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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.
Highlights
A structural analysis indicated that new water molecules were recruited into the spaces created by the alanine substitutions, facilitating protein–water hydrogen-bonded interactions as well as interactions with the methyl group and creating hydration networks around the substitution sites
The enthalpy stabilization introduced to a protein with multiple alanine substitutions is novel and unexpected
A comparison of the thermodynamic parameters and structural data suggests that the enthalpy stabilization of the simplified bovine pancreatic trypsin inhibitor (BPTI) probably arises from improved interactions between water molecules and the protein
Summary
The alanine substitutions exerted a small unexpected enthalpy stabilization, and the magnitude of which increased in a nearly additive fashion along with the number of alanine substitutions, which was, over-compensated with entropy destabilization. A structural analysis indicated that new water molecules were recruited into the spaces created by the alanine substitutions, facilitating protein–water hydrogen-bonded interactions as well as interactions with the methyl group and creating hydration networks around the substitution sites. This observation suggested that enthalpy stabilization originates from hydration rather than from chain enthalpy. This is the very first molecular perspective on hydration enthalpy/entropy, and it suggests a generic strategy for a water-mediated enthalpy stabilization of a protein
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