Toward Nanodisc Tailoring for SANS Study of Membrane Proteins
Round 1
Reviewer 1 Report
Comments and Suggestions for Authorsreview bioengineering-4050902
The problem of studying the structure of membrane proteins has been known for quite some time. A number of methods, as the authors note, have significant limitations. In this context, developing SANS techniques for this purpose is undoubtedly relevant.
Lines 47-48: Among solution methods, small-angle neutron scattering (SANS) is especially powerful for interrogating macromolecular complexes and protein lipid interactions.
This statement requires references.
Line 213: SANS was performed at 14 to 24 ºC, i.e below the lipid melting temperature.
The authors chose ripple phases (in particular, DMPC) for studies.
Why wasn't the temperature chosen when the lipids were in the gel phase? Analysis of small-angle scattering curves for determining structural parameters would have yielded a clear solution.
Distances in Table 1 should be changed as a Parameters.
The inner and outer leaflets of the lipid bilayers are asymmetrical. From this perspective, it is necessary to analyze the SLD profiles for the studied systems.
It's also worth comparing the calculated SLD values from the SANS and SAXS curves with theoretically calculated values. This will allo to assess how the protein integrates into the lipid bilayer by altering the polar head region of its outer layer.
The presented conclusions about the reliability of the method for studying the behavior of membrane proteins in solution must be confirmed by complementary methods.
Author Response
Thank you for your constructive comments
Comment 1:
Lines 47-48: Among solution methods, small-angle neutron scattering (SANS) is especially powerful for interrogating macromolecular complexes and protein lipid interactions. This statement requires references.
Answer 1:
References have been added
Comment 2:
Line 213: SANS was performed at 14 to 24 ºC, i.e below the lipid melting temperature.
The authors chose ripple phases (in particular, DMPC) for studies.
Why wasn't the temperature chosen when the lipids were in the gel phase? Analysis of small-angle scattering curves for determining structural parameters would have yielded a clear solution.
Answer 2:
You are completely right. Actually, we aimed for 10 ºC to be below the melting temperature for all lipids. But unfortunately, due to a technical problem, the temperature control failed. That is why we do not have a wisely chosen temperature. We decided to report it anyway for accuracy, but it is clearly a weakness that limits our interpretation of the data.
Comment 3:
Distances in Table 1 should be changed as a Parameters.
Answer 3:
This has been changed
Comment 4:
The inner and outer leaflets of the lipid bilayers are asymmetrical. From this perspective, it is necessary to analyze the SLD profiles for the studied systems.
Answer 4:
By our technique: extraction of full polar lipids and nanodiscs reconstitution, we cannot maintain leaflet asymmetry. The sentence : “The availability of these natural lipid mixtures allows the study of membrane proteins in an environment closer to their native context, although certain membrane properties, such as leaflet asymmetry, are still not fully recapitulated.” has been added to the conclusion (line 504) to clarify this point.
Comment 5:
It's also worth comparing the calculated SLD values from the SANS and SAXS curves with theoretically calculated values. This will allo to assess how the protein integrates into the lipid bilayer by altering the polar head region of its outer layer.
Answer 5:
This paragraph has been added (line 377): “The presented conclusions about the reliability of the method for studying the behavior of membrane proteins in solution must be confirmed by complementary methods.” Together with corresponding references.
Reviewer 2 Report
Comments and Suggestions for Authors The research considers the structural characterization of the nanodiscs made from three membrane proteins and three different lipids by SAXS and SANS. The study forms a systematic design of the experiments by a well-motivated series of samples and experimental conditions. The results of the study will add a relatively small but crucial part of structural information on the biomembrane mimicking nanodiscs, which will be interesting for a wide field of scientific audience (incl. soft matter physics, biophysics and chemistry, pharmacy, medicine, biology) Here are some minor comments which could be checked:Introduction:
One more paragraph about the examples of the previous studies, and especially some more recent references could be added after the line 62. There are not many recent (younger than 5 years) references given in the introduction. Besides, in the end of the introduction, the specific novelties of this study should be explained and highlighted more clearly for the reader: i.e. which if these scientific aims and samples have not been studied previously Results Line 279: the labels of the subfigures should be checked in Figure 1: there is now figure 1g (instead 1h), also check line 461: 1h <-> 1i (or change Figure 1) Lines 355-359: considering language, the sentences here should be checked and written in a more clearly way. Line 472: here could be added some more discussion on the results; e.g. How is this about 100% deuteration rate (at the matchpoint) comparable to the previous results on similar kinds of nanodisc samples? Was this result expected? Conclusion Lines 492-496: These sentences of the conclusions could be moved into results/discussion section (more reference/comparison to other studies would be nice to have in the results part) Overall note: The language is very fine but the proof reading should be done still more carefully (errors were find in several places and words of the manuscript)Author Response
Thank you very much for your constructive comments. I detail bellow the way I followed them to improve the manuscript:
Comments 1:
Introduction:
One more paragraph about the examples of the previous studies, and especially some more recent references could be added after the line 62. There are not many recent (younger than 5 years) references given in the introduction.
Answer 1: This sentence, with relevant references, was added after line 62: As examples, contrast variation SANS has been used to study protein-protein complexes [12], protein-nucleic acid complexes [13] and protein-surfactants complexes [7,9,14]
Comment 2: Besides, in the end of the introduction, the specific novelties of this study should be explained and highlighted more clearly for the reader: i.e. which if these scientific aims and samples have not been studied previously
Answer 2:
This sentence was added to the end of the introduction: “In particular, the natural lipid nanodiscs are presented for the first time and constitute a major step toward the development of tools for mem-brane protein study in their natural environment.”
Comment 3:
Results Line 279: the labels of the subfigures should be checked in Figure 1: there is now figure 1g (instead 1h),
Answer 3:
This has been corrected
Comment 4:
also check line 461: 1h <-> 1i (or change Figure 1)
Answer 4:
This has been corrected
Comment 5:
Lines 355-359: considering language, the sentences here should be checked and written in a more clearly way.
Answer 5:
The whole text has been polished using Chat-GPT.
Comment 6:
Line 472: here could be added some more discussion on the results; e.g. How is this about 100% deuteration rate (at the matchpoint) comparable to the previous results on similar kinds of nanodisc samples? Was this result expected?
Answer 6:
This comment made us realized that we did not really clarify why we were measuring deuterated nanodiscs. We added this paragraph at the beginning of the section 3.3 to overcome this point: “To study membrane proteins reconstituted in nanodiscs by SANS, the most favorable contrast—maximizing the signal-to-noise ratio—is achieved when the protein of interest is protonated, while the nanodisc components are deuterated to match the SLD of a D2O-based buffer. Under these conditions, the nanodisc becomes effectively invisible, providing the highest contrast between the protein and its environment while minimizing incoherent scattering. Optimal contrast is obtained when the nanodisc belt protein is approximately 72% deuterated and the lipids are fully deuterated”
Comment 7:
Conclusion Lines 492-496: These sentences of the conclusions could be moved into results/discussion section (more reference/comparison to other studies would be nice to have in the results part) Overall note: The language is very fine but the proof reading should be done still more carefully (errors were find in several places and words of the manuscript)
Answer 7:
The whole text has been polished using Chat-gpt, so these mistakes were hopefully removed.
