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Amino Acids

pp 1–10 | Cite as

Fragments of the second transmembrane helix of three G-protein-coupled receptors: comparative synthetic, structural and conformational studies

  • Douglas D. Lopes
  • Jamille H. Cuvero
  • Mariana M. L. Ferreira
  • Rogério L. Silva
  • Sinval E. G. Souza
  • Luciana Malavolta
  • Shirley Schreier
  • Clovis R. Nakaie
Original Article
  • 36 Downloads

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.

Keywords

Peptide Peptide synthesis Electron paramagnetic resonance Spin label Receptor GPCR 

Abbreviations

Ac

Acetyl

AII

Angiotensin II

AN

Electron acceptor number

BHAR

Benzhydrylamine resin

Boc

Tert-butyloxycarbonyl

Bz

Benzyl

C18

Octadecyl

2BrZ

2-Bromobenzyloxycarbonyl

For

Formyl

tBu

t-Butyl

DCM

Dichloromethane

DIC

N,N′-Diisopropylcarbodiimide

DIEA

Diisopropylethylamine

DMF

N,N-Dimethylformamide

DMS

Dimethyl sulfide

DMSO

Dimethylsulfoxide

DN

Electron donor number

EPR

Electron paramagnetic resonance

EtOH

Ethanol

Fmoc

9-Fluorenylmethyloxycarbonyl

HBTU

N-[(1H-benzotriazol-1-yl)-(dimethylaminomethylene)]-N-methylmethanaminium hexafluorophosphate N-oxide

HOAt

1-Hydroxy-7-azabenzotriazole

HOBt

1-Hydroxybenzotriazole

HPLC

High performance liquid chromatography

LC/ESI-MS

Liquid chromatography/eletrospray mass spectrometry

MBHAR

Methylbenzhydrylamine-resin

MeCN

Acetonitrile

MeOH

Methanol

NMP

N-Methylpiperidine

OcHex

Cyclohexyl

SPPS

Solid phase peptide synthesis

TEA

Triethylamine

TFA

Trifluoroacetic acid

TFE

trifluoroethanol

Toac

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid

Tos

p-Toluenesulfonyl

Notes

Acknowledgements

This work was supported by the Sao Paulo State Research Foundation (FAPESP) and the Brazilian National Council for Scientific and Technological Research (CNPq). SS and CRN are CNPq research fellows.

Compliance with ethical standards

Conflict of interest

The authors of this manuscript declare no conflict of interest, either financial or otherwise.

Ethical statement

This article does not contain any studies with human participants or animals.

Informed consent

Informed consent was obtained from all individual participants included in the study.

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Douglas D. Lopes
    • 1
  • Jamille H. Cuvero
    • 1
  • Mariana M. L. Ferreira
    • 1
  • Rogério L. Silva
    • 1
  • Sinval E. G. Souza
    • 1
  • Luciana Malavolta
    • 2
  • Shirley Schreier
    • 3
  • Clovis R. Nakaie
    • 1
  1. 1.Department of Biophysics, Escola Paulista de MedicinaUniversidade Federal de Sao PauloSão PauloBrazil
  2. 2.Department of Physiological Sciences, Santa Casa de Sao PauloSchool of Medical SciencesSão PauloBrazil
  3. 3.Department of Biochemistry, Institute of ChemistryUniversidade de Sao PauloSão PauloBrazil

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