Hydrogen-Deuterium Exchange Analysis of Phosphorylase Kinase, a Complex with 325 kDa of Unique Sequence

摘要

Overview: (1) Phosphorylase Kinase (PhK), at 1.3 MDa, is a large regulatory enzyme in the glycogenoltyic cascade composed of four 325 kDa protomers. (2) There is no known structure for the two largest subunits and little information on subunit interactions. (3) Hydrogen-Deuterium Exchange with Mass Spectrometry was used to analyze subunit structure. (4) Despite the large amount of data, good coverage and reproducible data were obtained. (5) Noteworthy regions were identified in three of the four subunits Introduction: PhK is a hexadecameric complex having four copies each of four subunits, with 325 kDa of unique sequence: α (138 kDa), β (125 kDa), γ (45 kDa) and δ (16.5 kDa). While there are high resolution structures of the catalytic core of γ and of δ, only predicted structures of α and β are known. Chemical crosslinking studies have provided some information about subunit interactions within the complex, but there is scant information on how the subunits are situated within that complex. Here we use hydrogen-deuterium exchange to identify exposed regions of the subunits, suggesting possible interactions among them. Methods: Following incubation in D_2O buffer at 0.4 mg/mL for various times, PhK was quenched at pH 2.3 and digested with a 1:1 ratio of pepsin for 5 min. Digested samples were subjected to reversed phase HPLC on a C18 column and directed to a LTQ-FT mass spectrometer. To minimize H/D back exchange, the sample loop, reversed phase column, delivery line, and valves were housed in a cooling chamber and the temperature was maintained at 2°C. Peptides were identified by automatic search ananylsis of their collision-induced dissociation spectra using Sequest. Data were analyzed using HDExaminer.