Analysis of the Structure of Phosphorylase Kinase by Mass Spectrometry
Rimmer, Mary Ashley
University of Kansas
Biochemistry & Molecular Biology
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Phosphorylase Kinase (PhK) is a large, 1.3 MDa, regulatory enzyme in the glycogenolysis cascade, made up by four copies each of four different subunits, α, β, γ, and δ, giving 325 kDa of unique sequence. Three of the four subunits are regulatory (α, β, and δ), leaving the γ subunit to have the only known catalytic function. Likely due to the size and complexity of PhK, high resolution structures are only available for the smallest subunit, δ, and the catalytic domain of the γ-subunit. The structure of both subunits either in the complex or individually has proved difficult to study. To address some of the questions about the structure of PhK, we have employed three techniques (partial proteolysis, hydrogen/deuterium (H/D) exchange, and chemical footprinting), each in conjunction with mass spectrometry, to elucidate information about the solvent exposure and dynamics of the subunits in complex. In this search for more information about the location and disposition of the subunits in the complex, we have also produced models using a structural prediction program for the largest subunit, α, and of the regulatory domain of the catalytic subunit, γ. These models have been assessed using the H/D exchange results, and were found to be consistent with the experimental exchange data. Taking these three techniques together, we have been able to identify potential regions of inter-subunit contact on the α and γ subunits, identify regions on the α, β, and γ subunits that are surface exposed, even in complex with the other subunits, and produce experimentally consistent models of α and the γ regulatory domain.
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