X-ray crystallographic studies on the hydrogen isotope effects of green fluorescent protein at sub-ångström resolutions

Abstract

PDB references: S65T/F99S/M153T/V163A variant of green fluorescent protein, perdeuterated, pD 8.5, 6kkz; perdeuterated, pD 7.0, 6kl0; nondeuterated, pD 8.5, 6kl1Supporting information: this article has supporting information at journals.iucr.org/dHydrogen 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 pK a of the chromophore became higher in D 2 O. Indeed, X-ray crystallographic analyses at sub-å ngströ m 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.