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X‐ray crystallographic studies on the hydrogen isotope effects of green fluorescent protein at sub‐ångström resolutions
Author(s) -
Tai Yang,
Takaba Kiyofumi,
Hanazono Yuya,
Dao Hoang-Anh,
Miki Kunio,
Takeda Kazuki
Publication year - 2019
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.374
H-Index - 138
ISSN - 2059-7983
DOI - 10.1107/s2059798319014608
Subject(s) - protonation , chromophore , deprotonation , chemistry , deuterium , fluorescence , green fluorescent protein , crystallography , kinetic isotope effect , proton , photochemistry , ion , organic chemistry , atomic physics , biochemistry , physics , quantum mechanics , gene
Hydrogen atoms are critical to the nature and properties of proteins, and thus deuteration has the potential to influence protein function. In fact, it has been reported that some deuterated proteins show different physical and chemical properties to their protiated counterparts. Consequently, it is important to investigate protonation states around the active site when using deuterated proteins. Here, hydrogen isotope effects on the S65T/F99S/M153T/V163A variant of green fluorescent protein (GFP), in which the deprotonated B form is dominant at pH 8.5, were investigated. The pH/pD dependence of the absorption and fluorescence spectra indicates that the protonation state of the chromophore is the same in protiated GFP in H 2 O and protiated GFP in D 2 O at pH/pD 8.5, while the p K a of the chromophore became higher in D 2 O. Indeed, X‐ray crystallographic analyses at sub‐ångström resolution revealed no apparent changes in the protonation state of the chromophore between the two samples. However, detailed comparisons of the hydrogen OMIT maps revealed that the protonation state of His148 in the vicinity of the chromophore differed between the two samples. This indicates that protonation states around the active site should be carefully adjusted to be the same as those of the protiated protein when neutron crystallographic analyses of proteins are performed.