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    Referee #3

    Evidence, reproducibility and clarity

    Recommendation: Publish after revisions

    Duart and colleagues have put forth a manuscript detailing the under-appreciated effect of intrahelical salt bridge formation to the insertion transmembrane helices. Utilizing an in vitro and in vivo applicable construct of vehicle protein leader peptidase (Lep) from Escherichia coli, the authors were able to use glycosylation patterns as a way to quantify the apparent free energy of membrane insertion for multiple transmembrane helices. These results have demonstrated the importance of taking salt bridge formation into account when developing membrane protein prediction tools; however, prior to publication, further analyses would be beneficial for supporting their quantitative conclusions.

    Major Comments:

    • It would be helpful if the authors detailed their process in deciding which of the 136 potential salt bridge-containing helices were chosen for further investigations.
    • Considering the data presented in Fig. 3c, it may be useful to also include charged pair mutations in the i, i+3 positions in the analyses of helix G and helix A, as these positions are the most likely to form salt bridges. This would act as a useful positive control, to see if the mutations would improve the insertion of the sequence.
    • Page 14, Line 255: Authors state "The salt bridge contributes approximately ~0.5 kcal/mol to the apparent experimental free energy of membrane insertion." Can this change in apparent free energy be attributed completely to the mutation? Are there any potential interactions between the inserted helix and the natural H2 transmembrane sequence of Lep that could be changing with the various mutations?
    • It is promising to see these results in the context of the Lep protein, but the authors should consider the effect these salt bridges may have in the context of the full protein. Creating mutants of Halorhodopsin or calcium ATPase would determine the impact of potential salt bridge disruption on protein folding, which would provide some context on the functional consequences of these mutations.
    • Authors have presented a strong argument for the inclusion of potential salt bridge formation in the prediction of transmembrane helices; however, they have not detailed the necessary steps for developing a new system. It would be encouraging to see recommendations on the next steps towards better prediction software.

    Minor comments:

    • Page 8, Line 118: Authors state "Our results showed a tendency to better insertion when charge pairs were placed in positions (i, i+1; i, i+3; i, i+4) that are permissive with salt bridge formation (Fig. 1c), actually an effect not observed in the predictions (Fig. 1b)." It is important to clarify that this "better" insertion in Fig. 1c is compared to each respective predicted value in Fig. 1b. Currently, it reads as if the authors are suggesting the introduction of the charged pair residues is helping the insertion of the unaltered L4/A15 sequence.
    • Figure 1a: Add cytoplasmic and lumen identifiers for clarity.
    • Figure 3b: Slashes for "Opp charge" and "Same charge" in the legend appear to be reversed according to the values presented in Table 2.

    Significance

    This paper increases our understanding of salt bridges in membrane protein structure and function, as performed systematically by a lab with major expertise in this research area.

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    Referee #2

    Evidence, reproducibility and clarity

    As the title states, Duart et al. have examined the energetic cost of the translocon-assisted insertion of TM helices containing salt bridges into membranes. The paper has three parts: (1) model studies using the methods of Hessa et al. (12,13), (2) statistical analysis of salt bridges in membrane proteins of known 3D structure, and (3) Hessa et al. measurements of selected i,i+4 salt-bridge containing TM helices from halorhodopsin (PDB ID = 3QBG) and calcium ATPase (1SU4). The major overall conclusion is that for i,i+4 salt bridges yield a more favorable insertion free energy of about 0.5 to 0.7 kcal/mol.

    The subject of the paper is broadly interesting, but it suffers from several problems that must be addressed before it can be considered seriously for publication. The comments below are described in terms of the three parts.

    (1) Figure 1b reports tabular values of predicted Hessa et al. DG values for sequences that contain K, D, or K & D substitutions in a parent L4A15 parent sequence, which has a favorable DG of about -0.5 kcal/mol. For all of the other sequences, DG is predicted to be unfavorable 1.4 kcal/mol to 3.5 kcal/mol. Figure 1c presents triplicate experimental measurements of DG for the sequences in Figure 1b shown as bar graphs. All of the sequences yield unfavorable DG values of about +0.2 kcal/mol except for the parent sequence that has a favorable value close to the predicted value.

    There are several problems with these data and their presentation. Fig. 1b should also include the measured DGs with standard deviations in addition to the predicted values. In Fig. 1c, the positive values are plotted on different scale than the sole negative value. This causes the authors to insert a break in the bar representing the sole negative value. The bars are color coded in a mysterious way that is not clearly described in the figure legend. In any case, the measured DG values are all about the same.

    A fundamental problem with the measurements is that the method of Hessa et al (12) should have been adhered to rigidly. As those authors noted "The quantification [of DG values]is maximally sensitive for H-segments with DGapp values close to zero (p < 0.5 in Fig. 1d); therefore, for each kind of residue we balanced the contribution from the central residue by varying the number of Leu residues until an H-segment with DG in the range -1.2 to 1.2 kcal/mol was found." The measurements reported Fig. 1b and 1c are far outside the maximum sensitivity range. The Western blots upon which the numbers are based should have been included (perhaps as an appendix).

    (2) The statical analysis seems fine and is useful.

    (3) Fig.4, halorhodopsin helix G measurements. The table of Fig. 4a should be expanded to include both in vitro (panel e) and in vivo data (panel f). It is not entirely clear where the values given in the table are from, but presumably from the in vitro data (panel e). Fig. 5, calcium ATPase helix A. Comments similar to those regarding Fig. 4 apply. The division of the long helix into a short greasy one and longer one carrying more charges is interesting, but it seems to add little to the main intent of the paper to assess the thermodynamic properties of helices containing salt-bridges.

    Overall, the paper would be stronger if it focused mostly on the part (1) experiments to arrive at definitive answer to the energetics of salt-bridge insertion. As it stands, it is a smash up of ideas and experiments.

    Significance

    Surprisingly,there have been no reported measurements that I am aware that examine the energetic cost of inserting TM helices containing salt bridge into membranes. This paper is a start in that direction.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In the manuscript „Intra-helical salt bridge contribution to membrane protein insertion" the authors investigate the effect of salt bridge formation between positively and negatively charged amino acids on the insertion behavior of α-helical protein segments into the membrane. Generally it is believed that polar or even charged residues prevent stable membrane insertion of α-helical protein segments, but some of these authors had already shown in a previous paper that such residues are more frequent than expected in transmembrane helices. In the current study, the authors investigate in detail the role of intra-helical salt bridge formation on stable membrane insertion. Using an in vitro membrane insertion assay based on the E. coli leader peptidase (Lep) protein, they found better membrane insertion for helical segments with opposite charge pairs placed at positions compatible with intra-helical salt bridge formation (positions i→i+1; i→i+3 and i→i+4). Furthermore, the authors performed a database screen which revealed that oppositely charged residues are overrepresented at these positions. Finally they picked two candidate membrane proteins from the database (Halorhodopsin and calcium ATPase 1) and proved the presence of an intra-helical salt bridge and determined the contribution of the salt bridge to the apparent free energy of membrane insertion (ΔGapp), which was in the range of 0,5-0,7 kcal/mol.

    Major comments

    1. It seems that the data in Fig. 3b has been mixed up, making it difficult to judge the conclusions. The bars with forward slash seem to represent the "same charge" data and the bars with backward slash seem to represent the "opposite charge" data (exactly contrary to the figure legend). In general the forward and backward slash representation is not easily distinguishable, and for the position i+4 both bars contain a forward slash (making it impossible to discriminate same and opposite charge). Please use filled and unfilled bars instead. Furthermore the bar diagram in Fig. 3a is stacked for opposite and same charge, whereas in Fig. 3b the respective bars are placed next to each other. Additionally the label of the y-axis in Fig. 3c is misleading, as it is not the "Frac. of opp. charged pairs" but the fraction of oppositely charged pairs that form salt bridges.
    2. The authors don´t give details no how the log odds ratios and the respective p-values have been determined. Please include this in the Materials and Methods section. What does a p-value of 0.00e+00 mean (see Table 2, Spacing: +3, "All Log odds")?
    3. What is the proof that for the isolated helix A from the calcium ATPase 1 the membrane embedded part is identical to the full-length protein? The authors investigated two different helix A peptides, the full-length helix ranging from L49-F78, and one short fragment ranging from L49-A69 containing the more hydrophilic N-terminal region, which is the membrane-embedded region in the full-length protein. The authors state: "In contrast, when only the membrane-embedded sequence was included, the Lep chimera was mainly doubly-glycosylated (Fig. 5c, lane 3), suggesting that helix A is properly inserted when the full helical sequence is present." In my opinion this conclusion cannot be drawn from the data presented. The authors used an isolated helical segment, so in my opinion it is much more likely that the isolated full-length helix inserted via its hydrophobic C-terminal part (L60-F78) into the membrane. The authors themselves state in their manuscript: "It has been previously shown that the position in the membrane of TM helices in protein folded structures does not always correspond to the thermodynamically favored positions in the membrane of the isolated helices." Also the i→i+5 mutant points into that direction, because the effect of disturbing the intra-helical salt bridge for the helix A is much less pronounced compared to the similar data in Fig. 4f for the Halorhodopsin protein. In my opinion this shows that most probably only one charged residue (R63) is embedded inside the membrane (with a membrane embedded part of L60-F78).

    Minor comments:

    1. line 151: ",see Figure 2)" Typo: Bracket missing.
    2. line 172: "Other known structural features can also be hinted at, including aromatic ring stacking by His-Trp pair [20] at i→i+6." Please give some more examples of important structural features of membrane proteins, which can be seen in your analysis (e.g. I think that also the glycine zipper can be seen in the i→i+4 data set).
    3. line 255: "The salt bridge contributes approximately ~0,5 kcal/mol to the apparent experimental free energy of membrane insertion." Please explain that this value was derived from the comparison of the ΔGexp between the wt and the i→i+5 mutant. Please comment also on the large difference between the predicted (ΔGpred) and the experimental values (ΔGexp), even if no salt-bridge is involved (e.g. for the DD mutant).
    4. line 348: "Asp-Lys pairs at position i, i+4 and Glu-Lys pairs at position i→ i+3 are the most prevalent as seen previously in Figure 2. They are both among the most prevalent oppositely charged pairs and the charged pairs that form the highest number of salt bridges in membrane protein structures. This is in stark contrast to Glu-Arg pair at position i→ i+1 that although as frequent in pairs as Asp-Lys and Glu-Lys at positions i→i+4 and i→i+3 respectively, only form salt bridges in one-fourth of the cases." Fig. 2 shows that each charge pair has a different prevalence depending on the order (e.g. Asp-Lys and Lys-Asp pairs). I think for this statement the sum of both prevalences should be taken into account, and as the sum is not easy discernible from Fig. 2, it would help to include a table containing the sums. Furthermore, it would be good to refer also to Fig. 3, which also contains a part of the discussed data.
    5. line 402: "c-myc tag (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, EQKLISEEDL) was added in Ct in hanging with de PCR primer before cloning." Please revise the sentence and I think the one letter code for the c-myc tag is sufficient (please correct this also in line 428).
    6. line 420: "A region's total ΔG is the sum of these individual scores weighted on where in the region the residue, a residue in the middle of the helix has a higher weight than residues at the ends." Please revise the sentence, the meaning is unclear.
    7. line 436: "Total protein was quantified and equal amounts of protein submitted to Endo H treatment or mock-treated, followed by SDS-PAGE analysis and transferred into a PVDF transfer membrane (ThermoFisher Scientific)." Please revise the sentence.
    8. line 498: "Topological files with sequence and membrane topology are created with the help of the RCSB secondary structure file and only membranes annotated as pure α-helices were retained." I assume that the description contains a typo (membranes annotated as pure α-helices?)
    9. line 507: typo "..., but we did not clustered the proteins" 14: line 560: "The individual value of each experiment in represented by a solid dot being represented as a green square the experimental ΔG value for the L4/A15 sequence from [2]." Please revise the sentence. 15: line 562: "The wt and simple mutants are shown in white bars." Typo: single mutants 16: line 563: "Charges at compatible distances with salt bridge formation (i→i+1; i→i+3; and i→i+4) are shown in yellow. Not compatible distances with salt bridge formation (i→i+2; and i→i+5) are shown in dark gray. Compatible distances but not compatible amino acid pair (i, i+4 DD pair) is shown in clear gray." The given colors don´t match with the figure (i→i+1 = brown; i→i+3 = orange; i→i+4 = yellow and i→i+4 DD pair = white) 17: line 597: "The different monomers are shown in transparent blue, purple and indigo." The different colors are hardly distinguishable in the figure. 18: Figure 4a: The table could be simplified. I think the column "charges" can be removed, as it contains not really charges and the names of the peptides already contain the same information. The column "Å" contains only a value for the wt (and not for the DK i,i+5 mutant) and as the distance for the wt is also given in Fig. 4g, this column can be also removed.
    10. Fig. 4f: The marker lane is hardly visible (completely dark lane)
    11. Fig. 5b: The column "Å" contains only values for the wt sequences (long and short). See also comment 18.
    12. Fig 5d: Why is in the SDS gel a mass shift between the wt and the i→i+5 mutant visible, even though the peptide mass is equal.
    13. There are several changes of font type or format changes (e.g. line 210-214). Please correct this.

    Significance

    As a structural biologist with a focus on membrane-proteins, I understand that the study is concerned on intra-helical salt bridges, but the implications of inter-helical salt bridges should also be discussed, at least in the introduction or outlook. The authors propose that their results are important for the improvement of membrane protein topology prediction methods, so for this aim it is also necessary to take any potential inter-helical salt bridges into account. In this context, it would be relevant to point point out that there even exist extended rows of salt bridges between transmembrane segments (charge-zippers), which serve an important structural element in several membrane proteins.

    The article is well written and most of the conclusions drawn from the experimental results are convincing. I agree with the authors that their results are relevant for future improvement of membrane protein topology prediction software, which so far does not take the possibility of salt bridge formation into account. Therefore, I recommend publication after clarification/revision of the abovementioned points.

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