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Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation
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The interplay between effector binding and allostery in an engineered protein switch.

Jay H Choi1, Tina Xiong1, Marc Ostermeier1

  • 1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles St, Baltimore, Maryland, 21218.

Protein Science : a Publication of the Protein Society
|June 9, 2016
PubMed
Summary

Engineering protein switches requires understanding allosteric regulation. This study characterized mutations in a maltose-activated enzyme, revealing key insights into ligand binding and allosteric control for designing biosensors.

Keywords:
allosteryligand bindingmaltose binding proteinmaltose transportprotein engineeringprotein switch

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Area of Science:

  • Protein Engineering
  • Biochemistry
  • Molecular Biology

Background:

  • Allosteric regulation is crucial for protein function but poorly understood for design.
  • Engineering protein switches and biosensors requires knowledge of ligand binding determinants.
  • The maltose-activated β-lactamase MBP317-347 serves as a model for studying allosteric regulation.

Purpose of the Study:

  • To investigate the effects of point mutations on the allosteric regulation of MBP317-347.
  • To identify mutations that enhance or disrupt the switch phenotype and maltose binding.
  • To gain fundamental insights into protein design rules for allosteric control.

Main Methods:

  • Extensive in vitro and in vivo characterization of 285 unique point mutations.
  • Analysis of mutations within the maltose-binding pocket of MBP317-347.
  • Assessment of maltose-dependent ampicillin resistance (switch phenotype) and maltose affinity.

Main Results:

  • The switch phenotype was robust to mutations, with most improving performance.
  • Fifteen mutations enhanced switch performance 2- to 22-fold, often by reducing basal catalytic activity.
  • Specific mutations altered maltose affinity and the ability of maltose to modulate activity, providing insights into MBP physiology.

Conclusions:

  • Protein design for allosteric regulation is complex, with intricate relationships between ligand binding and function.
  • Understanding mutation effects on basal activity and ligand-induced conformational changes is key for designing protein switches.
  • This study provides valuable data for the rational design of novel protein-based biosensors and regulatory systems.