Peer review of: Molecular design principles of Lysine-DOPA wet adhesion (2020)

Reviewers' Comments: 
Reviewer #1: 
Remarks to the Author: 

This manuscript, entitled “Topological Design Principles of Lysine-DOPA Wet Adhesion: Single-
Molecule Perspective”, reports an experimental study of the influence of chemical composition and 
structure on the adhesion properties of mussel-inspired peptides. Studied results show that as the 
number of repeating structural units increase within a specific range, with the peptide number 
from KY to (KY)3, the adhesion strength to TiO2 increases significantly, and with the further 
increase of peptide chain structural units, increase in peptide length to (KY)10, it does not show an 
additional adhesive increase. The effect of different adhesive moieties and molecular spacer 
incorporated between repeated units on the adhesion strength was also explored. The method 
provided in this work for exploring the molecular adhesion behavior is both convenient and 
practical. The selected system is interesting and some novel results got have a considerable 
practical significance. The present manuscript is suitable for publication in Nature Communications 
after a minor revision as pointed bellow. 
 
Comments 
For the case of (KY)10 peptide, the electrostatic repulsion between the repeating structural units 
(KY units) would likely affect the simultaneous interaction of each structural unit with the substrate 
surface, for which the author should give further discussion or explanation. 
Due to the presence of spacer groups (PEG spacer), the strength of the interaction is greatly 
affected. However, it is necessary to further explain why this group can have such a great 
influence, and make further explanation from the perspective of interaction force or steric 
hindrance effect. For example, the presence of spacer can reduce the electrostatic repulsion 
between repeating structural units (KY), thereby affecting the interaction behavior of these 
structural units and the substrate surface, etc.. 
 
 
Reviewer #2: 
Remarks to the Author: 
Recommendation: Minor corrections 
 
Comments: 
This manuscript by Messersmith and coworkers presents a single-molecule force spectroscopy 
(SMFS) study on the adhesion strength of mussel-inspired peptides to elucidate the effect of 
molecular topology over wet adhesion. Inspired by the sequence of the adhesive mussel foot 
protein 5 (mfp-5) that contains DOPA and Lys moieties, a library of peptides containing adhesive 
dipeptide units of Lys and either DOPA (KY) or Phe (KF) were synthesized with variable length, 
number of repeating units (1, 3 or 10), amino acids order in the dipeptide (KY vs YK) and presence 
of a non-adhesive and flexible oligoethylenglycol spacer between the repeating units. These 
peptides were covalently bound to an AFM cantilever and probed via SMFS under wet conditions 
(PBS) to two different surfaces: a hydrophilic charged inorganic surface (TiO2) and an organic 
hydrophobic substrate (polystyrene). The adhesion strength of the different peptide systems was 
measured and compared to reveal the effect of the molecular design over adhesion performance. 
Additionally, a 17-mer mimic of mfp-5 was synthesized as well and used for comparison. 
 
The authors found that: i) increasing the number of repeat units from 1 to 3 results in higher 
adhesion, while further increase to 10 does not noticeably enhance adhesion. ii) KY and KF 
derivatives showed similar adhesion to the hydrophobic polystyrene, while KY showed much higher 
adhesion onto TiO2. This was attributed to the molecular ability of Y to establish more and diverse 
interfacial adhesive interactions with surfaces of different kinds. This result points to a higher 
versatility of Y vs F as a functional component of adhesive formulations designed to perform over 
various substrates. iii) The incorporation of a flexible, non-adhesive spacer was found to reduce 
the overall peptide rigidity and allowed each KY unit to interact with the surface more efficiently. 
Although in this particular case the adhesion strength did not increment, the inclusion of a spacer 
provided a hidden length to the structure that could be useful to increase energy dissipation before 
rupture, which would ultimately enhance the adhesive performance of a formulation. 
 
The novelty of this study is somehow limited. However, the findings of this work are highly 
relevant to guide the rational molecular design of mussel inspired underwater adhesives and are 
interesting to the community of adhesive and bioinspired materials. Therefore, I rather support its 
publication. The article is well presented, is easy to follow and offers an adequate discussion in 
relation to the current literature. 
 
I recommend the following minor corrections: 
 
1. Page 1, abstract, line 2: “DOPA” should be defined at first mention. 
2. Page 2, line 23: “SFMS” should be defined at first mention. 
3. Page 4, lines 7-8: When mentioning the library of peptides synthesized, please, include a 
sentence to refer to Table 1. 
4. Page 4, lines 20-23: in the part related to the synthesis of the peptide library, what is the yield 
of such syntheses? No data is currently included about this point, neither in the main manuscript 
nor in the SI. Please, comment on that. 
5. Pages 4-5: when describing a typical force spectroscopy experiment, could you please mention 
what is the occurrence rate (in %) of observation of the rupture events? I would expect this to be 
quite low. 
6. Page 10, lines 10-12: “We used monodisperse PEG oligomers (P8) to mimic the ‘spacer’ 
residues”. To my point of view, describing the spacer as “PEG oligomers” is not accurate. It is 
recommended that this term is replaced by just “oligoethylene glycol spacer”. 
7. Page 10, lines 16-17: in relation to the statement “The energy dissipated in this process can 
further confer toughness and superior performance to the adhesive”, I recommend to add 
references to back this up. 
8. Page 19, caption of Table 1: “Calculated and measured molecular weight values (obtained from 
MALDI) are shown”. This is not accurate. Mass spec techniques provide information on mass-to-
charge ratio, where the “mass” refers to “exact mass” and not to molecular weight. Therefore, the 
correct terms should be “calculated exact mass” and “measured or found mass”. The same 
correction should be introduced to the caption of Figure S1 at the SI. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Review of "Topological Design Principles of Lysine-DOPA Wet Adhesion: Single-Molecule 
Perspective" 
 
This is an outstanding communication that reports single molecule force spectroscopy on a series 
of DOPA-Lysine peptides with comparison to Phe-Lys peptides. The paper demonstrates the 
synergistic effect of DOPA and Lys in wet adhesion to TiO2 and shows that increasing the length of 
the peptide increases adhesion strength, up to a point, but more intriguing is incorporating a 
region of nonadhesive spacing between Lys-DOPA units also increases adhesion. These are new, 
important and exciting results, that increase our understanding of mussel foot protein adhesion in 
significant ways. This communication differs substantially from a previous report in Science on the 
synergistic relationship between catechol and primary amines in aqueous adhesion, in that the 
current results use peptides with DOPA as opposed to a different catechol on a non peptidic base, 
and this communication uses SMFS to investigate adhesion to TiO2 versus the Science report using 
SFA on mica. The authors suggest that their work provides a solid foundation guiding the 
deliberate design of bioinspired wet adhesives, however they fail to bring out in the main text 
specifically the fact that their experiments are all carried out under anaerobic conditions to prevent 
oxidation of DOPA at neutral pH under their PBS conditions – This conclusion, as written, seems 
somewhat overstated – since wet adhesive materials would not likely be especially useful in only 
anaerobic environments. However, overall, I find this is well written communication, and I 
recommend that it be accepted with only very minor revisions. 
 
Since the experiments are carried out at neutral pH in PBS and since the manuscript indicates 
catechol interaction with TiO2 is via coordination to Ti(IV), what happens to the SMFS of these 
compounds at lower pH? Also one wonders how the adhesive properties of Lys-Tyr compare to 
Lys-DOPA and Lys-Phe – but these are not required to be addressed in this communication 
 
Minor points: 
In the Abstract, writing “(DOPA, Y)” is misleading since Y is for Tyrosine – Maybe HO(superscript)Y 
would be better to indicate hydroxylated Tyr – and elsewhere in the text. 
 
Page 7, define PS 
P 8 line 13 the word ‘the’ is missing: “almost the same” 
Conclusion: reword on of the two successive sentences beginning with “Moreover,”