Title
Fragments of the second transmembrane helix of three G-protein-coupled receptors: comparative synthetic, structural and conformational studies
Author
Douglas D. Lopes, Jamille H. Cuvero, Mariana M. L. Ferreira, Rogério L. Silva, Sinval E. G. Souza, Luciana Malavolta, Shirley Schreier, Clovis R. Nakaie
Year
2018
Journal
Amino Acids
Abstract
We compared the synthesis and structural/conformational details of the (66–97) segments of the second transmembrane helix of AT1, MAS and B2, all of which belong to the class of G-protein-coupled receptors (GPCR). Step-by-step monitoring of the coupling reactions during the growth of these transmembrane peptides revealed that the increase in the level of difficulty started at the 6–10 regions of the sequence. Possibly due to their long and hydrophobic sequences, the final estimated synthesis yields decreased progressively by up to 20–25%. Analytical high pressure liquid chromatography showed that the hydrophobicity indexes of each TM-8, -16, -24 and -32 segments correlated linearly with their retention time. Microscopic measurements of peptide–resin beads indicated that, in general, dichloromethane and dimethylsulfoxide were the best solvents for solvating resin beads in the initial and final stages of the synthesis, respectively. Results from electron paramagnetic resonance experiments with Toac (2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled peptide resins revealed that the level of peptide chain mobility throughout the polymer network was in agreement with their swelling data measured in different solvents. Initial results regarding conformational features determined by circular dichroism (CD) spectra revealed typical α-helicoidally structures for MAS and B2 TM32 fragments when in more than roughly 30% (v/v) trifluoroethanol (TFE). In contrast, the AT1-TM32 segment revealed CD spectra, more representatives of a mixture of other secondary helical conformers, regardless of the amount of TFE. These findings observed in different aspects of these receptors’ fragments support further investigations of GPCR-type macromolecules.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Chemical stability, Biochemistry