiro TJ et al (1997) Structural and kinetic description of cytochrome c unfolding induced by the interaction with lipid vesicles. Biochemistry 36:13122–13132

    Article  PubMed  CAS  Google Scholar 

  36. Davis-Searles PR et al (1998) Sugar-induced molten-globule model. Biochemistry 37:17048–17053

    Article  PubMed  CAS  Google Scholar 

  37. Sedlak E, Antalik M (1999) Molten globule-like state of cytochrome c induced by polyanion poly(vinylsulfate) in slightly acidic pH. Biochim Biophys Acta 1434:347–355

    Article  PubMed  CAS  Google Scholar 

  38. Das TK et al (1998) Characterization of a partially unfolded structure of cytochrome c induced by sodium dodecyl sulphate and the kinetics of its refolding. Eur J Biochem 254:662–670

    Article  PubMed  CAS  Google Scholar 

  39. Hiramatsu K, Yang JT (1983) Cooperative binding of hexadecyltrimethylammonium chloride and sodium dodecyl sulfate to cytochrome c and the resultant change in protein conformation. Biochim Biophys Acta 743:106–114

    Article  PubMed  CAS  Google Scholar 

  40. Takeda K et al (1985) Kinetic aspects of the interaction of horse heart cytochrome c with sodium dodecyl sulfate. Arch Biochem Biophys 236:411–417

    Article  PubMed  CAS  Google Scholar 

  41. Oellerich S et al (2003) Conformational equilibria and dynamics of cytochrome c induced by binding of sodium dodecyl sulfate monomers and micelles. Eur Biophys J 32:599–613

    Article  PubMed  CAS  Google Scholar 

  42. Moosavi-Movahedi AA et al (2003) Formation of the molten globule-like state of cytochrome c induced by n-alkyl sulfates at low concentrations. J Biochem 133:93–102

    Article  PubMed  CAS  Google Scholar 

  43. Xu Q, Keiderling TA (2004) Effect of sodium dodecyl sulfate on folding and thermal stability of acid-denatured cytochrome c: a spectroscopic approach. Protein Sci 13:2949–2959

    Article  PubMed  CAS  Google Scholar 

  44. Sosnick TR et al (1994) The barriers in protein folding. Nat Struct Biol 1:149–156

    Article  PubMed  CAS  Google Scholar 

  45. Margoliash E, Frohwirt N (1959) Spectrum of horse-heart cytochrome c. Biochem J 71:570–578

    PubMed  CAS  Google Scholar 

  46. Di Iorio EE (1981) Preparation of derivatives of ferrous and ferric hemoglobin. Methods Enzymol 76:57–72

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA

    Eefei Chen & David S. Kliger

Authors
  1. Eefei Chen
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  2. David S. Kliger
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Corresponding author

Correspondence to Eefei Chen .

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Editors and Affiliations

  1. Department of Molecular Medicine, College of Medicine, University of South Florida, Bruce B. Downs Blvd 12901, Tampa, 33612, Florida, USA

    Vladimir N. Uversky

  2. Dept. Biochemistry & Molecular Biology, Center for Computational Biology &, Indiana University, West 10th Street 410, Indianapolis, 46202, Indiana, USA

    A. Keith Dunker

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Chen, E., Kliger, D.S. (2012). Deconstructing Time-Resolved Optical Rotatory Dispersion Kinetic Measurements of Cytochrome c Folding: From Molten Globule to the Native State. In: Uversky, V., Dunker, A. (eds) Intrinsically Disordered Protein Analysis. Methods in Molecular Biology, vol 895. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-927-3_23

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  • DOI: https://doi.org/10.1007/978-1-61779-927-3_23

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-926-6

  • Online ISBN: 978-1-61779-927-3

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