title: Design, expression, and purification of de novo transmembrane "hairpin" peptides.
abstract: While our understanding of the folding and structure of water-soluble proteins has progressed to the point where they can be artificially designed and produced from first principles, there has been only limited work toward the de novo design of membrane proteins. Such studies have been hindered in large part due to the practical challenges in the production and characterization of multispanning transmembrane (TM) proteins that arise from their highly hydrophobic character. In this work, we used molecular biology cloning techniques to produce a library of partially randomized Ala- and Ile-rich de novo helix-loop-helix (hairpin) TM constructs as models for tertiary TM-TM folding. From this plasmid DNA library, we selected sequences corresponding to hairpins with 0, 1, or 2 putative TM segments. While purification protocols could be adapted for application with a broad range of designed protein hairpins, bacterial expression of constructs with multiple predicted TM segments was limited as it is with native membrane proteins. Examples of the peptide hairpins obtained were characterized by circular dichroism spectroscopy, tryptophan fluorescence, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We found that hairpins composed of two TM segments display characteristic behavior on detergent solubilization, such as an increase in helical structure (vs. that in aqueous buffer), and sequence-dependent migration rates in SDS-PAGE analysis-features that may serve as structural hallmarks to verify dual TM topology in hairpin sequences.
authors.affiliation: Division of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: David V
authors.initials: DV
authors.lastname: Tulumello
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Johnson
authors.forename: Inga
authors.initials: I
authors.lastname: Isupov
authors.forename: Charles M
authors.initials: CM
authors.lastname: Deber
mesh_terms: D000595:Amino Acid Sequence; D015870:Gene Expression; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019151:Peptide Library; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012995:Solubility; D013050:Spectrometry, Fluorescence
chemical_list: D008565:Membrane Proteins; D019151:Peptide Library