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Domains of Pyrococcus furiosus l-asparaginase fold sequentially and assemble through strong intersubunit associative forces

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Abstract

Here, we report the folding and assembly of a Pyrococcus furiosus-derived protein, l-asparaginase (PfA). PfA functions as a homodimer, with each monomer made of distinct N- and C-terminal domains. The purified individual domains as well as single Trp mutant of each domain were subjected to chemical denaturation/renaturation and probed by combination of spectroscopic, chromatographic, quenching and scattering techniques. We found that the N-domain acts like a folding scaffold and assists the folding of remaining polypeptide. The domains displayed sequential folding with the N-domain having higher thermodynamic stability. We report that the extreme thermal stability of PfA is due to the presence of high intersubunit associative forces supported by extensive H-bonding and ionic interactions network. Our results proved that folding cooperativity in a thermophilic, multisubunit protein is dictated by concomitant folding and association of constituent domains directly into a native quaternary structure. This report gives an account of the factors responsible for folding and stability of a therapeutically and industrially important protein.

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Abbreviations

PfA:

Pyrococcus furiosus l-asparaginase

NPfA:

N-terminal domain of PfA

CPfA:

C-terminal domain of PfA

SEC:

Size-exclusion chromatography

CD:

Circular dichroism

WT:

Wild type

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Acknowledgments

DKG and RT acknowledge CSIR-INDIA and AS acknowledges ICMR Govt. of INDIA for their Research Fellowship. BK acknowledges the financial and infrastructural support of IIT Delhi.

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Authors and Affiliations

  1. Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, 110016, New Delhi, India

    Dushyant K. Garg, Rachana Tomar, Ankit Srivastava & Bishwajit Kundu

  2. CSIR Institute of Microbial Technology Chandigarh, Chandigarh, India

    Reema R. Dhoke

Authors
  1. Dushyant K. Garg
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  2. Rachana Tomar
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  3. Reema R. Dhoke
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  4. Ankit Srivastava
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  5. Bishwajit Kundu
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Corresponding author

Correspondence to Bishwajit Kundu.

Additional information

Communicated by F. Robb.

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792_2015_748_MOESM1_ESM.tif

Fig. 1 Structure of WT PfA. a Each monomer of dimeric PfA consists of an N- (green) and C-terminal domain (magenta), connected by a linker (dark red). The two active sites of PfA (dotted circles) are formed at the interface of the N-terminal domain of one subunit (marked as I) and the C-terminal domain of the other subunit (marked as II) and vice versa. Each domain harbors one Trp residue indicated in blue. b The residues surrounding the Trps in each domain are represented (TIFF 894 kb)

792_2015_748_MOESM2_ESM.tif

Fig. 2 Unfolding and refolding curves of the proteins monitored after incubation at two different conditions. Percentage unfolding (solid squares) and refolding (open circles) of proteins monitored by Trp fluorescence against increasing GdnCl concentrations. The solid lines are sigmoidal fit to the data a WT, W301F and NPfA and b WT, W60F and CPfA after overnight incubation at 25 °C. While the unfolding and refolding curves of W301F and NPfA were non-overlapping, they were overlapping for the W60F and CPfA, even after overnight incubation at 25 °C. c Unfolding/refolding of WT, W301F and NPfA after 3 days incubation at 60 °C, showing overlapping curves. Regardless of variation in incubation condition, the refolding curves did not change for any sample (TIFF 379 kb)

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Garg, D.K., Tomar, R., Dhoke, R.R. et al. Domains of Pyrococcus furiosus l-asparaginase fold sequentially and assemble through strong intersubunit associative forces. Extremophiles 19, 681–691 (2015). https://doi.org/10.1007/s00792-015-0748-z

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  • DOI: https://doi.org/10.1007/s00792-015-0748-z

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