CHEMICAL SYNTHESIS AND CHARACTERIZATION OF PEPTIDES AND OLIGOMERIC PROTEINS DESIGNED TO FORM TRANSMEMBRANE

A strategy for the synthesis of peptides and oligomeric proteins desig ned to form transmembrane ion channels is described. A folding motif t hat exhibits a functional ionic pore encompasses amphipathic alpha-hel ices organized as a four-helix bundle around a central hydrophilic por e. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides s elf-assemble into conductive oligomers. The covalent attachment of cha nnel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detai led protocols for the total synthesis of peptides and template-assembl ed four-helix bundle proteins, exemplified with the sequence of M2 del ta (EKMSTAISVLLAQAVFLLLTSQR), considered involved in lining the pore o f the nicotinic acetylcholine receptor channel. For comparison, the sy nthesis of a second four-helix bundle, T(4)CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fo urth 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 anal ytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid anal ysis and sequencing. Optimization of synthetic procedures for hydropho bic molecules include reducing resin substitution to avoid steric hind rance and aggregation of the final product. Protocols for the preparat ion of the samples prior to HPLC purification as well as the condition s 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 templat e directed helical bundle proteins. (C) Munksgaard 1994.