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. 2001 Feb;80(2):1013–1023. doi: 10.1016/S0006-3495(01)76080-8

Time-resolved hole-burning study on myoglobin: fluctuation of restricted water within distal pocket.

Y Shibata 1, H Ishikawa 1, S Takahashi 1, I Morishima 1
PMCID: PMC1301299  PMID: 11159468

Abstract

We have studied the equilibrium fluctuation dynamics of Zn-substituted myoglobin and its His64-->Leu (H64L) mutant in the pH range from 5 to 9 by using time-resolved transient-hole-burning (TRTHB) spectroscopy. In the H64L mutant, we have observed a largely reduced width of the absorption spectrum and only a slight temporal shift of the hole-burning spectrum. These observations both reflect the suppressed conformational fluctuation in the mutant. On the other hand, the pH-dependent change in the absorption spectrum could not be solely explained by the change in the protonation state of His64 induced by the pH change. These results suggest that although the fluctuation dynamics observed by the TRTHB experiment of the native sample mainly reflects the conformational motion around His64, the interconversion process of His64 between its protonated and unprotonated states has a minor contribution. Instead, we have proposed a tentative interpretation that the motion of the water molecule around His64 is the main source of the observed dynamics in the TRTHB technique.

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Selected References

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  1. Alben J. O., Beece D., Bowne S. F., Doster W., Eisenstein L., Frauenfelder H., Good D., McDonald J. D., Marden M. C., Moh P. P. Infrared spectroscopy of photodissociated carboxymyoglobin at low temperatures. Proc Natl Acad Sci U S A. 1982 Jun;79(12):3744–3748. doi: 10.1073/pnas.79.12.3744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ansari A., Berendzen J., Bowne S. F., Frauenfelder H., Iben I. E., Sauke T. B., Shyamsunder E., Young R. D. Protein states and proteinquakes. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5000–5004. doi: 10.1073/pnas.82.15.5000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Austin R. H., Beeson K. W., Eisenstein L., Frauenfelder H., Gunsalus I. C. Dynamics of ligand binding to myoglobin. Biochemistry. 1975 Dec 2;14(24):5355–5373. doi: 10.1021/bi00695a021. [DOI] [PubMed] [Google Scholar]
  4. Barboy N., Feitelson J. Diffusion of small molecules through the structure of myoglobin. Environmental effects. Biochemistry. 1989 Jun 27;28(13):5450–5456. doi: 10.1021/bi00439a020. [DOI] [PubMed] [Google Scholar]
  5. Barboy N., Feitelson J. Quenching of the zinc-protoporphyrin triplet state as a measure of small-molecule diffusion through the structure of myoglobin. Biochemistry. 1987 Jun 2;26(11):3240–3244. doi: 10.1021/bi00385a046. [DOI] [PubMed] [Google Scholar]
  6. Beece D., Eisenstein L., Frauenfelder H., Good D., Marden M. C., Reinisch L., Reynolds A. H., Sorensen L. B., Yue K. T. Solvent viscosity and protein dynamics. Biochemistry. 1980 Nov 11;19(23):5147–5157. doi: 10.1021/bi00564a001. [DOI] [PubMed] [Google Scholar]
  7. Carlson M. L., Regan R. M., Gibson Q. H. Distal cavity fluctuations in myoglobin: protein motion and ligand diffusion. Biochemistry. 1996 Jan 30;35(4):1125–1136. doi: 10.1021/bi951767k. [DOI] [PubMed] [Google Scholar]
  8. Carver T. E., Rohlfs R. J., Olson J. S., Gibson Q. H., Blackmore R. S., Springer B. A., Sligar S. G. Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques. J Biol Chem. 1990 Nov 15;265(32):20007–20020. [PubMed] [Google Scholar]
  9. Frauenfelder H., Petsko G. A., Tsernoglou D. Temperature-dependent X-ray diffraction as a probe of protein structural dynamics. Nature. 1979 Aug 16;280(5723):558–563. doi: 10.1038/280558a0. [DOI] [PubMed] [Google Scholar]
  10. Frauenfelder H., Sligar S. G., Wolynes P. G. The energy landscapes and motions of proteins. Science. 1991 Dec 13;254(5038):1598–1603. doi: 10.1126/science.1749933. [DOI] [PubMed] [Google Scholar]
  11. Kleinert T., Doster W., Leyser H., Petry W., Schwarz V., Settles M. Solvent composition and viscosity effects on the kinetics of CO binding to horse myoglobin. Biochemistry. 1998 Jan 13;37(2):717–733. doi: 10.1021/bi971508q. [DOI] [PubMed] [Google Scholar]
  12. Müller J. D., McMahon B. H., Chien E. Y., Sligar S. G., Nienhaus G. U. Connection between the taxonomic substates and protonation of histidines 64 and 97 in carbonmonoxy myoglobin. Biophys J. 1999 Aug;77(2):1036–1051. doi: 10.1016/s0006-3495(99)76954-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nienhaus G. U., Chu K., Jesse K. Structural heterogeneity and ligand binding in carbonmonoxy myoglobin crystals at cryogenic temperatures. Biochemistry. 1998 May 12;37(19):6819–6823. doi: 10.1021/bi972843h. [DOI] [PubMed] [Google Scholar]
  14. Quillin M. L., Arduini R. M., Olson J. S., Phillips G. N., Jr High-resolution crystal structures of distal histidine mutants of sperm whale myoglobin. J Mol Biol. 1993 Nov 5;234(1):140–155. doi: 10.1006/jmbi.1993.1569. [DOI] [PubMed] [Google Scholar]
  15. Sastry G. M., Agmon N. Trehalose prevents myoglobin collapse and preserves its internal mobility. Biochemistry. 1997 Jun 10;36(23):7097–7108. doi: 10.1021/bi9626057. [DOI] [PubMed] [Google Scholar]
  16. Shibata Y., Kurita A., Kushida T. Real-time observation of conformational fluctuations in Zn-substituted myoglobin by time-resolved transient hole-burning spectroscopy. Biophys J. 1998 Jul;75(1):521–527. doi: 10.1016/S0006-3495(98)77541-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shibata Y., Kurita A., Kushida T. Solvent effects on conformational dynamics of Zn-substituted myoglobin observed by time-resolved hole-burning spectroscopy. Biochemistry. 1999 Feb 9;38(6):1789–1801. doi: 10.1021/bi9815694. [DOI] [PubMed] [Google Scholar]
  18. Srajer V., Teng T., Ursby T., Pradervand C., Ren Z., Adachi S., Schildkamp W., Bourgeois D., Wulff M., Moffat K. Photolysis of the carbon monoxide complex of myoglobin: nanosecond time-resolved crystallography. Science. 1996 Dec 6;274(5293):1726–1729. doi: 10.1126/science.274.5293.1726. [DOI] [PubMed] [Google Scholar]
  19. Tian W. D., Sage J. T., Champion P. M., Chien E., Sligar S. G. Probing heme protein conformational equilibration rates with kinetic selection. Biochemistry. 1996 Mar 19;35(11):3487–3502. doi: 10.1021/bi952474u. [DOI] [PubMed] [Google Scholar]
  20. Uchida T., Ishimori K., Morishima I. The effects of heme pocket hydrophobicity on the ligand binding dynamics in myoglobin as studied with leucine 29 mutants. J Biol Chem. 1997 Nov 28;272(48):30108–30114. doi: 10.1074/jbc.272.48.30108. [DOI] [PubMed] [Google Scholar]
  21. Varadarajan R., Szabo A., Boxer S. G. Cloning, expression in Escherichia coli, and reconstitution of human myoglobin. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5681–5684. doi: 10.1073/pnas.82.17.5681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Vojtechovský J., Chu K., Berendzen J., Sweet R. M., Schlichting I. Crystal structures of myoglobin-ligand complexes at near-atomic resolution. Biophys J. 1999 Oct;77(4):2153–2174. doi: 10.1016/S0006-3495(99)77056-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yang F., Phillips G. N., Jr Crystal structures of CO-, deoxy- and met-myoglobins at various pH values. J Mol Biol. 1996 Mar 8;256(4):762–774. doi: 10.1006/jmbi.1996.0123. [DOI] [PubMed] [Google Scholar]

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