279726 NMR Characterization of an Engineered Allosteric Enzyme

Wednesday, October 31, 2012: 10:00 AM
Washington (Westin )
Abigail H. Laurent1, Yuchao Chen2, Luke Arbogast3, Jason Labonte1, Ananya Majumdar4, Jeffrey J. Gray1, Joel Tolman3 and Marc Ostermeier1, (1)Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, (2)Biophysics, Johns Hopkins University, Baltimore, MD, (3)Chemistry, Johns Hopkins University, Baltimore, MD, (4)Biomolecular NMR Center, Johns Hopkins University, Baltimore, MD

Allosteric enzymes have activity that is modulated by the presence of an effector that binds to the protein at a site distal from the active site and is transmitted by a conformational change.  Engineered allosteric enzymes, or protein-based molecular switches, have many potential applications including as biosensors and locally targeted therapeutics.  One way of developing switches is by fusing an enzyme with a ligand binding proteins in such a manner that the ligand modulates the enzyme activity. 

RG13 is a maltose activated beta-lactamase (BLA) enzyme identified from a combinatorial library in which circular permutants of TEM-1 BLA were inserted into maltose binding protein (MBP).   RG13’s catalytic activity is compromised in the absence of maltose and increases 25-fold in the presence of maltose, approaching that of wild-type TEM-1. The primary structure of RG13 consists of a circularly permuted TEM-1 (that has a GSGGG linker between the original N- and C-termini and new termini W227 and G226 respectively) inserted in place of residues 317 and 318 of MBP, with a serine inserted at the end of the TEM-1 sequence.  This corresponds to the insertion of TEM-1 near the hinge region of the MBP domain, opposite the maltose binding pocket.

In this work, nuclear magnetic resonance (NMR) is used to study the allosteric mechanism of RG13.  NMR spectra of 15N-2H-labeled RG13 confirmed the similarity of the MBP and TEM-1 domains to the spectra of their wild-type parental proteins.  They also provided evidence for larger structural differences in the TEM-1 domain in RG13 in the absence of maltose, consistent with the depressed enzymatic activity of RG13.  Further work on 13C-15N-2H-labeled protein is allowing for assignments of NMR measurements to specific residues.   Mutations at the linkage points between the TEM-1 and MBP that affect the switching provide evidence for the linker’s role in the transmission of the conformational change from the MBP domain to the BLA domain.  Results have implications for the natural evolution of allostery and for construction of engineered protein switches.

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