NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates.

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D.

Structural characterization of triple transmembrane domain containing fragments of a yeast G protein-coupled receptor in an organic : aqueous environment by solution-state NMR spectroscopy.

This report summarizes recent biophysical and protein expression experiments on polypeptides containing the N-terminus, the first, second, and third transmembrane (TM) domains and the contiguous loops of the α-factor receptor Ste2p, a G protein-coupled receptor. The 131-residue polypeptide Ste2p(G31-R161), TM1-TM3, was investigated by solution NMR in trifluoroethanol/water. TM1-TM3 contains helical TM domains at the predicted locations, supported by continuous sets of medium-range NOEs.

Invited review: GPCR structural characterization: Using fragments as building blocks to determine a complete structure.

The structural characterization of G protein-coupled receptors has surged since the development of methodologies to facilitate the crystallization of these highly helical, seven transmembrane, integral membrane receptors. In the past seven years, eighteen GPCR structures were determined by X-ray crystallography. The crystal structures represent a static picture of these conformationally flexible signal transducers.

Guided reconstitution of membrane protein fragments.

Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs.