Mutation of active site residues in the chitin-binding domain ChBDChiA1 from chitinase A1 of Bacillus circulans alters substrate specificity: use of a green fluorescent protein binding assay

2004 ◽  
Vol 426 (2) ◽  
pp. 286-297 ◽  
Author(s):  
Markus Hardt ◽  
Roger A. Laine
Keyword(s):  
Green Fluorescent Protein ◽  
Substrate Specificity ◽  
Protein Binding ◽  
Active Site ◽  
Fluorescent Protein ◽  
Binding Assay ◽  
Active Site Residues ◽  
Chitin Binding Domain ◽  
Protein Binding Assay ◽  
Green Fluorescent

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

2019 ◽  
Vol 75 (12) ◽  
pp. 1096-1106 ◽  
Author(s):  
Yang Tai ◽  
Kiyofumi Takaba ◽  
Yuya Hanazono ◽  
Hoang-Anh Dao ◽  
Kunio Miki ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Active Site ◽  
Hydrogen Isotope ◽  
Fluorescent Protein ◽  
Isotope Effects ◽  
Protonation State ◽  
X Ray ◽  
Two Samples ◽  
Deuterated Proteins ◽  
Green Fluorescent

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 H2O and protiated GFP in D2O at pH/pD 8.5, while the pK a of the chromophore became higher in D2O. 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.

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