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Highly Hydrophilic Segments Attached to Hydrophobic Peptides Translocate Rapidly across Membranes Jamie LeBarron and Erwin London* Stony Brook University Stony Brook, New York 11794-5215, United States S Supporting Information *
ABSTRACT: Hydrophilic segments attached to transmembrane helices often cross membranes. In an increasing number of cases, it has become apparent that this occurs in a biologically relevant post-translational event. In this study, we investigate whether juxta-membrane (JM) hydrophilic sequences attached to hydrophobic helices are able to rapidly cross lipid bilayers via their ability or inability to block hydrophobic helix interconversion between a transmembrane (TM) and non-TM membrane-associated state. Interconversion was triggered by changing the protonation state of an Asp residue in the hydrophobic core of the peptides, and peptide configuration was monitored by the fluorescence of a Trp residue at the center of the hydrophobic sequence. In POPC vesicles, conversion of the TM to non-TM state at high pH and the non-TM to TM state at low pH was rapid (seconds or less) for KK, KKNN, and the KKNNNNNN flanking sequences on both N- and C-termini and the KLFAGHQ sequence that flanks the spontaneously TM-inserting 3A protein of polio virus. In vesicles composed of 6:4 (mol/mol) POPC/cholesterol, interconversion was still rapid, with the exception of the peptide flanked by KKNNNNNN sequences, for which the half time of interconversion slowed to minutes. This behavior suggests that, at least in membranes with low levels of cholesterol, movement of hydrophilic JM segments (and analogous hydrophobic loops in multipass TM proteins) across membranes may be more facile than previously thought. This may have important biological implications.
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hydrophilic segments.4 Inversion of 6 of the 12 helices in lactose permease is observed when E. coli lipid composition is altered by blocking the synthesis of PE.5−7 Some of these events might involve the translocon. However, for lactose permease it has been shown that inversion can be induced after reconstituting the purified protein in liposomes containing no other proteins, when lipid composition is altered in situ using cyclodextrin-induced lipid exchange.8,9 Another case in which spontaneous topological changes occur in proteins without the aid of the translocon involve bacterial toxin proteins.10 Many AB type toxins have developed methods to insert themselves across membranes, often triggered by the protonation of acidic residues when the toxin enters an acidic vacuole.11−15 The TM elements and connecting loops of pore-forming protein toxins cross membranes after binding to the appropriate receptor and/or ligand molecule.16 In yet another set of examples, single hydrophobic helix Ctail anchored proteins and the hydrophilic residues beyond the hydrophobic helix also must cross membranes post-translationally (reviewed in ref 17). Although some steps in this process are catalyzed by proteins, whether the actual membrane
INTRODUCTION In order for multipass membrane proteins to be properly inserted in membranes, hydrophilic loops that link the hydrophobic transmembrane (TM) segments must be moved across the bilayer. In most cases, organisms use some form of a proteinaceous translocon complex to sidestep the energetic cost of passing hydrophilic segments through the nonpolar membrane.1 Although more than one path has been proposed for the detailed route followed by the translocating protein,2 the consensus is that the growing protein is passed through the translocon such that hydrophilic segments crossing the membrane pass through the translocon without being exposed to the full hydrophobicity of the core of the lipid bilayer.3 For at least some membrane proteins, major topological rearrangements involving inversion of TM helices have been observed. These rearrangements also involve movement of hydrophilic residues through the lipid bilayer. In the case of aquaporin, after the last TM helix was synthesized multiple hydrophilic loops in the middle of the protein were found to translocate through the membrane due to changes in protein topology (reviewed in ref 1). The four TM segment drug pump EmrE protein has been shown to work as an antiparallel homodimer. Considering that both protein chains must insert from the same direction, half of the protein molecules must completely invert their orientation at some point, including inversion of three hydrophilic loops and N and C terminal © 2016 American Chemical Society
Received: July 15, 2016 Revised: September 14, 2016 Published: September 20, 2016 10752
DOI: 10.1021/acs.langmuir.6b02597 Langmuir 2016, 32, 10752−10760
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Langmuir
Lange Sensors (Vaulx-en-Velin, France). Acrylamide was purchased from Serva (Heidelberg, Germany). Glacial acetic acid was purchased from Pharmco-Aaper (Shelbyville, KY). Tris base was obtained from Roche (Nutley, NJ). Hydrochloric acid was purchased from Fisher Scientific (Pittsburgh, PA). Sodium hydroxide (NaOH) was purchased from Mallinckrodt Baker (Phillipsburg, NJ). All other chemicals (isopropanol, trifluoroacetic acid) were obtained from Sigma-Aldrich (St. Louis, MO). Peptides were purchased either from W.M. Keck Small Scale Peptide Synthesis Facility (New Haven, CT) or from Anaspec (San Jose, CA). Peptide abbreviations and sequences are as follows: N0K2flanked peptide, KKLALALLLDWLLLLALALKK; N2K2-flanked peptide, NNKKLALALLLDWLLLLALALKKNN; N6K2-flanked peptide, NNNNNNKKLALALLLDWLLLLALALKKNNNNNN; and polioflanked peptide, KLFAGHQLALALALDWLALALALKLFAGHQ. Except for the polio-flanked peptide, which was unblocked, all peptides were blocked on their N-terminus with an acetyl group and Cterminus with an amide group. Peptides were suspended in water, and isopropanol was titrated until peptides dissolved, typically at ∼30− 45% (by volume) isopropanol. Peptides were stored at this isopropanol/water ratio at 4 °C until use. Peptide concentrations were determined at an absorbance of 280 nm on a Beckman (Indianapolis, ID) DU-650 spectrophotometer using the extinction coefficient of Trp as 5,560 cm −1 M −1. Peptide purity was determined by matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry at the Center for Analysis and Synthesis of Macromolecules (Stony Brook, NY) and was determined to be 70−88% pure. To check if purity influenced results, crude N2K2 peptide (72% pure) was purified via reverse-phase HPLC processing as previously described.44 Trifluoroacetic acid from HPLC was removed with a modified protocol of that in ref 45, as previously described.44 No difference in behavior between purified and crude peptide was noted. Vesicle Preparation. Ethanol dilution vesicles containing 1 mol % peptide when desired, were made by ethanol dilution as described previously.44 800 μL samples containing 200 μM PC were prepared in PBS or PBS that had been preadjusted to a pH of ∼4 or 10 with either NaOH or acetic acid, respectively (pH adjustment involved
