FRET‐based Measurements of Protein Multimerization in Pyruvate Carboxylase

Abstract

Changes in environmental conditions can affect protein multimerization which, in turn, may alter an enzyme's activity. The multi‐domain homotetrameric enzyme pyruvate carboxylase (PC) serves as a model to study protein multimerization. Past studies on PC have shown that it equilibrates between different oligomeric forms, but that it is active exclusively as a tetramer. When diluted to low concentrations, the PC equilibrium shifts to favor inactive monomeric and dimeric oligomerization states[1],[2]. Since the in vitro activity of PC depends on its oligomerization state, it stands to reason that regulation of oligomerization states offers a way to control the enzyme activity in vivo.In a heterogeneous population, smaller PC oligomers can reassociate into a population containing mixed heterotetramers. Controlled manipulation of PC multimerization will aid in developing hybrid tetramers that would serve as a tool to reveal key mechanistic insights into inter‐subunit coordination during catalysis. Past remixing studies have failed to precisely control the assembly of heterogeneous tetramers. This research will contribute to a more complete description of the factors influencing PC multimerization by using a FRET‐based approach to measure the assembly of PC tetramers under a wide array of remixing conditions. A non‐conserved residue (Leu 339) on the surface of the biotin carboxylase (BC) domain of Staphylococcus aureus PC (SaPC) was mutated to a Cys for site‐specific maleimide‐based labeling with either a Cy3 or Cy5 fluorophore. These site‐specifically incorporated fluorophores will form a FRET pair when the BC domain of one PC population dimerizes with a labeled BC domain of another PC population en route to re‐associating into a heterogeneous tetramer. Preliminary characterization revealed no significant difference in the maleimide‐based labeling of WT SaPC and L339C in SaPC. However, the addition of ADP along with the BC domain transition state analogues, phosphonoacetate (PPA), dramatically reduced the labeling of WT SaPC without affecting labeling of SaPC L339C. The site‐specifically labeled FRET pair of Cy3 and Cy5 offers a method to measure PC multimerization, which enables the investigation of conditions that alter the formation of the heterogeneous PC tetramer. These studies represent the first efforts to carefully control remixing of PC through manipulation of PC oligomerization states and may provide insight into ways to control the oligomerization states in other oligomeric proteins.Support or Funding InformationThis work was supported by the National Institutes of Health grant GM117540.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.