Bioluminescence Resonance Energy Transfer (BRET) Allows Monitoring the Barnase-Barstar Complex In Vivo

Round 1

Reviewer 1 Report

This is interesting paper about the application of BRET technique to structural studies of complexes.

Publication can be accepted in present form, however, there is additional conclusion which could be added. As the distance is 56A and the deviation is only 2A this suggest that this complex is rigid and there is no major structural changes in the linker region.

Author Response

Response to Reviewer 1 Comments

Point 1: Publication can be accepted in present form, however, there is additional conclusion which could be added. As the distance is 56A and the deviation is only 2A this suggest that this complex is rigid and there is no major structural changes in the linker region.

Response 1: Dear Reviewer, Thank you for the valuable remark about the rigidity of the mutual orientation of the photoproteins within the complex barnase•barstar. Certainly, Fig.2(b) represents one of the possible orientations of photoproteins that satisfies the estimated distance (56 ± 2) Å. The error ± 2 Å does not signify the rigidity of this orientation but shows only the accuracy of the average distance determination by several experiments. The same distance may be realized at other orientations, as far as allowed by the linkers. The corresponding explanation is inserted in the text.

Sincerely yours,

Gennady V. Semisotnov,

Corresponding author

Author Response File: Author Response.pdf

Reviewer 2 Report

The work is focused on the interaction of barnase and barstar using BRET. The work is interesting, and the presentation is neat and easily understandable. However, I have few concerns about the results.

If the proteins are not folded properly, does it show bioluminescence at some other wavelength or the bioluminescence property is lost? Some citation regarding this will be helpful.

Please include some references for the alternative square donor - square acceptor concept as mentioned in line 168?

Reference 21 doesn’t contain the quantum yield information of RLuc

Is there any difference in quantum yield between Luciferase and Barnase-Luciferase? If yes, then what it is? can it be the reason why the cell lysate in Figure 1a doesn’t show EGFP activity?

The quantum yield 0.053 seems very low to me. I strongly recommend including some references (preferably in the introduction) in support of the fact that luciferase can act as donor even with such low quantum yield.

Why does the overlap integral doesn’t cover the entire area under the common region of donor emission and acceptor absorption? Is there room to explain it with the mathematical calculation?

The distance calculated exceeds the usually acceptable range of R0±(0.5*R0). The FRET efficiency is 3% which is of serious concern to me. I would require more control experiments to show that it is actually due to the BRET and not any other noise.

If the authors can take care of the abovementioned concerns, I would recommend the article for publication.

Author Response

Response to Reviewer 2 Comments

Dear Reviewer,

The authors are grateful for the careful reading of the manuscript and valuable remarks. The answers you can find below.

Point 1: If the proteins are not folded properly, does it show bioluminescence at some other wavelength or the bioluminescence property is lost? Some citation regarding this will be helpful.

Response 1: Bioluminescence of luciferases is caused by oxidation of the specific cofactor (for example, coelenterazine), while the protein is the matrix that provides specific binding and oxidation of this cofactor. The bioluminescence spectrum position is conditioned rather by the structure of the cofactor than by the protein structure. If the protein is not folded properly it cannot bind cofactor and hence emit bioluminescence.

Point 2: Please include some references for the alternative square donor - square acceptor concept as mentioned in line 168?

Response 2: Included.

Point 3: Reference 21 doesn’t contain the quantum yield information of RLuc.

Response 3: Thank you. The reference is changed.

Point 4: Is there any difference in quantum yield between Luciferase and Barnase-Luciferase? If yes, then what it is? can it be the reason why the cell lysate in Figure 1a doesn’t show EGFP activity?

Response 4: On the basis of literature data and our own experiments, we can confirm that the luciferase does not essentially change its quantum yield after fusion with other proteins (see, for example, [21]). The spectra of the mixture of cell lysates containing the luciferase alone and barstar●EGFP fusion (represented in Fig.1a,b) only confirm that the interaction of these proteins is absent while the BRET (Fig 1c) occurs due to interaction of barnase with barstar.

Point 5: The quantum yield 0.053 seems very low to me. I strongly recommend including some references (preferably in the introduction) in support of the fact that luciferase can act as donor even with such low quantum yield.

Response 5: Several additional references are included in the introduction.

Point 6: Why does the overlap integral doesn’t cover the entire area under the common region of donor emission and acceptor absorption? Is there room to explain it with the mathematical calculation?

Response 6: When calculating the overlap integral, the products of the intensities of donor emission and acceptor absorption peaks are summarized. As the distance from the maximum increases, the product of two decreasing values leads to a sharper decline in the multiplied value.

Point 7: The distance calculated exceeds the usually acceptable range of R0±(0.5*R0). The FRET efficiency is 3% which is of serious concern to me. I would require more control experiments to show that it is actually due to the BRET and not any other noise.

Response 7: One of the BRET advantages is that in the absence of external excitation practically the whole luminescence of the acceptor is arisen due to energy transfer from the donor. This event essentially decreases the errors at BRET efficiency measurements. Besides, in our case, the emission areas of the donor and acceptor are similar (Fig.1c) that increasing the accuracy of the measurements.

Sincerely yours,

Gennady V. Semisotnov,

Corresponding author

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have addressed the questions satisfactorily. I would recommend accepting the paper.