The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

Round 1

Reviewer 1 Report

The authors described the application of EPR spectroscopy for the characterization of different Cu(I) binding proteins. In general, this review is clear and different examples are provided to evidence the strength of the technique.

Major points

1) The explanation of SDSL should be introduced prior the description of cw-EPR and DEER experiments in section 1.2. Since the authors reported studies about "diamagnetic" systems, SDSL allows to investigate these proteins.

2) Alternative SDSL strategies, such as Tyr labelling or unnatural aminoacid, should be cited in the text for non-expert scientists

3) Spin labels different from nitroxide labels, such as Gd(III) tags or trityl radicals, should be mentioned in the review

4) The sentence at line 126-128 is not correct. Natural Cys can be exploited as labelling site if this aminoacid is not implicated in structural and/or funtional functions. For this reasons, this sentence should be rephrased

5) Basing on the guidelines of DEER spectroscopy (see ref: J. Am. Chem. Soc. 2021, 143, 43, 17875–17890), the background corrected DEER traces should be reported for each experiments

Minor point

1) (lines 122-123) This sentence is misleading and it should be rephrased in a correct form

2)  (lines 112-116) the explanation of the type of information achievable with cw-EPR experiments should be better explained

3) (legend of Fig. 10) The number of the reference should be added

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

General comment:

The review “The advantages of EPR spectroscopy in exploring diamagnetic metal ion binding and transfer mechanisms in biological systems” by Meron, Shenberger, and Ruthstein describes their recent advances in the use of electron paramagnetic resonance spectroscopy in studying conformational changes of biomolecules triggered by Cu(I) binding and transfer. The manuscript is well written and results are presented concisely; in some instances some additional elaboration could be beneficial, see specific points below.

Line 111/112 “nanoscale fluctuations” – can the authors specify? I assume they are talking about structural changes on the nanoscale but could be worded more clearly.

Line 117 “within a distance of 2.0-10. nm” – this suggests a very clear-cut distance range defined for PDS measurements, however distances beyond 10 nm have been measured previously.

Lines 116-120: The authors refer to DEER as the most common pulse EPR approach for acquiring nanoscale structure information without mentioning hyperfine spectroscopy. The authors might want to elaborate on this.

Figure 2: It would be helpful to also indicate the leaving group.

Line 158: “MTSSL-CMTGMHKMSC-MTSSL” – this representation seems unusual and I believe is, strictly speaking, wrong as not the full MTSSL molecule is attached as spin label. Introducing the R1 side chain might be appropriate.

Lines 204-208: Could the authors provide the version of MMM and the temperature used for the modelling, please. The way the authors describe the MMM modelling approach seems to differ from my recollections – the authors may want to check on this.

Also, could the authors provide some basic information on the processing for their DEER data (software; Tikhonov regularization or Gaussian fitting)?

Figure 5A: not readable, seems to be low resolution.

Line 219: It is not clear what the authors mean with “comparable small dynamics proteins”.

Line 221: MBDs could be named “subdomain” to avoid having domains within domains.

Line 251: cysteine?

Lines 254-256: “Pep1...” – The sequences of Pep1 to Pep4 are shown in Figure 7, however from this sentence it is not clear what has been mutated and where; this sentence should be clearer.

Line 318: Change “One questin asks” to “One question is”

Line 329: It should be clarified if the four methionine residues were replaced individually.

Line 332: Could the authors elaborate on what is the effect of having more than two MTSSL spin labels present in one protein complex? Do the authors expect multi-spin effects, and if so what would be their effects on the time traces and distance distributions, and did the authors take any measures into account to suppress such effects (e.g. power-scaling)?

Figure 10: Reference needs updating.

Lines 388-394: The authors should expand on their conclusions section. Specifically, the second paragraph seems rather short. When using dHis-Cu(II) as spin label, recent publications employing the RIDME method could demonstrate a sensitivity down to nanomolar protein concentrations, even for Cu(II)-Cu(II) measurements, as well as robustness against competitor ions.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

See Attached.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have dealt with all my comments.

Minor point - wording: amended text in line 140 should be corrected from 'refereed' to 'referred to'.