High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic‐aqueous medium

Abstract

The NMR properties of seven peptides representing the transmembrane domains of the alpha-factor receptor from Saccharomyces cerevisiae were examined in trifluoroethanol/water (4:1) at 10 to 55 degrees C. The parameters extracted indicated all peptides were helical in this membrane mimetic solvent. Using chemical shift indices as the criterion, helicity varied from 64 to 83%. The helical residues in the peptides corresponded to the region predicted to cross the hydrocarbon interior of the bilayer. A study of a truncated 25-residue peptide corresponding to domain 2 gave evidence that the helix extended all the way to the N-terminus of this peptide, indicating that sequence and not chain end effects are very important in helix termination for our model peptides. Both nuclear Overhauser effect spectroscopy (NOESY) connectivities and chemical shift indices revealed significant perturbations around prolyl residues in the helices formed by transmembrane domains 6 and 7. Molecular models of the transmembrane domains indicate that helices for domains 6 and 7 are severely kinked at these prolyl residues. The helix perturbation around proline 258 in transmembrane domain 6 correlates with mutations that cause phenotypic changes in this receptor.

Peptide design and structural characterization of a GPCR loop mimetic

“…This approach builds on prior efforts by multiple research groups to characterize GPCR loop sequences in organic and aqueous solutions both without [16][17][18][19] and with 18,20 internal structural constraints. NMR structures have been determined for various segments of GPCRs including TMs of the a-factor receptor in 80% trifluoroethanol (TFE) 21 ; the N-terminus, 22 first extracellular loop 23 and third cytoplasmic loop 24 of the parathyroid hormone receptor (PTH1R) in aqueous solution or SDS/DPC micelles; the third intracellular loop of the human cannabinoid 1 receptor (CB1) in SDS micelles 16 ; and bovine rhodopsin loops in dimethylsulfoxide (DMSO). 17 Structural studies on unconstrained peptide mimics of GPCR loops indicated that the native conformation in the intact protein cannot be obtained in aqueous solution and SDS micelles.…”

Estudos conformacionais de seqüências hidrofóbicas de domínios de receptores acoplados a proteínas G (GPCR) e da proteína G. Um estudo de CD e espectroscopia de fluorescência

“…Mesmo em relação a fragmentos transmembranares, grupos como o de Naider et aI. (64)(65)(66)(67) mostraram a importância de se estudar domínios de…”

“…(64)(65)(66)(67) observou que alguns dos fragmentos transmembranares não adotam conformação a-helicoidal nem mesmo em TFE. É possível que isso também seja verdade para outros receptores.…”

“…O mesmo grupo também observou por espectroscopia de fluorescência que esse peptídeo era capaz de atravessar a membrana (63), O grupo de Naider et aI. (64)(65)(66)(67) também têm tido uma contribuição relevante na literatura ao estudar fragmentos sintéticos correspondentes a TMs de um GPCR de Saccharomyces cerevisiae. Esse grupo observou que com exceção das TMs 3 e 6 na presença de modelos de membrana, todos os outros peptídeos adotaram conformação ahelicoidal na presença de TFE, misturas de TFE-água e na presença de micelas de detergentes e de vesículas fosfolipídicas (64).…”

“…Estudos de NMR da TM6 e TM7 na presença de TFE mostraram que as a-hélices formadas por esses fragmentos possuem dobras (kinks) ao redor dos resíduos de prolina (67). Albert et aI.…”