Ramesh, Balasubramaniyam; (1995) The chemical synthesis and characterization of biomembrane polypeptides. Doctoral thesis (Ph.D), UCL (University College London).

Text out.pdf Download (8MB)

Abstract

Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy have been applied to the characterization of chemically synthesized polypeptides corresponding to transmembrane proteins. A new disaggregation process was introduced and applied to these hydrophobic polypeptides to enable them to be incorporated into membrane mimetic environments without exhibiting the aggregated form. This is illustrated by a comparison of the FTIR spectrum of the natural Pf1 coat protein with that of synthetic Pf1 coat protein when incorporated into lipid vesicles and detergent micelles, before and after disaggregation. The natural Pf1 coat protein and the disaggregated synthetic protein exhibit an α-helical arrangement, whilst the aggregated synthetic protein exhibits a β-sheet type structure. The existence of these aggregated forms of protein can be shown to make spectral analysis difficult and unreliable. The characterization of synthetic polypeptides corresponding to sequences from (a)ion-selective pore H5, (b) the proposed voltage "sensor" S4 and (c) the region including the transmembrane domains S4-S5-H5-S6 of the voltage-sensitive potassium channel protein was carried out. The FTIR spectra of the disaggregated monomeric, extended, tetrameric version of H5, and the S4-S5-H5-S6 synthetic polypeptides exhibit predominantly an α -helical structure when incorporated into membrane mimetic environments. CD spectroscopy also shows an α-helical conformation of the disaggregated monomeric polypeptide H5 in lipid micelles. When incorporated into lipid planar bilayers the monomeric and the tetrameric H5 polypeptides exhibit ion-channel activity. The FTIR spectra of the synthetic S4 polypeptide were examined in aqueous buffer, trifluoroethanol solvent and lipid vesicles. The polypeptide in trifluoroethanol and lipid vesicles adopts predominantly an α-helical conformation, whilst in aqueous solution adopts a random coil arrangement. CD spectroscopy also shows an a-helical conformation in lipid micelles and a random coil structure in aqueous buffer. The coil to helix transition observed for the S4 polypeptide when transferred from an aqueous to a lipid environment indicates a high degree of conformational flexibility in response to its environment. This property may have important implications for its proposed role as a voltage-"sensor" during channel activation in response to an electric potential across the membrane.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as XMLCSVJSON

Archive Staff Only

View Item