Membrane Depth Measurements of E Protein by ²H ESEEM Spectroscopy in Lipid Bilayers

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The current manuscript presents a topological analysis of site-specifically ^8^-labeled E protein from SARS-CoV-2 using ESEEM measurements. The authors describe the ESEEM methodology and perform benchmarks of distance-modulated echoes arising from couplings between the electronic and nuclear spins. The results demonstrate that the protein is in close contact with the lipid environment. Interestingly, the C-terminal residues 40 and 68 show clear modulation originating from the deuterated lipids. Another notable observation is that the residue located in the middle of the transmembrane region, L19 shows a decrease in solvent accessibility, yet still retains a surprising degree of water exposure—despite being expected to reside in a region largely devoid of solvent.

These findings are highly significant, highlighting the topological flexibility of transmembrane proteins and offering new insights into the existing structural model. I recommend that this paper be accepted after minor revision.

Specifically, since the results suggest that L19C retains considerable solvent accessibility, could this be attributed to a tilt of the transmembrane helix within the bilayer? If so, the authors should include a comment on this possibility in the Discussion section.

It would also improve readability to include a Conclusion section summarizing the key findings and their implications.

Finally, note that there are two full stops at the end of the sentence on line 130 after the word “absent..” this should be corrected.

Author Response

Please see Cover letter

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a concise study on a site-resolved view of how the SARS-CoV-2 E protein engages a membrane. The combination of MTSL labeling with ²H ESEEM, benchmarked against aqueous TEMPOL and depth-calibrated doxyl lipids, yields a coherent picture in which the transmembrane segment is lipid-embedded yet hydrated, while the C-terminal domain resides at or near the interface. The figures communicate the key comparisons cleanly, the narrative is focused, and the experimental design is straightforward. 

Beyond the specific system, the manuscript showcases ²H ESEEM as a practical depth-sensing readout that other groups can adopt to complement NMR or fluorescence approaches. That methodological accessibility, together with the biologically relevant insights into E-protein positioning, makes this contribution both timely and useful. 

Author Response

See Cover Letter

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Your work represents an adequate application of ESEEM spectroscopy for clarification of the envelope protein (E) topology within the membrane. The article is well written and can be recommended for publication in Biophysica after minor adjustments concerning mostly the abbreviations (see below).

Please note the following:

1. Keywords – Viroporin, ESEEM is not in the abbreviations.
2. Introduction – line 26: ER is not in the abbreviations; lines 32, 33 – if this structure was in a lipid……

Legend to Fig. 1 ²H-DMPC is not in the abbreviations. 

Author Response

 

See Cover Letter

 

Author Response File: Author Response.pdf