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Methodology for Further Thermostabilization of an Intrinsically Thermostable Membrane Protein Using Amino Acid Mutations with Its Original Function Being Retained
journal contribution
posted on 2020-03-10, 15:39 authored by Satoshi Yasuda, Tomoki Akiyama, Sayaka Nemoto, Tomohiko Hayashi, Tetsuya Ueta, Keiichi Kojima, Takashi Tsukamoto, Satoru Nagatoishi, Kouhei Tsumoto, Yuki Sudo, Masahiro Kinoshita, Takeshi MurataWe develop a new methodology best suited to the identification
of thermostabilizing mutations for an intrinsically stable membrane
protein. The recently discovered thermophilic rhodopsin, whose apparent
midpoint temperature of thermal denaturation Tm is measured to be ∼91.8 °C, is chosen as a paradigmatic
target. In the methodology, we first regard the residues whose side
chains are missing in the crystal structure of the wild type (WT)
as the “residues with disordered side chains,” which
make no significant contributions to the stability, unlike the other
essential residues. We then undertake mutating each of the residues
with disordered side chains to another residue except Ala and Pro,
and the resultant mutant structure is constructed by modifying only
the local structure around the mutated residue. This construction
is based on the postulation that the structure formed by the other
essential residues, which is nearly optimized in such a highly stable
protein, should not be modified. The stability changes arising from
the mutations are then evaluated using our physics-based free-energy
function (FEF). We choose the mutations for which the FEF is much
lower than for the WT and test them by experiments. We successfully
find three mutants that are significantly more stable than the WT.
A double mutant whose Tm reaches ∼100
°C is also discovered.