Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion

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Hydrophobic clusters raise the threshold hydrophilicity for insertion of transmembrane sequences in vivo Tracy A. Stone, Nina Schiller, Natalie Workewych, Gunnar von Heijne, and Charles M. Deber Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00650 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 15, 2016

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Biochemistry

Hydrophobic clusters raise the threshold hydrophilicity for insertion of transmembrane sequences in vivo

Tracy A. Stone1,2#, Nina Schiller3,4#, Natalie Workewych1, Gunnar von Heijne3,4, and Charles M. Deber1,2,*

1

Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children, Toronto

M5G 0A4; and 2Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada. 3

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; and

4

Science for Life Laboratory, Stockholm University, Box 1031, SE-171 21 Solna, Sweden.

#These authors contributed equally to the work. *Address correspondence to: Charles M. Deber, Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children, Peter Gilgan Center for Research and Learning, 686 Bay Street, Toronto, Ontario, Canada M5G 0A4. Tel. (01) 416 813-5924; Fax (01) 416 813-5005; E-mail: [email protected].

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Biochemistry

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ABBREVIATIONS

TM, transmembrane; Fmoc, N-(9-fluorenyl)methoxycarbonyl; PAL-PEG, Peptide amide linker polyethylene glycol; TFA, trifluoroacetic acid; RP-HPLC, reverse phase high-performance liquid chromatography; TFE, trifluoroethanol; POPC, 1-palmitoyl-2-oleoylglycero-3-phosphocholine; CD, Circular Dichroism; SDS, sodium dodecyl sulfate; MRE, mean residue ellipticity; CMC, critical micelle concentration; ER, endoplasmic reticulum; FRET, Förster resonance energy transfer.


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ABSTRACT

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; and by extent of membrane insertion in a translocon-mediated assay using microsomal membranes from dog pancreas endoplasmic reticulum (ER). 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.


Biochemistry

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Although major advancements have been made in understanding the compositional and positional requirements of amino acids for the insertion of transmembrane (TM) domains1–4, the detailed roles of both amino acid patterning and the lipid bilayer’s tolerance to polar amino acids remain to be understood. The ease with which a soluble secreted protein may be altered to a membrane protein and vice versa implies that a fine balance exists between the secretion and insertion of protein segments on the hydrophobic edge.5 In vivo, most eukaryotic membrane proteins pass through an aqueous protein-conducting channel prior to insertion into the lipid bilayer, an environment devoid of water. The translocon channel (Sec61αβγ in eukaryotes, SecYEG in bacteria) provides a conduit through the membrane for proteins destined for secretion, and a pathway into the lipid bilayer for membrane integration.6 Protein sequences transiting through the aqueous channel of the translocon are exposed to the interior of the lipid bilayer through an opening, the ‘lateral gate’, in the side wall of the channel.7–9 At this point, TM segments may laterally diffuse out of the translocon channel, partition into the lipid bilayer, and become membrane-embedded, positioning the translocon as a ‘stepping stone’ into the bilayer.10 As well, alterations to the translocon protein (i.e., mutations to the lateral gate or amino acids that line the channel) have been shown to affect the hydrophobicity threshold for membrane insertion.11–14 Yet the insertion and folding of membrane proteins from the translocon into the bilayer is complicated due to the diverse environments these proteins encounter. Thus, partially ordered water is present within the translocon channel15,16,17. The hydrophobic effect is therefore operative to some extent within the channel. However, there is little to no water present in the bilayer, and once each segment adopts its TM helical form upon insertion, the hydrophobic effect is no longer a major force in the tertiary and quaternary folding of a protein within the receiving


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lipid environment; rather, side chain-side chain helix/helix packing and side chain-lipid interactions play these roles. Further, while it is generally accepted that TM domains are composed of mostly apolar amino acids, highly compatible with the hydrophobic interior of the lipid bilayer, native TM helices nevertheless contain on average 30% polar amino acids18–21 – the latter of which may function as electrostatic side chain-side chain helix-helix interaction/stabilization sites, and/or as water-interactive linings of aqueous channels. A subset of naturally-occurring TM helices, common to multi-pass membrane proteins, exhibit even higher than average content of polar character.19 These ‘marginally hydrophobic’ TM helices can lead to ambiguous TM domain prediction and may represent ‘hot spots’ for misfolding. As such, the degree to which the lipid bilayer can accommodate non-compatible components, such as polar and charged amino acids - and what level qualifies as ‘sufficient’ hydrophobic character in the bilayer – are influential factors. An additional question is whether hydrophobicity is measured as an average value along the length of a potential TM helix, or whether local areas of high hydrophobic character promote insertion. Native TMs contain de facto a preponderance of hydrophobic residues that regularly occur consecutively in the sequence; a typical example is the Ile-Leu-Ile-Leu-Leu stretch found in TM helix 2 of the mammalian TRPV2 ion channel (PDB ID: 5AN8). Interestingly, a comparison of poly-Ala sequences supplemented with three or four Leu residues showed a noticeable increase in membrane insertion when hydrophobic residues were contiguous.3 Similarly, in a series of Leu/Ser peptides of equal Leu and Ser content, we previously found that sequences with Leu residues placed consecutively in the sequence (‘blocks’) increased both the partitioning of peptide sequences into lipid environments and their membrane integration22, i.e., the Leu-block peptides were more helical in membrane-mimetics, and readily underwent


Biochemistry

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translocon-mediated membrane insertion relative to compositionally-identical (‘scrambled’) sequences lacking a contiguous stretch of hydrophobic amino acids. While we now understand how such clustering of hydrophobic amino acids affects the insertion and folding of TMs on an intentionally exaggerated scale (50/50 polar/apolar design)22, the subtle effects of hydrophobic and helical patterning within more native-like TMs remain to be elucidated. To address these issues systematically, here we have designed a library of peptides that mimic the amino acid content of natural TM domains, including those with a range of polar character. An assessment of peptide properties across different membrane mimetics, utilizing a combination of biophysical and biological techniques, enabled us to isolate the features of protein sequence and environment(s) that influence peptide partitioning into lipid systems in vitro and membrane insertion in vivo.

MATERIALS AND METHODS Peptide synthesis and purification. Peptides were synthesized on a PS3 peptide synthesizer using standard solid state Fmoc [N-(9-fluorenyl)methoxycarbonyl] chemistry on a low-load PAL−PEG resin that produced an amidated C-terminus after cleavage. Peptides were purified using high-performance liquid chromatography (HPLC) with a C4 semipreparative column. Typically, linear acetonitrile/water gradients were employed with initial conditions of 80% solvent A (95% water, 5% acetonitrile, and 0.1% TFA) and 20% solvent B (95% acetonitrile, 5% water, and 0.1% TFA). Peptides were quantified using the absorbance at 280 nm in water. Liposome preparation. TFE-solubilized peptide was added to chloroform-solubilized 1palmitoyl-2-oleoylglycero-3- phosphocholine (POPC) (10 µM peptide and 2.5 mM lipid) and


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dried under N2. The lipid−peptide film was washed with water prior to resuspension in aqueous buffer [10 mM Tris-HCl and 10 mM NaCl (pH 7.4)] and underwent three freeze− thaw cycles. Samples were then passed through a 0.2 µm filter until the solution became clear. Samples were equilibrated overnight. Circular dichroism spectroscopy. Peptide was added to buffer [10 mM Tris- HCl and 10 mM NaCl, pH 7.4] with or without 175 mM SDS to a final peptide concentration of 25 µM. Samples were allowed to equilibrate overnight at room temperature. POPC-solubilized samples (10 µM peptide and 2.5 mM lipid) were equilibrated overnight after extrusion. CD spectra were recorded on a Jasco J-810 CD spectropolarimeter at room temperature in a 0.1 cm path length cuvette. Spectra represent the average of at least three independently-prepared samples. Spectra were background subtracted and converted to mean residue molar ellipticity (MRE) using standard formulas. HPLC retention times. Reverse phase high performance liquid chromatography (RPHPLC) on a Zorbax StableBond C-18 analytical column was performed using 20 µg of peptide dissolved in 1 mL of water. Day-to-day variations in elution were corrected using the elution of uracil (5 µg in 50 µL of water), injected prior to addition of peptide to the column. The mobile phase composition was 60% solvent A (95% water, 5% acetonitrile, and 0.1% TFA) and 40% solvent B (95% acetonitrile, 5% water, and 0.1% TFA) for samples with 5 Leu and 65% solvent A and 35% solvent B for samples with 7 Leu. Tryptophan fluorescence. Fluorescence spectra were recorded on a Photon Technology International fluorimeter using a 1 cm path length quartz cuvette. Tryptophan was excited at 280 nm, and emission spectra were recorded between 300 and 400 nm. Peptide samples (5 µM in 10 mM Tris- HCl, 10 mM NaCl, pH 7.4) with (35 mM) and without detergent were equilibrated


Biochemistry

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overnight at room temperature prior to reading. POPC-solubilized samples (10 µM peptide and 2.5 mM lipid) were equilibrated overnight after extrusion. Samples were background subtracted, and the wavelength of maximal fluorescence emission intensity was recorded. Detergent titration assay. Procedures were adapted from Alvares et al., (2013).23 Increments of 0.2 mM SDS from a concentrated stock supplemented with peptide (to avoid changes in total peptide concentration over time) was added to peptide samples (15 µM in 10 mM Tris- HCl, 10 mM NaCl, pH 7.4) and allowed to equilibrate for 8 minutes prior to taking a CD reading. Titration were carried out until the detergent CMC was surpassed (3.46 mM SDS; SI Materials and Methods, Figure S1). Data points were fit with inhibitory-dose response curves using GraphPad Prism. Translocon-mediated free energies of insertion. LepB constructs encoding peptide sequences were generated by modifying the lepB gene in the pGEM-1 vector containing SpeI and KpnI restriction sites as previously described. 1,2,22 Lysine tags were omitted from sequences in the LepB construct. GGPG repeats insulated the core TM domain (e.g. 5L0Sbl : GGPGAAAAAALLLWLLAAAAAAA-GPGG); see Supplementary Fig. S2 for the full sequence of the LepB constructs. Constructs cloned in pGEM1 were transcribed and translated in the TNT Quick coupled transcription/translation system as described in the Supplementary material. An apparent equilibrium constant between the membrane-integrated and non-integrated forms was calculated as Kapp = f1g/f 2g, where f1g is the fraction of singly glycosylated LepB molecules and f2g is the fraction of doubly glycosylated LepB molecules. The results were then converted to apparent free energies of membrane insertion via the equation ∆Gapp = −RT ln Kapp.


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RESULTS Peptide design. To define the sequence context in which clustering hydrophobic amino acids together impacts the overall properties of a transmembrane domain, we designed compositionally identical sequences with hydrophobic amino acids either clustered together into a central, contiguous sequence (termed “block” (bl) peptides), or dispersed along the length of the TM (termed “scrambled” (scr) peptides), amid increasing average polar character (Table 1). The core sequences are each 19 residues long, composed of a poly-alanine background supplemented with 5 or 7 Leu residues and a single centrally-positioned Trp to enable fluorescent studies. Polar character was increased through the stepwise substitution of two Ala for Ser residues (2S peptides), up to a total of six Ser residues (6S peptides). Ala was chosen for its ‘neutral’ behaviour towards membrane insertion24; Leu is the most common hydrophobic amino acid (and most common amino acid overall) found in natural TM helices, while Ser is the most common polar amino acid.20 All peptides were Lys-tagged (three on each terminus) to increase their aqueous solubility and ease of handling.25 Addition of Lys tags to transmembrane peptides have been shown to have no effect on core sequence properties.26 Each peptide was designed with an average segmental hydrophobicity value above that required for spontaneous membrane insertion (> 0.4 on the Liu-Deber scale24) Peptide HPLC retention times. Peptide retention times on a C18 column were measured under isocratic mobile phase conditions to determine peptide effective hydrophobicity, viz., peptides that preferentially interact with the column alkyl chains relative to the mobile phase will be retained longer. Not unexpectedly, peptides of relatively high average hydrophobicity displayed the longest retention times (Table 2). Yet, large differences were observed in retention times of compositionally identical sequences when no or few polar amino acids were present (0,


Biochemistry

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2S) - a difference that diminished as polar character is increased. Thus, while 5-Leu block peptides with 0, 2 or 4 Ser eluted later than their corresponding scrambled peptides, the elution times of the 5-Leu 6S block and scrambled peptides were indistinguishable from one another. Comparison of the CD spectra of the 5-Leu and 7-Leu peptides in the isocratic solvent ratios used in the HPLC retention time experiments to peptide retention time revealed no correlation between peptide helicity and time of elution (data not shown). All the peptides (except 5L6Sscr) exhibit identical helical spectra in HPLC isocratic solvents (Figure S3), confirming that the wide variations in retention times measured for the 5-Leu and 7-Leu peptide series (3-10 min.; Table 2) reflect the perceived hydrophobicity of each segment as it partitions from the mobile phase into the acyl chain stationary phase of the column. Helicity and Trp burial in detergent micelles. Secondary structure content of each peptide was assessed in sodium dodecyl sulfate (SDS) detergent micelles using CD spectroscopy. All peptides adopted helical conformations in the presence of detergent micelles, with sequences of higher average hydrophobicity exhibiting more helical character than those with higher polarity (Figure 1). With the exception of the 5-Leu 2S peptides, the block peptide was always more helical than the corresponding scrambled peptide when comparing sequences of identical composition. We used Trp fluorescence as a measure of how ‘buried’ the centrally placed Trp residue was within the hydrophobic micelle core. While increases in blue shift were correlated with higher average hydrophobicity, variations were found between block and scrambled sequences when polar residues were present (Figure S4). 5-Leu peptides lacking polar amino acids (0S), exhibited identical strong blue shifts for both peptide variants, while sequences of intermediate


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polarity (2S and 4S), showed relatively greater blue shifts for block sequences. The 5-Leu peptides with the highest polarity (6S) had the most solvent-exposed block sequence. In control experiments with dabsyl- and dansyl-labelled peptides, Förster Resonance Energy Transfer (FRET) experiments established that the 5L0Sbl and 5L0Sscr peptides were monomeric species (data not shown); the remaining peptides travel at similar molecular weights as the 0S peptides on SDS-PAGE gels, implying similar monomeric states. Peptide interactions with detergent molecules below the critical micelle concentration. CD spectroscopy was used to measure increases in peptide helicity as detergent (below CMC) was titrated into peptide samples. Initial charge pairing between positively charged peptide Lystags and negatively charged sulfate head groups of SDS caused an interim loss in peptide helical structure at low concentrations of detergent.23 Subsequent small increments (0.2 mM) of detergent then induced folding of the peptides into helical conformations. In almost all cases, the peptides adopted full helical character prior to SDS concentrations surpassing the CMC. Using the concentration of detergent (mM) at which a peptide had reached half of its full helicity, noted as the helical mid-point, we obtained a measure of how sub-micellar detergent monomers (or finite aggregates) initially interact with exposed hydrophobic loci. We found that peptides with relatively high average hydrophobicity reached their helical mid-point at lower concentrations of detergent (Figure 2). Interestingly, the largest differences in helical mid-point values between pairs of block and scrambled sequences is observed when no polar amino acids are present (0S) and decreases as polarity is increased. At high polarity (5-Leu 4S and 6S), little to no difference in helical mid-point is observed between sequence pairs. The data are summarized in Table 2. Helicity and Trp burial in liposomes. The secondary structures of the peptides in the presence of POPC liposomes were determined using CD spectroscopy. All peptides - with the


Biochemistry

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exception of the 5-Leu 6S peptides - were helical, with sequences of higher average hydrophobicity displaying relatively greater helical content (Figure S5). The two 5-Leu 6S sequences did not adopt helical conformations, but rather exhibited CD spectra representative of unstructured peptides. Observation of significant blue shifts in the presence of liposomes, in conjunction with adoption of helical structure, is a positive indication of favourable lipid interaction. We found that the 5-Leu 0S and 2S sequences exhibit strong blue shifts, while the 4S and 6S sequences have fluorescence spectra indicative of solvent exposed Trp residues (Figure 3). Notably, no difference is observed between the blue shifts within each pair of compositionally identical sequences at all polarity levels. Translocon-mediated membrane insertion assay. The ability of the translocon to identify and insert the sequences of the present peptide library into native membranes was tested using the Lep construct and rough microsomes, as described previously.1 Differential glycosylation of the Lep construct reveals the relative inserted/translocated state(s) of the sequence of interest (Figure 4A), from which a Kapp for insertion may be derived and the apparent free energy of insertion (∆Gapp) calculated (see Materials and Methods). When polar character is absent (5L0Sbl and 5L0Sscr; Figure 4B, black bars) or high (5L6Sbl and 5L6Sscr; Figure 4B, green bars), no difference was observed in the membrane insertion efficiency of paired block and scrambled sequences, with the two inserting equally well (0S) or equally poorly (6S). However, with the incorporation of two Ser residues, the block sequence inserts significantly better than the scrambled sequence - a trend that continues for the 4S peptide pair (Figure 4B, blue and red bars. respectively). The ∆Gapp values calculated from the data in Figure 4B are summarized in Table 2. Comparison of the ∆Gapp experimental values to values predicted


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using the online ∆G Prediction Server (http://dgpred.cbr.su.se/) yields a strong correlation (R2 = 0.9, Figure S6). For a given sequence composition, predicted ∆Gapp values are always lower for the block sequence compared to the scrambled sequence because Leu residues are concentrated near the middle of the block sequences where they contribute more to the overall ∆Gapp value.2 Increasing the number of hydrophobic amino acids. Experiments were repeated using sequences with an increased number of Leu residues (seven), in combination with 2- and 4-Ser residues (Table 1). We found that the trend persists of minimizing differences between compositionally-identical block and scrambled sequences as polarity increases. A large difference in HPLC elution time was observed between block and scrambled 7-Leu 2S peptides, while the retention times of the 7-Leu 4S pair of peptides were equivalent in a manner reminiscent of the 5-Leu 6S peptides (Table 2). In SDS micelles, the 7-Leu block peptides were more helical than the corresponding scrambled peptides (Figure 1; Figure S7A, left). For all polarity levels, the block peptide was more blue-shifted than its scrambled counterpart (Figure. S7A, right). In detergent titration assays, the block versions of the 2S and 4S 7-Leu peptides reached helical mid-point at lower concentrations of detergent than the corresponding scrambled peptides (Table 2). Similar to the 5-Leu peptides, little to no differences are observed between the peptide pairs with respect to liposome interactions (Figure S7B). However, transloconmediated membrane insertion was found to favor the block sequence when 4 Ser residues are present (Figure 4, brown bars), while the 7-Leu 2S block and scrambled peptides insert into the membrane equally well (Figure 4, grey bars) - analogous to the results with the 5-Leu 0S sequences. Helical mid-point values, retention times, and ∆Gapp values are summarized in Table 2.


Biochemistry

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DISCUSSION Effective hydrophobicity depends on sequence, composition, and environment. We have explored the role(s) of protein sequence and environment on the membrane insertion and folding of TM domains using a variety of biophysical and biological techniques, in conjunction with systematic comparisons of Leu block vs. scrambled sequences, and from comparisons of peptides with low vs. higher polar (Ser) content. When we evaluated peptide interactions with a C18 HPLC column, and SDS detergent micelles and monomers (sub-micellar concentrations) environments that enable peptide polar residues to ‘escape’ to water (viz., the column/solvent interface or the micelle surface) - block peptides tended to have longer retention times, and be more helical and buried into the micelle’s effective interior than corresponding scrambled peptides. In these water-accessible membrane environments, local areas of high hydrophobic character, rather than an averaged value, tended to dictate peptide behaviour. As well, peptides containing a strong hydrophobic locus read out as more hydrophobic - reinforcing the affinity (or ‘compatibility’) of the Leu cluster for acyl chains - and are therefore more likely to undergo membrane integration both in vitro and in vivo. In liposomes, a notably more restrictive environment where embedded segments have little contact with water, no difference was seen in the helicity or Trp burial of each pair of compositionally-identical block and scrambled peptides at all polarity levels (Figure 3). Regarding prospective insertion into the bilayers of POPC liposomes, sequences containing 0 or 2 Ser interacted favorably with bilayers (highly helical, solvent shielded Trp), while sequences containing 4 or 6 Ser did not. Thus, the 0S and 2S block and scrambled peptides were found to be equally lipid compatible, while the 4S and 6S peptides were equally lipid ‘incompatible’, a result pointing to an inevitable ‘toleration limit’ of bilayer membranes for polar content of


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sequences. Thus, interaction with lipid bilayers was found to be an ‘all-or-nothing’ event: sequences with core hydrophobicity below a threshold may not become membrane-integrated without the intervention of the insertion machinery or neighboring proteins within the bilayer.27 Here, the comparison of direct incorporation of peptides into POPC liposomes (Figure 3) to their translocon-mediated membrane insertion (Table 2, Figure 4B) is particularly apt. We found that sequences with high lipid compatibility and average hydrophobicity (5L0Sbl and 5L0Sscr; Figure 3), readily underwent translocon-mediated membrane insertion, independent of hydrophobic patterning. However, upon addition of polar character, sequences lacking a strong hydrophobic locus did not readily undergo membrane integration (e.g., 5L2Sscr, 5L4Sscr, Figure 4B). Thus, within the water-filled translocon channel, favorable solvation of the Ser residues may compete with strong hydrophobic loci driven towards the membrane interior by the unfavourable entropy loss of water surrounding a hydrophobic surface, as depicted schematically in Figure 5. The energetic sum of these favorable and opposing interactions determines the final translocation/insertion state of the sequence. The presence of polar residues within an incipient TM strand can therefore impede partitioning of sequences lacking a strong hydrophobic locus, until ultimately, sequences that are deemed to be incompatible with the lipid bilayer do not undergo translocon-mediated membrane insertion even when a strong hydrophobic centre was present (e.g., 5L6Sbl, Figure 4B). Hydrophobic blocks promote membrane integration when segmental hydrophobicity is moderate. In the present work, we further undertook to assess the limiting influence of hydrophobic content in the presence of increasing polar content through design and synthesis of a second set of peptides with higher average segmental hydrophobicity (7-Leu peptides; Table 1). Indeed, we found that the increased Leu content resulted in a corresponding shift in the


Biochemistry

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number of polar amino acids required to observe a difference between pairs of block and scrambled sequences in biophysical and biological assays (Table 2; Figure 1, 4; Figure S7). Largely, the 7-Leu 2S sequences (average hydrophobicity above 5-Leu 0S) behave like the 5-Leu 0S peptides, with block and scrambled sequences undergoing translocon-mediated membrane insertion equally well (Table 2; Figure 4, grey bars). The 7-Leu 4S sequences (average hydrophobicity in-between 5-Leu 0S and 2S) behave in a manner paralleling the 5-Leu 2S sequences, with block sequences preferentially inserting into native membranes over corresponding scrambled sequences (Table 2; Figure 4, brown bars). We thus find that the extents of insertion determined experimentally in the Lep construct assay favor the block vs. scrambled sequences for the 5L2S, 5L4S, and 7L4S peptide pairs (viz., those of moderate hydrophilicity) (Fig. 4B). This trend is expected from our previous observation that a hydrophobic residues located centrally in a transmembrane segment promotes membrane insertion more strongly than does a peripherally located hydrophobic residue2, and is captured by the ∆G Prediction Server based on these data (Fig. S6). Our combined biological and biophysical results thus identify an overall hydrophobicity window within which the hydrophobic content and patterning play the decisive role in determining whether a given protein segment will be suitable for insertion into a lipid bilayer. We further find that when average segmental hydrophobicity exceeds or falls below given threshold values, hydrophobic patterning has little effect on the efficiency of membrane integration (Figure 6; 5-Leu 0S, 6S and 7-Leu 2S): if the scrambled sequence is sufficiently hydrophobic to give close to 100% integration, the corresponding block sequence will integrate to a similar degree (although the free energy of integration may still be lower for the block sequence); conversely, if the block sequence is too polar (≈ 0% integration), the corresponding


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scrambled sequence (that should have a higher free energy of integration) is also expected not to integrate. In protein-free lipid bilayers, a distinct “threshold hydrophilicity” is observed, in which sequences of higher polar character are deemed incompatible, independent of sequence hydrophobic patterning (Figure 3; 4S and 6S). However, sequences partitioning from the translocon channel into the lipid bilayer experience an increase in the “threshold hydrophilicity”, as lipid-incompatible sequences of higher polar content readily undergo membrane integration when a hydrophobic locus is present (Figure 4, 5L4Sbl). Our results suggest that when a segment’s average hydrophobicity falls within a given range (Figure 6, 0.7-1.5, highlighted orange), the clustering of hydrophobic amino acids near the middle of the sequence significantly affects initial interactions with the translocon channel and the bilayer during the in vivo insertion process, promoting membrane insertion and thereby increasing the membrane’s tolerance for polar residues (Figure 6, 5-Leu 2S, 4S; 7-Leu 4S). Native TM domains with average hydrophobicity within this “block dominance” range may insert more efficiently into the lipid bilayer when a contiguous stretch of hydrophobic amino acids is present.

CONCLUSION By defining a range of average hydrophobicity and polar residue content within which a hydrophobic block promotes membrane integration, our work provides insight into potential mechanisms for the membrane integration and folding of membrane protein sequences. As models for partitioning of potential TM sequences from water to lipid, detergent-peptide and column-peptide interactions present to the peptide chain the types of interactions similar to those experienced during translocon-mediated membrane insertion, while liposome studies reveal information about the integration of protein sequences into the receiving environment, the lipid


Biochemistry

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bilayer. Significantly, our results suggest that the change of a single amino acid within a protein sequence, altering average hydrophobicity and hydrophobic patterning, may easily tip the balance in the wrong direction, resulting in anomalous translocation/insertion - potentially triggering susceptibility to a disease state.


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FUNDING INFORMATION

Supported, in part, by grants to C.M.D. from the Natural Science and Engineering Research Council of Canada (NSERC Discovery Grant A2807) and from the Canadian Institutes of Health Research (CIHR FRN-5810); and by grants to G.vH. from the Swedish Cancer Foundation, the Swedish Research Council, and the Knut and Alice Wallenberg Foundation. N.W. was a participant in the Samuel B. Lunenfeld Summer Research Program at the Hospital for Sick Children.

SUPPORTING INFORMATION AVAILABLE

The ANS titration plot for determination of the CMC of SDS in the buffer used is provided, along with tryptophan fluorescence spectra and CD spectra for the 5-Leu and 7-Leu peptide series in SDS micelles and POPC lipid bilayers. CD spectra of peptides under HPLC column conditions are reported. A comparison between predicted and experimental free energies of membrane insertion and the complete sequence for the lepB construct (e.g. 5L0Sbl) is also provided.


Biochemistry

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REFERENCES

(1) Hessa, T., Kim, H., Bihlmaier, K., Lundin, C., Boekel, J., Andersson, H., Nilsson, I., White, S. H., and von Heijne, G. (2005) Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433, 377–381. (2) Hessa, T., Meindl-Beinker, N. M., Bernsel, A., Kim, H., Sato, Y., Lerch-Bader, M., Nilsson, I., White, S. H., and von Heijne, G. (2007) Molecular code for transmembrane-helix recognition by the Sec61 translocon. Nature 450, 1026–1030. (3) Demirci, E., Junne, T., Baday, S., Berneche, S., and Spiess, M. (2013) Functional asymmetry within the Sec61p translocon. Proc. Natl. Acad. Sci. U. S. A. 110, 18856–18861. (4) Baeza-Delgado, C., von Heijne, G., Marti-Renom, M. A., and Mingarro, I. (2016) Biological insertion of computationally designed short transmembrane segments. Sci. Rep. 6, 1–9. (5) Nørholm, M. H. H., Cunningham, F., Deber, C. M., and von Heijne, G. (2011) Converting a marginally hydrophobic soluble protein into a membrane protein. J. Mol. Biol. 407, 171–179. (6) Cymer, F., von Heijne, G., and White, S. H. (2015) Mechanisms of integral membrane protein insertion and folding. J. Mol. Biol. 427, 999–1022. (7) Van den Berg, B., Clemons, W. M., Collinson, I., Modis, Y., Hartmann, E., Harrison, S. C., and Rapoport, T. A. (2004) X-ray structure of a protein-conducting channel. Nature 427, 36–44. (8) du Plessis, D. J. F., Berrelkamp, G., Nouwen, N., and Driessen, A. J. M. (2009) The lateral gate of SecYEG opens during protein translocation. J. Biol. Chem. 284, 15805–15814. (9) Egea, P., and Stroud, R. (2010) Lateral opening of a translocon upon entry of protein suggests the mechanism of insertion into membranes. Proc. Natl. Acad. Sci. U. S. A. 107, 17182– 17187. (10) MacCallum, J. L., and Tieleman, D. P. (2011) Hydrophobicity scales: a thermodynamic looking glass into lipid-protein interactions. Trends Biochem. Sci. 36, 653–662. (11) Veenendaal, A. K. J., van der Does, C., and Driessen, A. J. M. (2004) The proteinconducting channel SecYEG. Biochim. Biophys. Acta 1694, 81–95. (12) Junne, T., Schwede, T., Goder, V., and Spiess, M. (2007) Mutations in the Sec61p channel affecting signal sequence recognition and membrane protein topology. J. Biol. Chem. 282, 33201–33209. (13) Bondar, A. N., Val, C. Del, Freites, J., Tobias, D., and White, S. (2010) Dynamics of SecY translocons with translocation-defective mutations. Structure 18, 847–857. (14) Junne, T., Kocik, L., and Spiess, M. (2010) The hydrophobic core of the Sec61 translocon defines the hydrophobicity threshold for membrane integration. Mol. Biol. Cell 21, 1662–1670. (15) Crowley, K. S., Reinhart, G. D., and Johnson, A. E. (1993) The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell 73, 1101–1115.


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(16) Crowley, K. S., Liao, S., Worrell, V. E., Reinhart, G. D., and Johnson, A. E. (1994) Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore. Cell 78, 461–471. (17) Capponi, S., Heyden, M., Bondar, A.-N., Tobias, D. J., and White, S. H. (2015) Anomalous behavior of water inside the SecY translocon. Proc. Natl. Acad. Sci. U. S. A. 112, 9016–9021. (18) Cunningham, F., Rath, A., Johnson, R. M., and Deber, C. M. (2009) Distinctions between hydrophobic helices in globular proteins and transmembrane segments as factors in protein sorting. J. Biol. Chem. 284, 5395–5402. (19) Hedin, L. E., Öjemalm, K., Bernsel, A., Hennerdal, A., Illergård, K., Enquist, K., Kauko, A., Cristobal, S., von Heijne, G., Lerch-Bader, M., Nilsson, I., and Elofsson, A. (2010) Membrane insertion of marginally hydrophobic transmembrane helices depends on sequence context. J. Mol. Biol. 396, 221–229. (20) Baeza-Delgado, C., Marti-Renom, M. A., and Mingarro, I. (2013) Structure-based statistical analysis of transmembrane helices. Eur. Biophys. J. 42, 199–207. (21) De Marothy, M. T., and Elofsson, A. (2015) Marginally hydrophobic transmembrane α helices shaping membrane protein folding. Protein Sci. 24, 1057–1074. (22) Stone, T. A., Schiller, N., von Heijne, G., and Deber, C. M. (2015) Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences. Biochemistry 54, 1465–1473. (23) Alvares, R. D. A., Tulumello, D. V, MacDonald, P. M., Deber, C. M., and Prosser, R. S. (2013) Effects of a polar amino acid substitution on helix formation and aggregate size along the detergent-induced peptide folding pathway. Biochim. Biophys. Acta - Biomembr. 1828, 373–381. (24) Liu, L. P., and Deber, C. M. (1998) Guidelines for membrane protein engineering derived from de novo designed model peptides. Biopolymers 47, 41–62. (25) Melnyk, R. A., Partridge, A. W., Yip, J., Wu, Y., Goto, N. K., and Deber, C. M. (2003) Polar residue tagging of transmembrane peptides. Biopolymers 71, 675–685. (26) Melnyk, R. A., Partridge, A. W., and Deber, C. M. (2001) Retention of native-like oligomerization states in transmembrane segment peptides: application to the Escherichia coli aspartate receptor. Biochemistry 40, 11106–11113. (27) Heinrich, S. U., Mothes, W., Brunner, J., and Rapoport, T. A. (2000) The Sec61p complex mediates the integration of a membrane protein by allowing lipid partitioning of the transmembrane domain. Cell 102, 233–244. (28) Liu, L., and Deber, C. M. (1998) Uncoupling Hydrophobicity and Helicity in Transmembrane Segments. J. Biol. Chem. 273, 23645–23648.


Biochemistry

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Table 1. Sequences and segmental average hydrophobicity of peptides synthesized in the present work. a

Ala, Leu, Trp and Ser are colored red, yellow, black and blue respectively. Peptides contain

three Lys residues on each terminus (not shown). The subscript bl denotes Leu block peptides; scr denotes peptides where Leu residues have been “scrambled”; note that Leu positions in the 5L0Sscr peptide are maintained in subsequent scrambled sequences, and similarly for 7S2Sscr and 7L4Sscr peptides. bPeptide average hydrophobicity is reported using values calculated from the Liu-Deber scale.28

Peptide

5L0Sbl 5L0Sscr 5L2Sbl 5L2Sscr 5L4Sbl 5L4Sscr 5L6Sbl 5L6Sscr 7L2Sbl 7L2Sscr 7L4Sbl 7L4Sscr

a

Sequence

Avg. b hydrophobicity

5-Leucine AAAAAALLLWLLAAAAAAA LAAAALAAAWLAALALAAA AAASAALLLWLLAAAASAA LAASALAAAWLAALALSAA ASASAALLLWLLAAASASA LSAAALSAAWLSALALSAA ASASASLLLWLLASASASA LSASALASAWLASLSLASA 7-Leucine AASAALLLLWLLLAAASAA LASLALAALWAAALALSLA ASASALLLLWLLLAASASA LSALALSALWASALALSLA


1.6 1.3 1.0 0.7

1.8 1.5

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Table 2. Peptide helical mid-point, HPLC retention values, and experimental free energies of insertion. a

Peptide helical mid-points = the concentration of SDS at which half of full helical character is

regained during a detergent titration. bRetention time within a C18 HPLC column under isocratic mobile phase conditions (see Materials and Methods). cExperimental free energies of membrane insertion were derived from the fraction of singly- and doubly-glycosylated constructs after treatment with translocon-containing microsomes (see Materials and Methods).

Peptide

5L0Sbl 5L0Sscr 5L2Sbl 5L2Sscr 5L4Sbl 5L4Sscr 5L6Sbl 5L6Sscr 7L2Sbl 7L2Sscr 7L4Sbl 7L4Sscr

Helical Retention mid-point Time a b (mM SDS) (min) 5-Leucine 0.6 10 1.1 5.3 1.0 4.9 1.3 3.5 2.0 3.7 1.8 3.2 2.7 3.4 2.4 3.2 7-Leucine 1.7 4.9 2.0 4.7 1.6 3.2 2.2 3.0

∆Gapp Experimental c (kcal/mol)


-0.80 -0.95 -1.11 -0.22 0.23 0.80 1.15 1.16 -1.01 -0.97 -0.65 -0.29

Biochemistry

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FIGURE LEGENDS

Figure 1. Helicity of peptides in detergent micelles. Mean Residue Ellipticity (MRE) values at 222 nm, taken from CD spectra of the indicated peptides in SDS micelles. Error bars are reported as the standard deviation; significant differences are denoted by (*), p < 0.05.

Figure 2. Peptide interaction with detergent monomers. Detergent monomers were added (0.2 mM increments) to the indicated peptides (15 µM), and helical character was recorded using CD spectroscopy. Data points were fit with inhibitory-dose response curves. Detergent CMC is represented by a black dashed, vertical line (3.46 mM, 0.54 = log(3.46)). Peptides containing block sequences are depicted as solid lines with data points as squares, and corresponding scrambled sequences as dashed lines and circles. Block and scrambled 0S and 2S peptides are depicted in black and blue, respectively. Data points represent the averaged results of at least two independent experiments.

Figure 3. Tryptophan fluorescence of peptides in lipid bilayers. Tryptophan fluorescence spectra of the peptides in POPC liposomes. 0S and 2S peptides exhibit strong blue shifts (black and blue lines, respectively). 4S and 6S have spectra similar to fully solvent exposed Trp (red and green lines, respectively). The fluorescence emission maxima of free Trp in water is denoted by a vertical black line (350 nm). Leu block peptides are depicted by solid lines, and scrambled peptides as dashed lines. Spectra shown represent an average from three independently prepared samples.


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Figure 4. Experimental free energies of translocon-mediated membrane insertion. (A) Peptide sequences were inserted into the Lep construct as depicted in the cartoon (sequence colored red, and the two glycosylation sites indicated as G1 and G2; see Supplemental material for details), and expressed in the presence of rough microsomes. Sequences that insert into the ER become monoglycosylated at position G1, and sequences that are translocated across the ER become diglycosylated at positions G1 and G2. (B) Differential glycosylation patterns were used to derive Kapp and ∆Gapp values as described in Materials and Methods. ∆Gapp values represent an average of three independent experiments. Significant differences are denoted by (*), p < 0.05. Error bars are reported as the standard deviation.

Figure 5. The hydrophobic effect drives membrane integration of Leu block sequences with moderate average hydrophobicity. Schematic of the comparative insertion/translocation of Leu block peptide 5L4Sbl with its scrambled counterpart 5L4Sscr. Three perspectives are provided: the interior of the translocon channel (grey), the channel-lipid interface, and the lipid bilayer. Phospholipid molecules are colored white (hydrogen), red (oxygen) and grey (carbon). 5L4Sbl and 5L4Sscr sequences are depicted as helices with Ala in red, Leu in yellow, and Ser in blue. The 5L4Sbl sequence has a ‘cylinder’ of oriented water molecules surrounding the Leu block (highlighted in light blue). In the water-filled translocon channel, the unfavorable loss in water entropy may drive membrane insertion of the Leu block upon which the 5L4Sbl sequence becomes fully embedded within the POPC bilayer. The 5L4Sscr peptide, having a significant amount of solvated polar amino acids and lacking a strong hydrophobic locus along its length, is translocated rather than membrane integrated.


Biochemistry

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Figure 6. Hydrophobic blocks promote membrane integration in TMs with moderate average hydrophobicity. The difference between ∆Gapp for block and scrambled sequences (BL ∆Gapp - SCR ∆Gapp) is plotted against peptide average segmental hydrophobicity. ∆∆Gapp values below 0 indicate the block sequence is preferentially inserted into the membrane. As ∆∆Gapp approaches zero, block and scrambled sequences of a given peptide pair are inserted/translocated equally well. 5-Leu peptides are represented by black circles, 7-Leu peptides as red squares, and the 9Leu-9Ser peptides22 as a blue triangle. The average hydrophobicity range for “block dominance” is outlined in light orange.


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Figure 1.


Biochemistry

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Figure 2.


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Figure 3.


Biochemistry

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Figure 4.


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Figure 5.


Biochemistry

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Figure 6.


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For table of contents use only.

Hydrophobic clusters raise the threshold hydrophilicity for insertion of transmembrane sequences in vivo


Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion

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