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Equilibrium unfolding of kinetically stable serine protease milin: the presence of various active and inactive dimeric intermediates

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Abstract

Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has α-helix and β-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.

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Abbreviations

ANS:

8-Anilino-1-naphthalenesulfonic acid

BSA:

Bovine serum albumin

CD:

Circular dichorism

DSC:

Differential scanning calorimetry

FPLC:

Fast protein liquid chromatography

GuHCl:

Guanidine hydrochloride

GuSCN:

Guanidine isothiocynate

UV:

Ultraviolet

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Acknowledgments

The financial assistance to SCY from CSIR, Government of India, in the form of a research fellowship and to MBU, IMS, BHU from UGC and DBT, Government of India, for infrastructure is acknowledged. Appreciation is also extended to the University of Delhi, New Delhi, and DBT for financial and infrastructural assistance to SK. We are thankful to Prof. Rajiv Bhat, JNU, New Delhi, for providing the DSC facility. We are especially thankful to Prof. Vinod Bhakuni, CDRI, Lucknow, for his help in the CD data verification.

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Authors and Affiliations

  1. Nanobiology Lab, Biotechnology Division, Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

    Subhash Chandra Yadav

  2. Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India

    Subhash Chandra Yadav & Medicherla V. Jagannadham

  3. Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India

    Suman Kundu

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  1. Subhash Chandra Yadav
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  2. Medicherla V. Jagannadham
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  3. Suman Kundu
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Correspondence to Subhash Chandra Yadav, Medicherla V. Jagannadham or Suman Kundu.

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Yadav, S.C., Jagannadham, M.V. & Kundu, S. Equilibrium unfolding of kinetically stable serine protease milin: the presence of various active and inactive dimeric intermediates. Eur Biophys J 39, 1385–1396 (2010). https://doi.org/10.1007/s00249-010-0593-z

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