Biophysical and structural characterization of the thermostable WD40 domain of a prokaryotic protein, Thermomonospora curvata PkwA

Scientific Reports
Chen ShenYun-Dong Wu

Abstract

WD40 proteins belong to a big protein family with members identified in every eukaryotic proteome. However, WD40 proteins were only reported in a few prokaryotic proteomes. Using WDSP ( http://wu.scbb.pkusz.edu.cn/wdsp/ ), a prediction tool, we identified thousands of prokaryotic WD40 proteins, among which few proteins have been biochemically characterized. As shown in our previous bioinformatics study, a large proportion of prokaryotic WD40 proteins have higher intramolecular sequence identity among repeats and more hydrogen networks, which may indicate better stability than eukaryotic WD40s. Here we report our biophysical and structural study on the WD40 domain of PkwA from Thermomonospora curvata (referred as tPkwA-C). We demonstrated that the stability of thermophilic tPkwA-C correlated to ionic strength and tPkwA-C exhibited fully reversible unfolding under different denaturing conditions. Therefore, the folding kinetics was also studied through stopped-flow circular dichroism spectra. The crystal structure of tPkwA-C was further resolved and shed light on the key factors that stabilize its beta-propeller structure. Like other WD40 proteins, DHSW tetrad has a significant impact on the stability of tPkwA-C. Considering its ...Continue Reading

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Methods Mentioned

BETA
gel-filtration
Gel filtration
size-exclusion chromatography
circular dichroism
Fluorescence

Software Mentioned

Pro
CCP4
Coot
WDSP
data viewer
HKL2000
WDSP protein Structure Predictor
Refmac
PyMol

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