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Discovery of Nicotinamide Adenine Dinucleotide Binding Proteins in the Escherichia coli Proteome Using a Combined Energetic- and Structural-Bioinformatics-Based Approach
journal contribution
posted on 2016-11-18, 00:00 authored by Lingfei Zeng, Woong-Hee Shin, Xiaolei Zhu, Sung Hoon Park, Chiwook Park, W. Andy Tao, Daisuke KiharaProtein–ligand
interaction plays a critical role in regulating
the biochemical functions of proteins. Discovering protein targets
for ligands is vital to new drug development. Here, we present a strategy
that combines experimental and computational approaches to identify
ligand-binding proteins in a proteomic scale. For the experimental
part, we coupled pulse proteolysis with filter-assisted sample preparation
(FASP) and quantitative mass spectrometry. Under denaturing conditions,
ligand binding affected protein stability, which resulted in altered
protein abundance after pulse proteolysis. For the computational part,
we used the software Patch-Surfer2.0. We applied the integrated approach
to identify nicotinamide adenine dinucleotide (NAD)-binding proteins
in the Escherichia coli proteome, which
has over 4200 proteins. Pulse proteolysis and Patch-Surfer2.0 identified
78 and 36 potential NAD-binding proteins, respectively, including
12 proteins that were consistently detected by the two approaches.
Interestingly, the 12 proteins included 8 that are not previously
known as NAD binders. Further validation of these eight proteins showed
that their binding affinities to NAD computed by AutoDock Vina are
higher than their cognate ligands and also that their protein ratios
in the pulse proteolysis are consistent with known NAD-binding proteins.
These results strongly suggest that these eight proteins are indeed
newly identified NAD binders.