Application of the Triangular Spatial Relationship Algorithm in Representing and Quantifying Conformational Changes in Chlorophylls and Protein Local Environments

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Application of the TSR algorithm in representing and quantifying conformational changes of chlorophylls and protein local environments” by Milton and co-authors, use 3D structures of Chl-binding proteins already existing in the PDB and the TSR-based method to investigate 3D structures of various types of Chls.

There are small modifications I suggest to the authors.

First of all, both in the abstract and the introduction the aim of the work is summarized in three different research questions. It could be useful, then, to divide “Results and Discussion” section into those three questions or at least recall those questions into the conclusion section followed by author’s answer/work. “Conclusion” section is structured very similar to an abstract, thus I suggest implementing with some more result outcomes. Moreover, I suggest renaming chapter 2 into “Result and Discussion”.

Line 48: “to” can be deleted;

Lines 93-95: for the sake of clarity, I suggest rephrasing the sentence;

Lines 119-120: for the sake of clarity, I suggest rephrasing the sentence;

Line 294: remove the bracket.

Author Response

Reviewer #1

 

The manuscript “Application of the TSR algorithm in representing and quantifying conformational changes of chlorophylls and protein local environments” by Milton and co-authors, use 3D structures of Chl-binding proteins already existing in the PDB and the TSR-based method to investigate 3D structures of various types of Chls.

 

The authors sincerely thank Reviewer #1 for your time for reviewing the revised manuscript. The comments are specific, insightful and helpful. The changes are highlighted in “blue”.

 

 

There are small modifications I suggest to the authors.

First of all, both in the abstract and the introduction the aim of the work is summarized in three different research questions. It could be useful, then, to divide “Results and Discussion” section into those three questions or at least recall those questions into the conclusion section followed by author’s answer/work. “Conclusion” section is structured very similar to an abstract, thus I suggest implementing with some more result outcomes. Moreover, I suggest renaming chapter 2 into “Result and Discussion”.

 

We revised the Conclusion section. We correlate the three conclusions to the three unresolved questions raised in the Introduction section.

 

 

Line 48: “to” can be deleted.

 

Done!

 

 

Lines 93-95: for the sake of clarity, I suggest rephrasing the sentence.

 

We rephrased the sentence.

 

 

Lines 119-120: for the sake of clarity, I suggest rephrasing the sentence.

 

We corrected the grammatical mistake.

 

 

Line 294: remove the bracket.

 

Done.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Comments paper: ”Application of the TSR algorithm in representing and quantifying conformational changes of chlorophylls and protein local environments.”

Milon et al., 2024, Photochem, 3428920

The structures of the chlorophyll protein complexes of the photosynthetic apparatus are based on intensive non-covalent interactions between multiple pigments and the protein matrix. Although all chlorophyll a molecules (Chl a), for instance, are the same, each of them has a different orientation and their interaction with the protein part depends on specific amino acids. These results in conformational changes of the Chl molecules when embedded in the protein matrix and modulate their spectroscopic characteristics. In this paper, the authors used the Triangular Spatial Relationship based method in combination with the available 3-D structures of Chl-binding proteins deposited in the Protein Data Bank to investigate systematically the 3D structures of various types of Chls. The outcome of the study is a structural foundation through quantifying a large amount of (B)Chl and histidine conformations for future mechanistic understanding of relationships between pigment-protein interactions and their corresponding spectroscopic data.

The main purposes of the research work are clearly described in the abstract under the formulated as four fundamental questions. The problem the authors wanted to tackle is to get more insight in the complex interactions between chlorophylls and their protein environment. Till now, these interactions have not been studied systematically. The authors introduced here the Triangular Spatial Relationship (TSR)-based method which has been described in studies on protein 3D structures and probing drug and target interactions. Applying this approach in analysing the complex interactions in photosynthetic pigment-protein-complexes is quite novel and will contribute in the understanding of mechanisms of energy/exciton transfer. In this way, this approach is complementary to the research already done and to elucidate the physical/molecular mechanism involved in the photosynthetic light absorption and energy conversion.

The research done is not an experimental one, but described the application of an TSR algorithm on an extended data set. Based on 3D structures of 22 different pigments involved in photosynthesis and histidine. The theoretical background of the algorithm is clearly explained and the clustering analysis were visualized based on Average Linkage Clustering. The sections of the methods, Key Generation, Protein structural similarity and distance calculations, Development of a version of the TSR-based method, Development of a TSR-algorithm, Data preparation, Hierarchical cluster and classification analysis and visualization and finally, the Statistical analysis are all enough informative and understandable. The only remark is one of layout, this can be improved: the formulas (equation 1 and 2) should be aligned in the centre. The data are available in Excel-files in the supplementary information.

The results are presented in detail and illustrated with figures which has been taken care of making the section informative, understandable, well supported by an extensive reference list and pleasant to read.

In conclusion: the paper is well written, the methods are sufficiently informative, the description of the results, the discussion and conclusion are well-founded. The figures has been taken care off.

After reading meticulously the paper, I have no specific comments and in my opinion, the paper can be accepted for publication.

Author Response

Reviewer #2

 

Comments paper: ”Application of the TSR algorithm in representing and quantifying conformational changes of chlorophylls and protein local environments.” Milon et al., 2024, Photochem, 3428920

 

The structures of the chlorophyll protein complexes of the photosynthetic apparatus are based on intensive non-covalent interactions between multiple pigments and the protein matrix. Although all chlorophyll a molecules (Chl a), for instance, are the same, each of them has a different orientation and their interaction with the protein part depends on specific amino acids. These results in conformational changes of the Chl molecules when embedded in the protein matrix and modulate their spectroscopic characteristics. In this paper, the authors used the Triangular Spatial Relationship based method in combination with the available 3-D structures of Chl-binding proteins deposited in the Protein Data Bank to investigate systematically the 3D structures of various types of Chls. The outcome of the study is a structural foundation through quantifying a large amount of (B)Chl and histidine conformations for future mechanistic understanding of relationships between pigment-protein interactions and their corresponding spectroscopic data.

 

The main purposes of the research work are clearly described in the abstract under the formulated as four fundamental questions. The problem the authors wanted to tackle is to get more insight in the complex interactions between chlorophylls and their protein environment. Till now, these interactions have not been studied systematically. The authors introduced here the Triangular Spatial Relationship (TSR)-based method which has been described in studies on protein 3D structures and probing drug and target interactions. Applying this approach in analysing the complex interactions in photosynthetic pigment-protein-complexes is quite novel and will contribute in the understanding of mechanisms of energy/exciton transfer. In this way, this approach is complementary to the research already done and to elucidate the physical/molecular mechanism involved in the photosynthetic light absorption and energy conversion.

 

The research done is not an experimental one, but described the application of an TSR algorithm on an extended data set. Based on 3D structures of 22 different pigments involved in photosynthesis and histidine. The theoretical background of the algorithm is clearly explained and the clustering analysis were visualized based on Average Linkage Clustering. The sections of the methods, Key Generation, Protein structural similarity and distance calculations, Development of a version of the TSR-based method, Development of a TSR-algorithm, Data preparation, Hierarchical cluster and classification analysis and visualization and finally, the Statistical analysis are all enough informative and understandable. The only remark is one of layout, this can be improved: the formulas (equation 1 and 2) should be aligned in the center. The data are available in Excel-files in the supplementary information.

 

We align Equation 1 and Equation 2 in the center.

 

 

The results are presented in detail and illustrated with figures which has been taken care of making the section informative, understandable, well supported by an extensive reference list and pleasant to read.

 

In conclusion: the paper is well written, the methods are sufficiently informative, the description of the results, the discussion and conclusion are well-founded. The figures have been taken care off.

 

After reading meticulously the paper, I have no specific comments and in my opinion, the paper can be accepted for publication.

 

The authors sincerely thank Reviewer #2 for your time for reviewing the revised manuscript. We appreciate your encouraging comments for our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Comments

 

In this study, the 3D structures of various types of chlorophyll were systematically investigated using the 3D structures of chlorophyll-binding proteins deposited in the Protein Data Bank and methods based on triangular spatial relationships. However, before considering publication, the following issues need to be addressed.

 

1. Can spectroscopic techniques such as circular dichroism spectroscopy, Raman, and molecular dynamics simulations study the effect of protein environment on chlorophyll conformation? The authors need to summarize the limitations of the existing research methods in terms of spatial conformation in the context of the literature.

 

2. Figure 2b is not cited in the text and it is recommended that the relevant discussion be added or the figure be removed; there are problems with the arrangement of the icons in Figure 7 and Figure 7h is missing, which the authors need to double-check and optimize.

 

3. The conclusion section mentions that several spectroscopic and theoretical methods have been available for analyzing pigment-protein interactions. The authors were asked to briefly describe the core principles of the triangular spatial relationship algorithm and to compare it with common analytical methods.

 

4. Is the algorithm only applicable to chlorophyll-protein interactions or can it be generalized to other biomolecule systems?

 

5. English needs to be touched up, and spelling and grammar need to be double checked.

 

 

 

 

Author Response

Reviewer #3

 

In this study, the 3D structures of various types of chlorophyll were systematically investigated using the 3D structures of chlorophyll-binding proteins deposited in the Protein Data Bank and methods based on triangular spatial relationships. However, before considering publication, the following issues need to be addressed.

 

The authors sincerely thank Reviewer #3 for your time for reviewing the revised manuscript. The comments are specific, insightful and helpful. The changes are highlighted in “blue”.

 

 

1. Can spectroscopic techniques such as circular dichroism spectroscopy, Raman, and molecular dynamics simulations study the effect of protein environment on chlorophyll conformation? The authors need to summarize the limitations of the existing research methods in terms of spatial conformation in the context of the literature.

 

A variety of spectroscopy techniques (e.g., FTIR, ultrafast, EPR) have been used to probe interactions between cofactors and surrounding amino acids. However, those methods cannot quantify the chlorophyll conformations. Now, a new technique, XFEL, can demonstrate the conformational changes of chlorophylls and protein environments. However, to quantify the conformational changes, the TSR algorithms or other popular methods (e.g., RMSD) are needed. We revised the Conclusion section. We changed this section title to “Conclusions, limitations and future directions”.

 

We stated the limitation of the TSR algorithm. It is a structure-based approach. It can define and quantify structural similarity and difference. However, it cannot tell their functions (e.g., binding energy). Therefore, functional data are needed to fully understand the structure results obtained from the TSR-based method.

 

 

2. Figure 2b is not cited in the text and it is recommended that the relevant discussion be added or the figure be removed; there are problems with the arrangement of the icons in Figure 7 and Figure 7h is missing, which the authors need to double-check and optimize.

 

One of Figure 2a in the text should be Figure 2b. We corrected this.

 

We have renumbered some of the Figure 7 panels.

 

 

3. The conclusion section mentions that several spectroscopic and theoretical methods have been available for analyzing pigment-protein interactions. The authors were asked to briefly describe the core principles of the triangular spatial relationship algorithm and to compare it with common analytical methods.

 

We have compared the TSR algorithms with other popular structure-based methods in our previously published work. We cited those references. In addition, we stated the differences between the TSR-based methods and theoretical approaches (e.g., MD simulations and QM/MM calculations). The TSR algorithm can complement MD simulations and QM/MM calculations using theoretical or modeled 3D structures as the input data.

 

 

4. Is the algorithm only applicable to chlorophyll-protein interactions or can it be generalized to other biomolecule systems?

 

The TSR algorithm is a structure-based method that can be used in studying any types of molecular interactions (e.g., cofactor and protein, pigment and pigment, protein and protein and DNA and protein interactions). We have add this point in the revised manuscript.

 

 

5. English needs to be touched up, and spelling and grammar need to be double checked.

 

We polished the languages and corrected typos.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

ACCEPT