1 3 Eur Biophys J DOI 10.1007/s00249-014-0971-z ORIGINAL PAPER Subtilase from Beauveria sp.: conformational and functional investigation of unusual stability Sayli A. Dalal · Snehal V. More · Shiv Shankar · R. Seeta Laxman · Sushama M. Gaikwad Received: 19 March 2014 / Revised: 21 May 2014 / Accepted: 26 May 2014 © European Biophysical Societies’ Association 2014 simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interest- ing structural transitions. Keywords Beauveria · Subtilase · Serine protease · Conformational transitions · Thermodynamic stability · Chemical denaturation · Thermal denaturation Abbreviations Bprot Beauveria protease GdnHCl Guanidine hydrochloride CD Circular dichroism DMSO Dimethyl sulfoxide Introduction Understanding the principles of protein stability is essen- tial for optimizing biological functions of proteins. Pro- tein thermodynamic stability and kinetic stability are two major aspects of protein stability (Sanchez-Ruiz 2010). Thermodynamic stability is the existence of native and small amount of partially unfolded protein in equilibrium under physiological conditions, whereas kinetic stability implies entrapment of the protein in a specific conforma- tion because of the high unfolding barrier that results in slow unfolding rates. Recently, various proteases like milin (Yadav and Jagannadham 2009), α-lytic protease (Cun- ningham et al. 1999), and Nocardiopsis protease (Roham- are et al. 2013) have been reported to be kinetically stable proteases due to their high-energy barrier for unfolding under harsh conditions. The kinetic stability is reported to improve longevity of the protein and optimize its biological function under harsh environments like extreme acidic pH Abstract Retention of total activity of the subtilisin- like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50 % methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the pro- tein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environ- ment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chroma- tography in the presence of GdnHCl (1.0–3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentra- tion of denaturant (1.0–4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k unf of native protein to be 5.407 × 10 -5 s -1 and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resist- ant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and Electronic supplementary material The online version of this article (doi:10.1007/s00249-014-0971-z) contains supplementary material, which is available to authorized users. S. A. Dalal · S. V. More · S. Shankar · R. Seeta Laxman · S. M. Gaikwad (*) Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr Homi Bhabha Road, Pune 411008, India e-mail: sm.gaikwad@ncl.res.in