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Infrequent cavity-forming fluctuations in HPr from Staphylococcus carnosus revealed by pressure- and temperature-dependent tyrosine ring flips

¹ Department of Molecular Science, Graduate School of Science and Technology and ² Department of Chemistry, Faculty of Science, Kobe University, Kobe 657-8501, Japan
³ School of Biology-Oriented Science and Technology, Kinki University, Wakayama 649-6493, Japan
⁴ Ruhr-University Bochum, Department of Biology, D-465780 Bochum, Germany
⁵ University of Regensburg, Institute of Biophysics and Physical Biochemistry, D-93040 Regensburg, Germany

(RECEIVED May 24, 2004; FINAL REVISION August 17, 2004; ACCEPTED August 21, 2004)

Infrequent structural fluctuations of a globular protein is seldom detected and studied in detail. One tyrosine ring of HPr from Staphylococcus carnosus, an 88-residue phosphocarrier protein with no disulfide bonds, undergoes a very slow ring flip, the pressure and temperature dependence of which is studied in detail using the on-line cell high-pressure nuclear magnetic resonance technique in the pressure range from 3 MPa to 200 MPa and in the temperature range from 257 K to 313 K. The ring of Tyr6 is buried sandwiched between a -sheet and -helices (the water-accessible area is less than 0.26 nm²), its hydroxyl proton being involved in an internal hydrogen bond. The ring flip rates10¹~10⁵ s^–1 were determined from the line shape analysis of H^{1, 2} and H^1,2 of Tyr6, giving an activation volume V of 0.044 ± 0.008 nm³ (27 mL mol^–1), an activation enthalpy H of 89 ± 10 kJ mol^–1, and an activation entropy S of 16 ± 2 JK^–1 mol^–1. The V and H values for HPr found previously for Tyr and Phe ring flips of BPTI and cytochrome c fall within the range of V of 28 to 51 mL mol^–1 and H of 71 to 155 kJ mol^–1. The fairly common V and H values are considered to represent the extra space or cavity required for the ring flip and the extra energy required to create a cavity, respectively, in the core part of a globular protein. Nearly complete cold denaturation was found to take place at 200 MPa and 257 K independently from the ring reorientation process.

Keywords: high pressure; NMR spectroscopy; ring flips; HPr; PTS

Abbreviations: DSS, 4,4-dimethyl-4-silapentane-sulphonic acid • HPr, histidine-containing phosphocarrier protein • HSQC, heteronuclear single quantum coherence • NOE, nuclear Overhauser effect • NOESY, nuclear Overhauser effect spectroscopy • PEP, phosphoenolpyruvate • PTS, phosphoenolpyruvate-dependent phosphotransferase system • S. carnosus, Staphylococcus carnosus • S. aureus, Staphylococcus aureus • TPPI, time proportional phase incrementation • TSP, 3-(trimethylsilyl)[3,3,2,2-2H] propionate-d₄

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04877104.

Reprint requests to: Hans Robert Kalbitzer, University of Regensburg, Institute of Biophysics and Physical Biochemistry, D-93040 Regensburg, Germany; e-mail: hans-robert.kalbitzer{at}biologie.uni-regensburg.de; fax: +49-941-943-2479.

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