The synthesis of recombinant membrane proteins in yeast for structural studies

Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P.; Bawa, Zharain; Wilks, Martin D.B.; Low, Floren; Hardy, David; Rothnie, Alice; Bill, Roslyn M.

doi:10.1016/j.ymeth.2015.09.027

Cited by 45 publications

(21 citation statements)

References 117 publications

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“…A current limitation of using yeast as sensors is that most medically relevant GPCRs do not port directly into S. cerevisiae without requiring optimization of expression, membrane translocation, and pathway coupling ( Sarramegna et al., 2003 ). Co-expression of mammalian accessory proteins ( Fukutani et al., 2015 ) and the humanization of the yeast membrane ( Routledge et al., 2016 ) have also been shown to improve porting of receptors from mammalian species, and at the last count at least 50 different foreign GPCRs have been demonstrated to function in yeast ( Table S1 ). These past successes provide a guide for those looking to generate yeast sensors for ligands and metabolites.…”

Section: Discussionmentioning

confidence: 99%

Engineering a Model Cell for Rational Tuning of GPCR Signaling

Shaw

Yamauchi

Mead

et al. 2019

Cell

171

225

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Summary G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.

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Section: Discussionmentioning

confidence: 99%

Engineering a Model Cell for Rational Tuning of GPCR Signaling

Shaw

Yamauchi

Mead

et al. 2019

Cell

171

225

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“…SMALPs are attracting great attention as a new membrane mimic because they can solubilise proteins from artificial18 or natural910111213 membranes while retaining a native-like environment in the form of a nanosized lipid bilayer. Owing to their small size, SMALPs are well suited for optical-spectroscopic13489121415 and chromatographic13151617 techniques. Likewise, a diisobutylene/maleic acid (DIBMA) copolymer has been found18 to solubilise membrane proteins and lipids in a mild fashion, that is, without major perturbation of the bilayer order in DIBMA/lipid particles (DIBMALPs).…”

mentioning

confidence: 99%

Fast Collisional Lipid Transfer Among Polymer-Bounded Nanodiscs

Arenas

Danielczak

Martel

et al. 2017

Sci Rep

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Some styrene/maleic acid (SMA) copolymers solubilise membrane lipids and proteins to form polymer-bounded nanodiscs termed SMA/lipid particles (SMALPs). Although SMALPs preserve a lipid-bilayer core, they appear to be more dynamic than other membrane mimics. We used time-resolved Förster resonance energy transfer and small-angle neutron scattering to determine the kinetics and the mechanisms of phospholipid transfer among SMALPs. In contrast with vesicles or protein-bounded nanodiscs, SMALPs exchange lipids not only by monomer diffusion but also by fast collisional transfer. Under typical experimental conditions, lipid exchange occurs within seconds in the case of SMALPs but takes minutes to days in the other bilayer particles. The diffusional and second-order collisional exchange rate constants for SMALPs at 30 °C are kdif = 0.287 s−1 and kcol = 222 M−1s−1, respectively. Together with the fast kinetics, the observed invariability of the rate constants with probe hydrophobicity and the moderate activation enthalpy of ~70 kJ mol−1 imply that lipids exchange through a “hydrocarbon continuum” enabled by the flexible nature of the SMA belt surrounding the lipid-bilayer core. Owing to their fast lipid-exchange kinetics, SMALPs represent highly dynamic equilibrium rather than kinetically trapped membrane mimics, which has important implications for studying protein/lipid interactions in polymer-bounded nanodiscs.

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“…A current limitation of using yeast for biosensors is that most medically-relevant GPCRs do not port directly into S. cerevisiae without requiring optimization of expression, membrane translocation and pathway coupling. Solutions to this have previously been explored with limited success, such as the co-expression of mammalian accessory proteins 61 and the humanization of the yeast membrane 62 .…”

Section: Discussionmentioning

confidence: 99%

Engineering a model cell for rational tuning of GPCR signaling

Shaw

Yamauchi

Mead

et al. 2018

Preprint

View full text Add to dashboard Cite

G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression.Using genome engineering in yeast, we here constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose-response.Using four different receptors, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites and hormones relevant to human health. This work enables rational tuning of cell sensing, while providing a framework to guide reprogramming of GPCR-based signaling in more complex systems.

show abstract

The synthesis of recombinant membrane proteins in yeast for structural studies

Cited by 45 publications

References 117 publications

Engineering a Model Cell for Rational Tuning of GPCR Signaling

Engineering a Model Cell for Rational Tuning of GPCR Signaling

Fast Collisional Lipid Transfer Among Polymer-Bounded Nanodiscs

Engineering a model cell for rational tuning of GPCR signaling

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