Design of a gold clustering site in an engineered apo-ferritin cage

Water-soluble and biocompatible protein-protected gold nanoclusters (Au NCs) hold great promise for numerous applications. However, design and precise regulation of their structure at an atomic level remain challenging. Herein, we have engineered and constructed a gold clustering site at the 4-fold symmetric axis channel of the apo-ferritin cage. Using a series of X-ray crystal structures, we evaluated the stepwise accumulation process of Au ions into the cage and the formation of a multinuclear Au cluster in our designed cavity. We also disclosed the role of key residues in the metal accumulation process. X-ray crystal structures in combination with quantum chemical (QC) calculation revealed a unique Au clustering site with up to 12 Au atoms positions in the cavity. Moreover, the structure of the gold nanocluster was precisely tuned by the dosage of the Au precursor. As the gold concentration increases, the number of Au atoms position at the clustering site increases from 8 to 12, and a structural rearrangement was observed at a higher Au concentration. Furthermore, the binding affinity order of the four Au binding sites on apo-ferritin was unveiled with a stepwise increase of Au precursor concentration.

1) How stable are the Au(I)-Apo-R168H/L169C-rHLFr composites in solution? Will the content of Au(I) change with time going by? 2) I noted that the authors spend less lines to describe the morphology of the Au12 cluster on the 4fold site. Are they icosahedral in shape? Icosahedral M12 or centered-icosahedral M13 (M denotes Au or Ag) are common kernel structure observed in thiolate protected Au or Ag nanoclusters.
3) The structure change of Au12 cluster induced by the dosage of Au(I) on the 4-fold site is interesting. What is the driving force for such structure change? The underlying chemistry governing such structure change rather than the phenomenon itself should be more attractive to the readers. 4) What is the possible application of these Au(I)-Apo-R168H/L169C-rHLFr composites? The authors may like to present their UV-vis absorption and photoluminescent spectra, which may provide hints for their potential applications. 5) Can the Au(I)-Apo-R168H/L169C-rHLFr composites be converted to Au(0) nanoclusters by reduction? 6) There are many typos or editorial errors which are annoying. The authors need to prepare their manuscript with greater care. Some examples are (this is not a complete list): a. Line 62: "reocnstructed" should be "reconstructed". b. Line 107: what does "relatively single" mean? c. Line 126: there is a repeating word "showing". d. Many unidentified symbols in Lines 140, 235, 240, etc.
Reviewer #2 (Remarks to the Author): In this paper, the authors constructed a novel gold clustering site at the 4-fold symmetric axis channel of the apo-ferritin cage. The stepwise accumulation process of Au ions into the cage and the formation of gold clusters in the cavity were evaluated. The role of key residues in the metal accumulation process was disclosed. As a result, the unique Au clustering site with up to 12 Au atoms in the cavity was obtained, and such a structure of the Au cluster can be designed. The binding affinity order of the four Au binding sites on apo-ferritin was further unveiled. The construction of this apo-ferritin cage and Au clusters in the cage are interesting, and the manuscript is well written with solid data to support their conclusions. I believe this work will be of interest to a broad scientific audience, especially for cluster material scientists. Thus I would like to suggest the acceptance of this paper after the authors have addressed the following minor issues.
(1) In the X-ray crystal structures of these gold nanoclusters, Au atoms are anchored into the designed cage via different Au-X interactions, including Au-N, Au-Cl, Au-S (and Au-C?). What is the underlying chemistry among these interactions (such as the interaction robustness, the reactivity, etc)? (2) Is that possible to evaluate the entry route of Au atoms to this cage? For example, as depicted in Figure 2A, are Au atoms first anchored onto several peripheral positions, and then transferred to the inner 4-fold symmetric cage? Or just occupy the 4-fold symmetric cage directly?
(3) In previously reported protein-stabilized gold or silver nanoclusters, the metal clusters are always emissive. Thus, are the Au clusters (especially, the Au12 cluster) in the designed cage emissive? If so, what is the relationship between the emission and the number of Au atoms in clusters?
Reviewer #3 (Remarks to the Author): Lu et al report an engineering and construction of a new-to-nature gold clustering site at the 4-fold symmetric axis channel of the apo-ferritin cage. The stepwise accumulation of Au(I) into the cage and the formation of Au clustering (up to 12 atoms) in the designed cavity are evaluated by a series of X-ray crystal structures. QC calculations are further carried out to obtain insights.
This work is quite interesting, and the results should benefit other researchers in the community. I suggest its acceptance for publication in CommsChem.

Improvements:
The figures are very far away from the paragraphs that discuss them! Pls move them to where they are first mentioned in the main text.

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(2) I noted that the authors spend less lines to describe the morphology of the Au12 cluster on 30 the 4-fold site. Are they icosahedral in shape? Icosahedral M12 or centered-icosahedral M13 31 (M denotes Au or Ag) are common kernel structures observed in thiolate protected Au or Ag 32

nanoclusters.
Reply: Thank you very much for your question. As the reviewer pointed out, we have now 34 discussed this in the revised manuscript (Page 16, line 314-319). We agree with the reviewer 35 that Icosahedral or centered-icosahedral are common kernel structures observed in thiolate-36 protected Au 12 and Au 13 nanoclusters. In our case, the asymmetric protein environment leads 37 to giving the unique facial square shaped structure which is uncommon in literature. As 38 demonstrated in Figure 4, the Au12 cluster on the 4-fold site exhibits a two-layer layered 39 morphology. It highlights the unique channel topologies that aided in the creation of this one-40 of-a-kind Au clustering site. Reply: We thank the reviewer for pointing out this interesting fact. Actually, we discussed a 47 little about this in the previous manuscript (Page 17, line 319-327). As pointed out, we 48 discussed more about the driving force of the structure change etc. in the revised manuscript 49 (Page 17, line 327-333). At higher gold precursor concentration, the equilibrium of metal-50 ligand (protein) interaction was changed. Since amino acid side chains are flexible and can 51 change their conformations according to the metal coordination structure, a more 52 energetically favourable Au cluster structure was thus formed at 400 equiv. Figure 3C shows 53 the conformational changes of L169H with increasing Au precursor concentrations which 54 were reflected in the overall cluster structure in the 4-fold channel as well as structural 55 transition. 56

(4) What is the possible application of these Au(I)-Apo-R168H/L169C-rHLFr composites? 58
The authors may like to present their UV-vis absorption and photoluminescent spectra, which 59 may provide hints for their potential applications. composites to Au(0) by addition of reducing agent and the changes can be measured by X-ray 76 crystal structure analysis. We did similar work using wild-type ferritin before (Nat. Commun. 77 2017, 8, 1-9). However, such an experiment is beyond the goal of this manuscript and might 78 be published separately.

Reviewer #2 90
The construction of this apo-ferritin cage and Au clusters in the cage are interesting, and the 91 manuscript is well written with solid data to support their conclusions. I believe this work 92 will be of interest to a broad scientific audience, especially for cluster material scientists. 93 Thus I would like to suggest the acceptance of this paper after the authors have addressed the 94 following minor issues. 95 Reply: Thanks to the reviewer for his/her positive comments. 96

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(1) In the X-ray crystal structures of these gold nanoclusters, Au atoms are anchored into the 98 designed cage via different Au-X interactions, including Au-N, Au-Cl, Au-S (and Au-C?).