Interactions of an Artificial Zinc Finger Protein with Cd(II) and Hg(II): Competition and Metal and DNA Binding

Round 1

Reviewer 1 Report

The work from Haijdu et al. reports the characterization of an artificial zinc finger protein (1MEY#) containing 3 Cys2His2 binding sites. The interaction with toxic metal ions and its consequences on DNA binding was investigated by several spectroscopic techniques. I believe that the study is well performed and the results are of interest to a broad readership, comprising both inorganic and biological chemists.

However, the manuscript needs to be revised before publication in Inorganics. Following is a list of concerns to be addressed:

1. The introduction is very precise and contains lots of literature on ZFPs. However, recent examples of designed Cys2His2 containing peptides are missing. Please, consider including the following references: La Gatta et al. Molecules 2021, 26(17), 5221. Rodriguez et al. Chem. Sci. 2016, 7, 3298. 

2. In competition experiments, it was not possible to completely substitute Zn(II) with Cd(II) in the applied concentration range. Is there a specific reason for which the authors did not explore higher concentrations? The CD signals in Figure 1 and 3 are quite low (the maxima reach around 5-6 mdeg) and appear a bit noisy.

2. In my opinion, ESI-MS experiments provide a good support to the observation of metal exchange, but they are not suitable for the determination of a binding constant for the same reasons that the authors explained in the manuscript (different ionization capability and stability during the electrospray). Therefore, I suggest to remove the value of log(beta) obtained by ESI-MS.

3. Too many experimental details are given in the figure captions. I suggest keeping only the essential information in the caption (eg. Concentrations, pH, buffer) while moving the description of experimental procedures in the “Materials and methods” section of the manuscript.

4. Errors should be presented with a single significant digit (only when it equals to 1, it is possible to report the errors with two significant digits). For example, 12.8 +-0.33 on page 8, line 310 is not correct.

5. There is improper use of arrows in Schemes I and II (). The kind of arrows reported, is commonly used to represent different resonance structures of the same molecule. The authors should replace them with equilibrium arrows ().

6. There are some grammar and spelling errors throughout the manuscript, which should be carefully revised.

As an additional comment, I believe it is quite surprising that the authors did not consider the possibility of using Co(II) as a UV-Vis probe for studying the binding of Cd(II) and Hg(II). Indeed, Co(II) metal complexes have peculiar visible absorption bands and are usually characterised by lower binding constants with respect to Cd(II) complexes. This would have made simple and straightforward the determination of Cd(II) and Hg(II) binding constants by competition experiments followed by UV-Vis.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper by Hajdu et al explores the interaction of an artificial zinc finger with Cd(II) and Hg(II)  and their DNA binding. The protein, 1MEY#ZFP, consists of three CP1-like ZF subunits; the authors assume that they have similar metal-binding properties (since the three fingers differ only in few a.a.) and present data on an average binding site. In other words, they do not characterize separately each finger.

The paper is really interesting and original, the English is fluent and the figures clear. I cannot say the same for schemes 1 and 2 that I suggest to change or delete.

Scheme 1 (a) (Cadmium binding to the protein) reports a schematic representation of a so called ‘sequential’ Zn(II)àCd(II) exchange, supported by ESI-MS spectra. Nevertheless, there are no indications that such exchange happens in the order indicated in the reported scheme. Indeed, since the assumed similar metal binding properties and the reported average affinity, I think that the first metal ion can be bound at the same time by anyone of the fingers (and so happens when two metals are bound) and so this hierarchical scheme can be misleading. These same considerations can be applied to scheme 2(a).

As far as the Hg binding, the authors report in table 2 four estimated stability constants for three ZF-based binding sites: this is also quite misleading and should be commented.

Finally, since they found, with ESI-MS experiments, that the protein can bind up to 13 Hg(II) metal ions and that the exact mode of coordination remains unknown, it should be interesting to know if they have considered the possibility that the protein can form oligomers that can share the coordinated ions.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript by Gyurcsik and his coworkers describes binding behavior of Cd and Hg ions for an artificial zinc finger protein engineered by authors' previous work. The bindings of these ion for the protein were mainly evaluated by the combination of CD, fluorescence titration and ESI MS.  Also some informations of conformation of the protein were obtained by gel-shift assay in DNA binding.  The results clearly show that Cd is similar to Zn in the binding for the protein, whereas significant structural changes by binding with Hg were observed. The findings are quite reasonable when the characteristics of Cd and Hg ions are considered for surfer ligand.  Hypothetic Schemes shown in Schemes 1 and 2 are plausible, but this reviewer wonder how the authors rule out the oligomerization of protein via unfavorable metal binding. Thus, this reviewer recommends the minor revision before acceptance.  The followings are concerns of this reviewer.

1. How does the author confirm the binding without oligomerization? Is there experimental size information by size exclusion chromatography or dynamic light scattering?

2. The authors just evaluate ion exchange experiments.  How about thermodynamic stability? This is analyzed by temperature increase and/or addition of denaturant for purified samples.  

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf