Skip to main content
SpringerLink
Log in

Complexity of aromatic ring-flip motions in proteins: Y97 ring dynamics in cytochrome c observed by cross-relaxation suppressed exchange NMR spectroscopy

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

Dynamics of large-amplitude conformational motions in proteins are complex and less understood, although these processes are intimately associated with structure, folding, stability, and function of proteins. Here, we use a large set of spectra obtained by cross-relaxation suppressed exchange NMR spectroscopy (EXSY) to study the 180° flipping motion of the Y97 ring of horse ferricytochrome c as a function of near-physiological temperature in the 288–308 K range. With rising temperature, the ring-flip rate constant makes a continuous transition from Arrhenius to anti-Arrhenius behavior through a narrow Arrhenius-like zone. This behavior is seen not only for the native state of the protein, but also for native-like states generated by adding subdenaturing amounts of guanidine deuterochloride (GdnDCl). Moderately destabilizing concentrations of the denaturant (1.5 M GdnDCl) completely removes the Arrhenius-like feature from the temperature window employed. The Arrhenius to anti-Arrhenius transition can be explained by the heat capacity model where temperature strengthens ground state interactions, perhaps hydrophobic in nature. The effect of the denaturant may appear to arise from direct protein-denaturant interactions that are structure-stabilizing under subdenaturing conditions. The temperature distribution of rate constants under different stability conditions also suggests that the prefactor in Arrhenius-like relations is temperature dependent. Although the use of the transition state theory (TST) offers several challenges associated with data interpretation, the present results and a consideration of others published earlier provide evidence for complexity of ring-flip dynamics in proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

EXSY:

Cross-relaxation suppressed exchange NMR spectroscopy

GdnDCl:

Guanidinium deuterochloride

cyt:

Cytochrome

ferricyt:

Ferricytochrome

References

  • Ansari A, Berendzen J, Bowne SF et al (1985) Protein states and proteinquakes. Proc Natl Acad Sci USA 82:5000–5004

    Article  ADS  Google Scholar 

  • Austin RH, Beeson KW, Eisenstein L (1975) Dynamics of ligand binding to myoglobin. Biochemistry 14:5355–5373

    Article  Google Scholar 

  • Baldwin RL (1986) Temperature dependence of the hydrophobic interaction in protein folding. Proc Natl Acad Sci USA 83:8069–8072

    Article  ADS  Google Scholar 

  • Benson SW, Dobis O (1998) Existence of negative activation energies in simple bimolecular metathesis reactions and some observations on too fast reactions. J Phys Chem A 102:5175–5181

    Article  Google Scholar 

  • Bhuyan AK (2002) Protein stabilization by urea and guanidine hydrochloride. Biochemistry 41:13386–13394

    Article  Google Scholar 

  • Bryngelson JD, Wolynes PG (1989) Intermdeiates and barrier crossing in a random energy model with applications to protein folding. J Phys Chem 93:6902–6915

    Article  Google Scholar 

  • Bryngelson JD, Onuchic JN, Socci ND, Wolynes PG (1995) Funnels, pathways and the energy landscape of protein folding: a synthesis. Proteins 21:167–195

    Article  Google Scholar 

  • Bushnell GW, Louie GV, Brayer GD (1990) High-resolution three-dimensional structure of horse heart cytochrome c. J Mol Biol 214:585–595

    Article  Google Scholar 

  • Campbell ID, Dobson CM, Moore GR, Perkins S.J, Williams RJP (1976) Temperature dependent molecular motion of a tyrosine residue of ferrocytochrome c. FEBS Lett 70:96–100

    Article  Google Scholar 

  • Chan B-l, Baase WA, Schellman JA (1989) Low-temperature unfolding of a mutant of phage T4 lysozyme. 2. Kinetic investigations. Biochemistry 28:691–699

    Article  Google Scholar 

  • Dill KA (1990) Dominant forces in protein folding. Biochemistry 29:7133–7155

    Article  Google Scholar 

  • Dill KA, Shortle D (1991) Denatured states of proteins. Annu Rev Biochem 60:795–825

    Article  Google Scholar 

  • Doan-Nguyen V, Loria JP (2007) The effects of cosolutes on protein dynamics: the reversal of denaturant-induced protein fluctuations by trimethylamine N-oxide. Protein Sci 16:20–29

    Article  Google Scholar 

  • Denisov VP, Peters J, Hörlein HD, Halle B (1996) Using buried water molecules to explore the energy landscape of proteins. Nat Struct Biol 3:505–509

    Article  Google Scholar 

  • Dunbar J, Yennawar HP, Banerjee S, Luo J et al (1997) The effects of denaturants on protein structure. Protein Sci 6:1272–1733

    Article  Google Scholar 

  • Elöve GA, Bhuyan AK, Roder H (1994) Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands. Biochemistry 33:6925–6935

    Article  Google Scholar 

  • Englander SW, Mayne L (1992) Protein folding studied using hydrogen-exchange labeling and two-dimensional NMR. Annu Rev Biophys Biomol Struct 21:243–265

    Article  Google Scholar 

  • Englander SW, Downer NW, Teitelbaum H (1972) Hydrogen exchange. Annu Rev Biochem 41:903–924

    Article  Google Scholar 

  • Ernst RR, Bodenhausen G, Wokaun A (1988) Principles of nuclear magnetic resonance in one and two dimensions. Clarendon Press, Oxford

    Google Scholar 

  • Fejzo J, Zolnai Z, Macura S, Markley JL (1990) Quantitative evaluation of two-dimensional cross-relaxation NMR spectra of proteins. Interprotein distances in turkey ovomucoid third domain. J Magn Reson 88:93–110

    Google Scholar 

  • Fejzo J, Westler WM, Macura S, Markley JL (1991) Strategies for eliminating unwanted cross-relaxation and coherence-transfer effects from two-dimensional chemical exchange spectra. J Magn Reson 92:20–29

    Google Scholar 

  • Fenimore PW, Frauenfelder H, McMahon BH, Young RD (2004) Bulk-solvent and hydration-shell fluctuations, similar to alpha- and beta-fluctuations in glass, control protein motions and fluctuations. Proc Natl Acad Sci USA 101:14408–14413

    Article  ADS  Google Scholar 

  • Gall CM, Cross TA, DiVerdi JA, Opella SJ (1982) Protein dynamics by solid state NMR: aromatic rings of the coat protein in fd bateriophage. Proc Natl Acad Sci USA 79:101–105

    Article  ADS  Google Scholar 

  • Gallego J (2004) Sequence-dependent nucleotide dynamics revealed by intercalated ring rotation in DNA-bisnaphthalimide complexes. Nucleic Acid Res 32:3607–3614

    Article  Google Scholar 

  • Grishaev A, Wu J, Trewhella J, Bax A (2005) Refinement of multidomain protein structures by combination of solution small-angle X-ray scattering and NMR data. J Am Chem Soc 127:16621–16628

    Article  Google Scholar 

  • Hattori M, Li H, Yamada H, Akasaka K et al (2004) Infrequent cavity-forming fluctuations in HPr from Staphylococcus carnosus revealed by pressure- and temperature-dependent tyrosine ring flips. Protein Sci 13:3104–3114

    Article  Google Scholar 

  • Karplus M (1986) Internal dynamics of proteins. Methods Enzymol 131:283–307

    Google Scholar 

  • Karplus M (2000) Aspects of protein reaction dynamics: deviations from simple behavior. J Phys Chem B 104:11–27

    Article  Google Scholar 

  • Kawahara K, Tanford C (1966) Viscosity and density of aqueous solutions of urea and guanidine hydrochloride. J Biol Chem 241:3228–3232

    Google Scholar 

  • Khare R, Paulaitis ME (1995) A study of cooperative phenyl ring flip motions in glassy polystyrene by molecular simulations. Macromolecules 28:4495–4504

    Article  ADS  Google Scholar 

  • Krasnoperov LN, Peng J, Marshall P (2006) Modified transition state theory and negative apparent activation energies of simple metathesis reactions: application to the reaction CH3 + HBr → CH4 + Br. J Phys Chem A 110:3110–3120

    Article  Google Scholar 

  • Kumar R, Bhuyan AK (2005) Two-state folding of horse ferrocytochrome c: analyses of linear free energy relationship, chevron curvature, and stopped-flow burst relaxation kinetics. Biochemistry 44:3024–3033

    Article  Google Scholar 

  • Kumar R, Prabhu NP, Yadaiah M, Bhuyan AK (2004) Protein stiffening and entropic stabilization in the subdenaturing limit of guanidine hydrochloride. Biophys J 87:2656–2662

    Article  Google Scholar 

  • Lam PC-H, Carlier PR (2005) Experimental and computational studies of ring inversion of 1,4-benzodiazepin-2-ones: implications for memory of chirality transformations. J Org Chem 70:1530–1538

    Article  Google Scholar 

  • Li H, Yamada H, Akasaka K (1998) Effect of pressure on individual hydrogen bonds in proteins. Basic pancreatic trypsin inhibitor. Biochemistry 37:1167–1173

    Article  Google Scholar 

  • Maity H, Rumbley JN, Englander SW (2006) Functional role of a protein foldon- an omega-loop foldon controls the alkaline transition in ferricytochrome c. Proteins 63:349–355

    Article  Google Scholar 

  • Makhatadze GI, Privalov PL (1992) Protein interactions with urea and guanidinium chloride. A calorimetric study. J Mol Biol 226:491–505

    Article  Google Scholar 

  • McCammon JA, Wolynes PG, Karplus M (1979) Picosecond dynamics of tyrosine side chains in proteins. Biochemistry 18:927–942

    Article  Google Scholar 

  • Nall BT, Zuniga EH (1990) Rates and energetics of tyrosine ring flips in yeast iso-2-cytochrome c. Biochemistry 29:7576–7584

    Article  Google Scholar 

  • Okazaki K-i, Koga N, Takada S, Onuchic JN et al (2006) Multiple-basin energy landscapes for large-amplitude conformational motions of proteins: structure-based molecular dynamics simulations. Proc Natl Acad Sci USA 103:11844–11849

    Article  ADS  Google Scholar 

  • Oliveberg M, Tan Y-J, Fersht AR (1995) Negative activation enthalpies in the kinetics of protein folding. Proc Natl Acad Sci USA 92:8926–8929

    Article  ADS  Google Scholar 

  • Otting G, Liepinsh E, Wüthrich K (1993) Disulfide bond isomerization in BPTI and BPTI(G36S): an NMR study of correlated mobility in proteins. Biochemistry 32:3571–3582

    Article  Google Scholar 

  • Pellicena P, Kuriyan J (2006) Protein–protein interactions in the allosteric regulation of protein kinase. Curr Opin Struct Biol 16:702–709

    Article  Google Scholar 

  • Pike ACW, Acharya R (1994) A structural basis for the interaction of urea with lysozyme. Protein Sci 3:706–710

    Article  Google Scholar 

  • Portman JJ, Takada S, Wolynes PG (2001) Microscopic theory of protein folding rates. II. Local reaction coordinates and chain dynamics. J Chem Phys 114:5082–5096

    Article  ADS  Google Scholar 

  • Roder H (1989) Structural characterization of protein folding intermediates by proton magnetic resonance and hydrogen exchange. Methods Enzymol 176:446–473

    Article  Google Scholar 

  • Roder H, Elöve GA, Englander SW (1988) Structural characterization of folding intermediates in cytochrome c by H-exchange labeling and proton NMR. Nature 335:700–704

    Article  ADS  Google Scholar 

  • Scalley ML, Baker D (1997) Protein folding kinetics exhibit an Arrhenius temperature dependence when corrected for the temperature dependence of protein stability. Proc Natl Acad Sci USA 94:10636–10640

    Article  ADS  Google Scholar 

  • Shaw BF, Valentine JS (2007) How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein. Trends Biochem Sci 32:78–85

    Article  Google Scholar 

  • Shokhirev NV, Shokhireva TK, Polam JR, Watson CT et al (1997) 2D NMR investigations of the rotation of axial ligands in six-coordinate low-spin iron(III) and cobalt(III) tetraphenylporphyrinates having 2,6-disubstituted phenyl rings: quantitation of rate constants from 1H EXSY cross-peak intensities. J Phys Chem A 101:2778–2786

    Article  Google Scholar 

  • Skalicky JJ, Mills JL, Sharma S, Szyperski T (2001) Aromatic ring-flipping in supercooled water: implications for NMR-based structural biology of proteins. J Am Chem Soc 123:388–397

    Article  Google Scholar 

  • Stangler T, Hartmann R, Willbold D, Koenig BW (2006) Modern high resolution NMR for the study of structure, dynamics and interactions of biological macromolecules. Z Phys Chem 220:567–613

    Google Scholar 

  • Steinbach PJ, Ansari A, Berendzen J et al (1991) Ligand binding to heme protein: connection between dynamics and function. Biochemistry 30:3988–4001

    Article  Google Scholar 

  • Truhlar DG, Kohen A (2001) Convex Arrhenius plots and their interpretation. Proc Natl Acad Sci USA 98:848–851

    Article  ADS  Google Scholar 

  • Wagner G, DeMarco A, Wüthrich K (1976) Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI). Biohys Struct Mechanism 2:139–158

    Article  Google Scholar 

  • Wagner G, Wüthrich K (1978) Dynamic model of globular protein conformations based on NMR studies in solution. Nature 275:247–248

    Article  ADS  Google Scholar 

  • Wallace MI, Ying L, Balasubramanian S, Klenerman D (2001) Non-Arrhenius kinetics for the loop closure of a DNA hairpin. Proc Natl Acad Sci USA 98:5584–5589

    Article  ADS  Google Scholar 

  • Whittaker SB-M, Boetzel R, MacDonald C, Lian L-Y et al (1998) NMR detection of slow conformational dynamics in an endonuclease toxin. J Biomol NMR 12:145–159

    Article  Google Scholar 

  • Wu LC, Laub PB, Elöve GA, Carey J et al (1993) A noncovalent peptide complex as a model for an early folding intermediate of cytochrome c. Biochemistry 32:10271–19276

    Article  Google Scholar 

  • Wüthrich K, Wagner G (1975) NMR investigations of the dynamics of the aromatic amino acid residues in the basic pancreatic trypsin inhibitor. FEBS Lett 50:265–268

    Article  Google Scholar 

  • Zarrine-Afsar A, Mittermaier A, Kay LE, Davidson AR (2006) Protein stabilization by specific binding of guanidinium to a functional arginine-binding surface on an SH3 domain. Protein Sci 15:162–170

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Department of Biotechnology (BRB/15/227/2001), the Department of Science & Technology (4/1/2003-SF), and the University Grants Commission (UPE Funding), Government of India. AKB is the recipient of a Swarnajayanti Fellowship from the DST.

Author information

Authors and Affiliations

  1. School of Chemistry, University of Hyderabad, Hyderabad , 500046, India

    D. Krishna Rao & Abani K. Bhuyan

  2. School of Life Sciences, University of Hyderabad, Hyderabad, 500046, India

    Abani K. Bhuyan

Authors
  1. D. Krishna Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abani K. Bhuyan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Abani K. Bhuyan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Krishna Rao, D., Bhuyan, A.K. Complexity of aromatic ring-flip motions in proteins: Y97 ring dynamics in cytochrome c observed by cross-relaxation suppressed exchange NMR spectroscopy. J Biomol NMR 39, 187–196 (2007). https://doi.org/10.1007/s10858-007-9186-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-007-9186-2

Keywords

Search

Navigation