Dissociation behavior of microbial nitrilase in temperature-pressure plane studied by using high pressure near-ultraviolet circular dichroism spectroscopy

ABSTRACT

High pressure circular dichroism (HP-CD) spectroscopy below 200 MPa, especially in the near-ultraviolet region, has long been expected to probe the protein self-association phenomena. However, its experimental difficulties have hampered obtaining the data available for quantitative analyses. In this paper, near-ultraviolet CD spectra measurements under various temperatures and pressures were demonstrated on nitrilase from Rhodococcus rhodochrous J1 (J1-NTase), which thermally self-associates from inactive protomer to active oligomer at atmospheric pressure. The exploration of instrumental conditions and simple raw data correction enabled us to complete the spectra in a temperature-pressure plane. The quality of data was checked by multivariate curve resolution with the alternating least squares method (MCR-ALS), which gave model-free decomposition into component spectra and their concentration profiles. The resultant two components pointed out that the obtained data were self-consistent and appeared to reflect free and bound protomers.

KEYWORDS:

• High pressure circular dichroism (HP-CD) spectroscopy
• near-ultraviolet CD spectroscopy
• nitrilase
• association/dissociation equilibrium
• multivariate curve resolution with the alternating least squares method (MCR-ALS)

Acknowledgements

The authors wish to acknowledge Dr. Toyokazu Yoshida of Gifu University, for kindly providing E. coli transformed to express nitrilase from Rhodococcus rhodochrous J1.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the JSPS KAKENHI [grant number JP23K04788].