Understanding the basis of thermostability for enzyme “Nanoluc” towards designing industry-competent engineered variants

Abstract

As a leading contender in the study of luminescence, nanoluciferase has recently attracted attention and proven effective in a wide variety of research areas. Although numerous attempts have been made to improve activity, there has yet to be a thorough exploration of further possibilities to improve thermostability. In this study, protein engineering in tandem with molecular dynamics simulation at various temperatures (300 K, 400 K, 450 K and 500 K) was used to improve our understanding of nanoluciferase dynamics and identification of factors that could significantly enhance the thermostability. Based on these, three novel mutations have been narrowed down, which were hypothesised to improve thermostability. Root mean square deviation and root mean square fluctuation studies confirmed higher stability of mutant at high temperature. Solvent-accessible surface area and protein unfolding studies revealed a decreased tendency of mutant to unfold at higher temperatures. Further free energy landscape and principal component analysis was adapted to get deeper insights into the thermodynamic and structural behavior of these proteins at elevated temperature. Thus, this study provides a deeper insight into the dynamic factors for thermostability and introduces a novel, enhanced nanoluciferase candidate with potential use in industry.

Communicated by Ramaswamy H. Sarma

Keywords:

• luminescence
• nanoluciferase
• thermostability
• free energy landscape
• salt bridge
• protein engineering

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Author contributions

A.S.R.N: visualization, system preparation, methodology, software, data curation, manuscript writing and formal analysis; A.S: investigation, review and editing and project administration; S.H: conceptualization, supervision, validation, funding acquisition and resources. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

The authors thankfully acknowledge Centre for Nanotechnology, IIT Roorkee, Translational and Structural Bioinformatics (BIC) facility from DBT funded project (BT/PR40141/BTIS/137/16/2021) at Department of Biotechnology, IIT Roorkee and Indian Institute of Technology, Roorkee for the support and facilitating necessary infrastructure. National Supercomputing Mission (NSM) is acknowledged for providing computing resources for 'PARAM Ganga’ at Indian Institute of Technology Roorkee, which is implemented by C–DAC and supported by the Ministry of Electronics and Information Technology (MeitY) and Department of Science and Technology (DST) of the Government of India. A.S.R.N acknowledges the Ministry of Education, Government of India for her doctoral fellowship through the Prime Minister’s Research Fellowship (PMRF) scheme.

Disclosure Statement

The authors have no potential conflicts of interest (financial or non-financial) to disclose. No study-specific approval or consent by the appropriate ethics committee was required for this study.

Data availability statement

The data that support the findings of this study are available within the article and its supplementary materials.

Additional information

Funding

The research was supported by Indian Council of Medical Research (ICMR) sanction number BIC/12(15)/2014, the Science and Engineering Research Board, India (SERB) sanction number YSS/2014/000492, Board of Research in Nuclear Sciences sanction number 54/14/03/2023-BRNS and Technology Innovation Hub (TIH) sanction number TIH/CS/04.