Abstract
Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.
Highlights
The conformational stability of proteins and the methods of its evaluation have been the subject of intense interest for researchers from various fields, including molecular and cellular biophysics, biochemistry, food chemistry, biomedicine and many others
The present study has been carried out in the field of molecular biophysics and was directed at identifying factors affecting the conformational stability of proteins, as well as at developing methods of assessment and comparison of their structural stability
We have investigated the change of spectroscopic properties during urea-induced unfolding of two homologous proteins—V. harveyi and P. leiognathi luciferases—with special attention to the time-resolved fluorescence of these proteins
Summary
The conformational stability of proteins and the methods of its evaluation have been the subject of intense interest for researchers from various fields, including molecular and cellular biophysics, biochemistry, food chemistry, biomedicine and many others. The disturbance of protein stability due to gene mutations or the influence of external physical and chemical factors can lead to the appearance of misfolded species or structural intermediates which cause various disease states in cells and tissues [1,2]. Fluorescence spectroscopy techniques are widely used to probe the conformational transitions of proteins [3]. There is a downside to the use of tryptophan residues as natural fluorophores to monitor the conformational states of proteins. It seems difficult to compare the unfolding pathways of two proteins bearing different numbers of tryptophans, which are located
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