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Title: Engineering allostery

Allosteric proteins have great potential in synthetic biology, but our limited understanding of the molecular underpinnings of allostery has hindered the development of designer molecules, including transcription factors with new DNA-binding or ligand-binding specificities that respond appropriately to inducers. Such allosteric proteins could function as novel switches in complex circuits, metabolite sensors, or as orthogonal regulators for independent, inducible control of multiple genes. Advances in DNA synthesis and next-generation sequencing technologies have enabled the assessment of millions of mutants in a single experiment, providing new opportunities to study allostery. Using the classic LacI protein as an example, in this paper we describe a genetic selection system using a bidirectional reporter to capture mutants in both allosteric states, allowing the positions most crucial for allostery to be identified. Finally, this approach is not limited to bacterial transcription factors, and could reveal new mechanistic insights and facilitate engineering of other major classes of allosteric proteins such as nuclear receptors, two-component systems, G protein-coupled receptors, and protein kinases.
Authors:
 [1] ;  [1] ;  [1] ;  [2] ;  [1]
  1. Harvard Univ., Boston, MA (United States); Harvard Medical School, Boston, MA (United States)
  2. Univ. of Washington, Seattle, WA (United States)
Publication Date:
Grant/Contract Number:
FG02-02ER63445
Type:
Accepted Manuscript
Journal Name:
Trends in Genetics
Additional Journal Information:
Journal Volume: 30; Journal Issue: 12; Journal ID: ISSN 0168-9525
Publisher:
Elsevier
Research Org:
Harvard Univ., Boston, MA (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1344516
Alternate Identifier(s):
OSTI ID: 1227554