Zusammenfassung
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 ...
Zusammenfassung
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 P-sheet and alpha-helices (the water-accessible area is less than 0.26 mm(2)), its hydroxyl proton being involved in an internal hydrogen bond. The ring flip rates 10(1)similar to10(5) s(-1) were determined from the line shape analysis of H-delta1,H-delta2 and H-epsilon1,H-epsilon2 of Tyr6, giving an activation volume DeltaV(double dagger) of 0.044 +/- 0.008 nm(3) (27 mL mol(-1)), an activation enthalpy DeltaH(double dagger) of 89 +/- 10 kJ mol(-1), and an activation entropy DeltaS(double dagger) of 16 +/- 2 JK(-1) mol(-1). The DeltaV(double dagger) and DeltaH(double dagger) values for HPr found previously for Tyr and Phe ring flips of BPTI and cytochrome c fall within the range of DeltaV(double dagger) of 28 to 51 mL mol(-1) and DeltaH(double dagger) of 71 to 155 kJ mol(-1). The fairly common DeltaV(double dagger) and DeltaH(double dagger) 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.
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