Protein Fusion Strategies for Membrane Protein Stabilization and Crystal Structure Determination
Round 1
Reviewer 1 Report
This review discusses fusion protein strategies to improve the stability of membrane proteins and eventually to improve their crystallisation properties. While the paper is reasonably structured and the content is valid, the English has so many issues that it significantly hampers readability. Therefore a thorough language check is strongly recommended. The paper makes heavy use of acronyms and not all are defined (e.g. BRIL). Others are not defined at the first instance (e.g. VKOR). This should be corrected. A few other points are listed below:
l.123 Surely the authors mean endoplasmic reticulum; endothelium is the inside of blood vessels...
l.169 What is SFX34? SFX should be defined anyway.
ll. 232-234 What do the authors mean with "both type I 233 and type II properties"? Please clarify. Also a reference to type I and II membrane protein crystals might be in order as not all readers are familiar with the concept.
ll.427-432 I have a hard time imagining where it might be helpful to use a membrane protein as a scaffold for a soluble protein. The authors could motivate this suggestion a bit more.
Author Response
We appreciate the insightful comments from the reviewers and their careful reading of the manuscript. Please see our point-by-point responses below and tracked change text in the revised manuscript.
Reviewer 1:
While the paper is reasonably structured and the content is valid, the English has so many issues that it significantly hampers readability. Therefore a thorough language check is strongly recommended.
Response: We have thoroughly revised the language in this manuscript.
The paper makes heavy use of acronyms and not all are defined (e.g. BRIL). Others are not defined at the first instance (e.g. VKOR). This should be corrected.
Response: All abbreviations have been defined at the first occurrence.
l.123 Surely the authors mean endoplasmic reticulum; endothelium is the inside of blood vessels...
Response: Corrected.
l.169 What is SFX34? SFX should be defined anyway..
Response: We have spelled this out to “serial femtosecond crystallography”.
l. 232-234 What do the authors mean with "both type I 233 and type II properties"? Please clarify. Also a reference to type I and II membrane protein crystals might be in order as not all readers are familiar with the concept.
Response: We have explained this sentence as “the crystals have both type I (interactions between membrane regions) and type II (interactions between soluble regions) properties”. The reference for this definition has been added.
ll.427-432 I have a hard time imagining where it might be helpful to use a membrane protein as a scaffold for a soluble protein. The authors could motivate this suggestion a bit more.
Response: Agreed. We have removed this idea.
Reviewer 2 Report
This review concerns an interesting and actual topic of studying the structures of membrane proteins by protein crystallography. The authors describe the most critical problems in this area: the production of proteins, their purification and crystallization, and the use of fusion proteins in overcoming these problems. Three different strategies for constructing such fusion proteins are discussed in detail with specific use cases. The authors place particular emphasis on the use of the so-called "termini restraining" by sfGFP. This technique was developed by the authors of the review, successfully applied and actively promoted by them. Indeed, good results have been obtained using this technique.
In general, the review turned out to be interesting, informative, but not overloaded with unnecessary details. The manuscript deserves publication in Crystals.
However, the authors should carefully check the spelling due to the abundance of typos. In many places the names of organisms are not in italics: lines 49, 65,66, 68, 202, and also in the bibliography.
Line 53 the name of the author YELISEEV is in all capitals.
Line 142 - the comma is in wrong place.
Line 367 - in silico should be italic.
The inscription "Figure" in all cases is written with an error "Figureure".
And, it would be better to move the figures itself closer to the first mention in the text. So, the figure 2 may move to the beginning of part 3.2, figure 3 - at the beginning of section 5, and figure 4 - at the and of this section.
Author Response
Reviewer 2:
However, the authors should carefully check the spelling due to the abundance of typos. In many places the names of organisms are not in italics: lines 49, 65,66, 68, 202, and also in the bibliography.
Response: We have thoroughly corrected the typos and language. All names of organisms have been changed to italic.
Line 53 the name of the author YELISEEV is in all capitals.
Response: Corrected.
Line 142 - the comma is in wrong place.
Response: Corrected.
Line 367 - in silico should be italic.
Response: Deleted after the revision.
The inscription "Figure" in all cases is written with an error "Figureure".
Response: All corrected.
And, it would be better to move the figures itself closer to the first mention in the text. So, the figure 2 may move to the beginning of part 3.2, figure 3 - at the beginning of section 5, and figure 4 - at the and of this section.
Response: Sequence of figure references has been corrected.
