【For INTERNATIONAL COLLABORATIVE RESEARCH 2017】

SUMMARY of

DATE: Day 18 Month 5 Year 20182017 RESEARCH RESULTS REPORTFor International Collaborative Research with IPR, Osaka University
Research Title

Probing G protein-coupled receptor (GPCR) structural dynamics using selective aromatic labels

ApplicantName

Joshua Ziarek

Affiliation

Harvard Medical School

Present Title

Assistant Professor; Indiana University

Research Collaborator (Host PI)

Yohei Miyanoiri

Summary
G protein-coupled receptors (GPCRs) are the largest signaling protein superfamily –regulating diverse processes from vision, smell and taste to immune, neurologic and reproductivefunctions. Residing at cellular membranes they are the primary means of translating extracellular
stimuli into intracellular responses. Nonetheless, the molecular details of signal transduction remain
obscure despite an explosion of new GPCR crystal structures.
Crystallization has been impossible without additional modifications and very high-affinity
ligands that force receptors into a subset of nearly indistinguishable conformations – regardless ofthe presumed activation state. Yet, in contrast to other proteins, GPCRs are highly dynamic andpossess multiple active states rather than a binary, ‘on or off’, mode. Nuclear magnetic resonances(NMR) spectroscopy remains the only technique capable of quantifying protein dynamics at atomicresolution, but requires isotope labeling (e.g. 2H, 13C, 15N). Proteins recombinantly produced inbacteria are amenable to the multitude of isotopic labeling schemes; however, overexpression of
membrane proteins results in non-functioning, insoluble aggregates. In 2013, directed evolution ofneurotensin receptor 1 (NTR1) made possible large-scale prokaryotic expression of functional
receptors, but NTR1’s membrane environment greatly restricting traditional NMR methods.
Cell-free protein synthesis presents a unique solution as selectively-labeled amino acids canbe polymerized, in a H2O-based reaction, without scrambling from biosynthetic pathways. E. coliderived cell-free systems are the most efficient, but previous attempts at GPCR expression failed.
We hypothesized that our evolved NTR1 construct would be more amenable to E. coli-based cellfree methods. In collaboration with Prof. Yohei Miyanoiri, we pursued cell-free protein synthesis ofNTR1 for NMR-based structural dynamics studies. Our initial structural characterization at IPR did
not produce meaningful results as the protein yields were too low to be detected. We maintain ourhypothesis that receptors evolved to express in E. coli are far more likely to yield properly-folded
constructs in vitro. Using a mixed detergent-lipid micelle system, our preliminary studies of theevolved NTR1 construct produce high yields (> 2 mg/1 ml reaction) of folded receptor. We are
currently supplementing cell-free reactions to improve the yield of functional receptor. We anticipate
our method will be generally applicable to other membrane protein targets.
*Deadline: May 18, 2018

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