[NMR paper] Pressure-denatured state of Escherichia coli ribonuclease HI as monitored by Fourier

[nmrlearner]

Pressure-denatured state of Escherichia coli ribonuclease HI as monitored by Fourier transform infrared and NMR spectroscopy.

Related Articles Pressure-denatured state of Escherichia coli ribonuclease HI as monitored by Fourier transform infrared and NMR spectroscopy.

Biochemistry. 1998 Dec 22;37(51):18001-9

Authors: Yamasaki K, Taniguchi Y, Takeda N, Nakano K, Yamasaki T, Kanaya S, Oobatake M

Pressure denaturation of Escherichia coli ribonuclease HI (RNase HI) was studied by Fourier transform infrared (FTIR) and two-dimensional NMR spectroscopy at pD* 3.0 and 25 degrees C. A reversible transition in the pressure range of 0.1-1090 MPa was observed with second-derivative FTIR experiments. A cooperative and gradual denaturation, involving both the secondary and tertiary structures, was observed between 240 and 450 MPa. The two peaks at 1629 and 1652 cm(-1), due to beta-strands and alpha-helices, respectively, did not fully disappear after the denaturation, and are different from the spectra of the random coil peptides. The hydrogen-deuterium exchange rates of the individual backbone amide protons were determined by heteronuclear NMR combined with the pressure-jump technique at 500, 650, and 850 MPa. Although most of the amides protected in the native structure are also highly protected in the pressure-denatured state, the rate constants (0.048 +/- 0.007 min(-1)) for the amide protons at 500 MPa are similar regardless of their locations, which is an indication of the EX1 mechanism of hydrogen-deuterium exchange. The pressure-denatured state of RNase HI at 500 MPa represents a novel denatured state, which is different from a typical molten globule state at atmospheric pressure (0.1 MPa), from the viewpoint of the homogeneous rate constants. The observations at 650 MPa are essentially the same as those at 500 MPa. However, at 850 MPa, the amide exchange rates for the highly hydrophobic C-terminal half of alpha-helix I are significantly slower than those for the other part of the protein, which can be interpreted as a hydrophobic collapse centered at the C-terminal half of alpha-helix I.

PMID: 9922168 [PubMed - indexed for MEDLINE]



Source: PubMed