Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo
Number of Authors: 5
2016 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 40, 5772-5779 p.Article in journal (Refereed) Published
Insertion of a nascent membrane protein segment by the translocon channel into the bilayer is naturally promoted by high segmental hydrophobicity, but its selection as a transmembrane (TM) segment is complicated by the diverse environments (aqueous vs lipidic) the protein encounters and by the fact that most TM segments contain a substantial amount (similar to 30%) of polar residues, as required for protein structural stabilization and/or function. To examine the contributions of these factors systematically, we designed and synthesized a peptide library consisting of pairs of compositionally identical, but sequentially different, peptides with 19-residue core sequences varying (i) in Leu positioning (with five or seven Leu residues clustered into a contiguous block in the middle of the segment or scrambled throughout the sequence) and (ii) in Ser content (0-6 residues). The library was analyzed by a combination of biophysical and biological techniques, including HPLC retention times, circular dichroism measurements of helicity in micelle and phospholipid bilayer media, and relative blue shifts in Trp fluorescence maxima, as well as by the extent of membrane insertion in a translocon-mediated assay using microsomal membranes from dog pancreas endoplasmic reticulum. We found that local blocks of high hydrophobicity heighten the translocon's propensity to insert moderately hydrophilic sequences, until a threshold hydrophilicity is surpassed whereby segments no longer insert even in the presence of Leu blocks. This study codifies the prerequisites of apolar/polar content and residue positioning that define nascent TM segments, illustrates the accuracy in their prediction, and highlights how a single disease-causing mutation can tip the balance toward anomaloug translocation/insertion.
Place, publisher, year, edition, pages
2016. Vol. 55, no 40, 5772-5779 p.
IdentifiersURN: urn:nbn:se:su:diva-136147DOI: 10.1021/acs.biochem.6b00650ISI: 000385336200013PubMedID: 27620701OAI: oai:DiVA.org:su-136147DiVA: diva2:1050792