DOI: 10.1111/j.1399-3011.1994.tb00562.x. Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels

10.1111/j.1399-3011.1994.tb00562.x

Crossref journal-article

Wiley

International Journal of Peptide and Protein Research (311)

Abstract

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic α‐helices organized as a four‐helix bundle around a central hydrophilic pore. The channel‐forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self‐assemble into conductive oligomers. The covalent attachment of channel‐forming peptides to the lysine ε‐amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template‐assembled four‐helix bundle proteins, exemplified with the sequence of M2δ (EKM‐STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four‐helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the l‐type voltage‐gated calcium channel, is included. Peptides and proteins are synthesized step‐wise by solid‐phase methods, purified by reversed‐phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

Bibliography

IWAMOTO, T., GROVE, A., MONTAL, M. O., MONTAL, M., & TOMICH, J. M. (1994). Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels. International Journal of Peptide and Protein Research, 43(6), 597â607. Portico.

Authors 5

TAKEO IWAMOTO (first)
ANNE GROVE (additional)
MYRTA OBLATT MONTAL (additional)
MAURICIO MONTAL (additional)
JOHN M. TOMICH (additional)

References 32 Referenced 28

{'key': 'e_1_2_2_2_2', 'first-page': '121', 'volume': '83', 'author': 'Numa S.', 'year': '1989', 'journal-title': 'Harvey Lect.'} / Harvey Lect. by Numa S. (1989)
10.1016/0014-5793(85)80136-8
10.1007/978-1-4615-7305-0_1
10.1096/fasebj.4.9.1693348
10.1073/pnas.87.18.6929
10.1073/pnas.88.15.6418
10.1002/prot.340080305
10.1021/ar00028a009
10.1039/fs9821700109
10.1146/annurev.bi.53.070184.003115
10.1007/978-1-4613-1571-1_19
10.1146/annurev.pa.31.040191.000345
10.1126/science.1373907
10.1006/jmbi.1993.1107
10.1073/pnas.85.7.2393
10.1016/S0021-9258(18)82375-4 / J. Bid. Chem. by Oblatt‐Montal M. (1993)
10.1073/pnas.85.22.8703
{'key': 'e_1_2_2_19_2', 'first-page': '89', 'volume-title': 'Serono Symposium Series', 'author': 'Grove A.', 'year': '1991'} / Serono Symposium Series by Grove A. (1991)
10.1002/anie.198905353
10.1002/prot.340050104
10.1016/S0021-9258(18)82376-6 / J. Biol. Chem. by Reddy G.L. (1993)
10.1007/BF01877228
Gesell J. Opella S.J. Sun W.&Montal M.(1992)Sixth Symp. The Protein Society p.52 Abstr. 833.
10.1021/ja00897a025
10.1126/science.150.3693.178
10.1146/annurev.bi.57.070188.004521
10.1016/0003-2697(81)90704-1
10.1016/0006-291X(89)92003-2
10.1021/ja00359a014
10.1111/j.1399-3011.1990.tb01294.x
10.1021/ja00030a049
10.1021/ja00067a004

Dates

Type	When
Created	15 years ago (Aug. 9, 2010, 8:17 p.m.)
Deposited	1 year, 10 months ago (Oct. 25, 2023, 4:41 a.m.)
Indexed	2 months, 1 week ago (June 26, 2025, 9:10 a.m.)
Issued	31 years, 3 months ago (June 1, 1994)
Published	31 years, 3 months ago (June 1, 1994)
Published Online	16 years, 7 months ago (Jan. 12, 2009)
Published Print	31 years, 3 months ago (June 1, 1994)

Funders 0

None

BibTeX

@article{IWAMOTO_1994, title={Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels}, volume={43}, ISSN={0367-8377}, url={http://dx.doi.org/10.1111/j.1399-3011.1994.tb00562.x}, DOI={10.1111/j.1399-3011.1994.tb00562.x}, number={6}, journal={International Journal of Peptide and Protein Research}, publisher={Wiley}, author={IWAMOTO, TAKEO and GROVE, ANNE and MONTAL, MYRTA OBLATT and MONTAL, MAURICIO and TOMICH, JOHN M.}, year={1994}, month=jun, pages={597–607} }

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