title: Functional stability and structural transitions of Kallikrein: spectroscopic and molecular dynamics studies.
abstract: Kallikrein, a physiologically vital serine protease, was investigated for its functional and conformational transitions during chemical (organic solvents, Gdn-HCl), thermal, and pH induced denaturation using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The enzyme was exceptionally stable in isopropanol and ethanol showing 110% and 75% activity, respectively, after 96 h, showed moderate tolerance in acetonitrile (45% activity after 72 h) and much lower stability in methanol (40% activity after 24 h) (all the solvents [90% v/v]). Far UV CD and fluorescence spectra indicated apparent reduction in compactness of KLKp structure in isopropanol system. MD simulation studies of the enzyme in isopropanol revealed (1) minimal deviation of the structure from native state (2) marginal increase in radius of gyration and solvent accessible surface area (SASA) of the protein and the active site, and (3) loss of density barrier at the active site possibly leading to increased accessibility of substrate to catalytic triad as compared to methanol and acetonitrile. Although kallikrein was structurally stable up to 90 °C as indicated by secondary structure monitoring, it was functionally stable only up to 45 °C, implicating thermolabile active site geometry. In GdnHCl [1.0 M], 75% of the activity of KLKp was retained after incubation for 4 h, indicating its denaturant tolerance. A molten globule-like structure of KLKp formed at pH 1.0 was more thermostable and exhibited interesting structural transitions in organic solvents. The above results provide deeper understanding of functional and structural stability of the serine proteases at molecular level.
authors.affiliation: a Division of Biochemical Sciences , CSIR-National Chemical Laboratory , Dr. Homi Bhabha Road, Pune 411008 , India.
authors.forename: Sayli
authors.initials: S
authors.lastname: Dalal
authors.affiliation: b Division of Physical Chemistry , CSIR-National Chemical Laboratory , Dr. Homi Bhabha Road, Pune 411008 , India.
authors.forename: Anil
authors.initials: A
authors.lastname: Mhashal
authors.affiliation: a Division of Biochemical Sciences , CSIR-National Chemical Laboratory , Dr. Homi Bhabha Road, Pune 411008 , India.
authors.forename: Narendra
authors.initials: N
authors.lastname: Kadoo
authors.affiliation: a Division of Biochemical Sciences , CSIR-National Chemical Laboratory , Dr. Homi Bhabha Road, Pune 411008 , India.
authors.forename: Sushama M
authors.initials: SM
authors.lastname: Gaikwad
mesh_terms: D020134:Catalytic Domain; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D012997:Solvents; D013057:Spectrum Analysis; D013696:Temperature
chemical_list: D012997:Solvents; D007610:Kallikreins
keywords: Kallikrein; MD simulation; isopropanol tolerance; molten globule; serine protease; thermostability