title: Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy.
abstract: We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19-24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein-water hydrogen bonds and improving the hydration shells around the alanine's methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.|Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong-4331, Bangladesh.
authors.forename: Mohammad Monirul
authors.initials: MM
authors.lastname: Islam
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yohda
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Kamitomioka-cho, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Shun-Ichi
authors.initials: SI
authors.lastname: Kidokoro
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kuroda
mesh_terms: D000409:Alanine / Q000737:chemistry*; D019943:Amino Acid Substitution; D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics*; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011489:Protein Denaturation; D055550:Protein Stability; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
chemical_list: D014867:Water; D007611:Aprotinin; D000409:Alanine