Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo.
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 (∼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 anomalous translocation/insertion.
Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children , Toronto M5G 0A4, Ontario, Canada.|Department of Biochemistry, University of Toronto , Toronto M5S 1A8, Ontario, Canada.
Tracy A
TA
Stone
Department of Biochemistry and Biophysics, Stockholm University , SE-106 91 Stockholm, Sweden.|Science for Life Laboratory, Stockholm University , Box 1031, SE-171 21 Solna, Sweden.
Nina
N
Schiller
Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children , Toronto M5G 0A4, Ontario, Canada.
Natalie
N
Workewych
Department of Biochemistry and Biophysics, Stockholm University , SE-106 91 Stockholm, Sweden.|Science for Life Laboratory, Stockholm University , Box 1031, SE-171 21 Solna, Sweden.
Gunnar
G
von Heijne
Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children , Toronto M5G 0A4, Ontario, Canada.|Department of Biochemistry, University of Toronto , Toronto M5S 1A8, Ontario, Canada.
Charles M
CM
Deber
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D057927:Hydrophobic and Hydrophilic Interactions; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D008081:Liposomes; D008565:Membrane Proteins; D008823:Micelles