This is a highly knowledgeable report on an exhaustive study of the effects of fluorine atoms substituted in methyl groups of valine residues in a protein. The authors have carefully investigated the effects of the bulky fluorine atom on the local conformations and overall protein structure.  Heat denaturation monitored by circular dichroism indicates melting temperatures about 10 degrees lower than for the wild-type protein. There seems to be a slight bias for the fluorine atoms to sit near positively polarized carbons of carbonyl groups. The inverse correlation of 3JHF couplings with 19F chemical shifts is a manifestation of the gamma-gauche effect. “The broadest signals were observed for the most deeply buried residues, indicating that the peak heights are sensitive indicators of the side-chain mobilities”: why should deeply buried amino acids necessarily be rigid?

The expression “installed” seems a bit unfortunate. How about “incorporated”?

Why not write out “gamma-gauche effect” without using a Greek letter, for the convenience of data bases?

One of the curious observations is the existence of through-space scalar 19F-19F couplings due to transientfluorine-fluorine contacts between two remote residues, despite the polarity of the C-F bonds that may discourage direct 19F-19F contacts.

From the point of view of an ‘aficionado’ of long-lived states and coherences involving two or more 19F nuclei, it would be interesting to know if a similar study could be conducted with di-fluorinated methyl groups. Are di-fluorinated amino acids also commercially available?

Rarely has a naive question triggered such a rich reply !

Rarely has a naive question triggered such a rich reply !

This reviewer concurs with the views of the first reviewer that this is a thorough study demonstrating an angular dependence of the gamma-gauche effect of 19F on 13C chemical shifts. The ability to observe CH2F rotameric populations without 19F NMR via the chemical shift of the opposite methyl group is intriguing.

The observation of unique 1H chemical shifts for the CH2F groups as shown in Figure S3 is interesting. The authors have quite obviously spent a significant amount of time considering the structural environments of the four valine groups in this study. Do they care to speculate further about why some show unique 1H chemical shifts (particularly GB1-2 valine 54), while others do not?

The authors note qualitative analysis of the 3J(HA-HB) couplings via analysis of COSY cross peak intensities. Is there any evidence that the intensity of those cross peaks varies between the WT, GB-1, GB-2, and GB1-d samples, which would suggest that fluorination is affecting the chi1 populations?

Minor comments:

- On line 314, there appears to be an incomplete sentence at the beginning of the paragraph starting with a lowercase “using”.

- Perhaps change “little destabilized” to “slightly destabilized” (as an adverb seems more appropriate here than an adjective) on line 538.

- The melting temperatures were described to be about 10 °C lower than WT. However, the data for WT was not shown in Figure S1, nor given in the cited reference (Tan et al., 2024). Could the WT melting temperature be given and the source of that information be clarified? Are the reported melting temperatures from reversible or irreversible unfolding?

The manuscript by Abdelkader et al. presents a detailed and insightful investigation into how fluorination of valine residues in the GB1 protein can be used to study side-chain conformations. The work is interesting, carefully executed, and suitable for publication in Magnetic Resonance after minor revision. I have several questions and suggestions that may help to clarify and strengthen the manuscript.

1. Additional species in GB1-d (Fig. 2c):
   
   Could you expand the discussion regarding the presence of additional species observed in Fig. 2c? For instance, why are these species not present in GB1-1 and GB1-2? You write, “Therefore, the low-intensity peaks in the 19F-NMR spectrum of GB1-d most likely originate from the species with one valine and three diFVal residues.” Does this imply that the absence of one diFVal residue causes a significant change in the chemical shifts of the other 19F-labeled valines? Additionally, could you specify which residue is missing, since the small number of low-intensity peaks suggests that not all possible combinations are present in solution?
2. Differences in 19F chemical shifts between variants:
   
   Could you briefly discuss potential reasons why the 19F chemical shifts differ between GB1-1 and GB1-d, and between GB1-2 and GB1-d? This may become clearer later in the manuscript when you discuss how rotamers are affected by fluorine substitution, but at this earlier stage the distinction is not immediately obvious to the reader.
3. Formatting issue (page 17, line 314):
   
   There appears to be a paragraph break and an unfinished sentence at this location. Please check and correct this formatting issue.
4. Fig. 12 and DFT calculations:
   
   If I understand correctly, the DFT calculations do not indicate a strictly linear dependence of the FC coupling on the chemical shift, but rather a more complex relationship. Could you comment on this in the text, clarifying that the linear fit is used primarily to illustrate the presence of a correlation?
   
   In addition, could you discuss the relevance of the DFT calculations performed on (2R)-1-fluoro-2-methylpropane(3-13C) to the conformations of valine residues in GB1? A brief justification of this model system would be helpful.
5. Fig. 13:
   
   It might be useful to include the structures of the valine residues identified in GB1-d alongside those from GB1-1 and GB1-2, to facilitate direct comparison.
6. Clarification of 3JFC coupling statement:
   
   The sentence “Assigning preferential rotamers in GB1-d is more difficult, as the diFVal residues contain no CH3 group, which makes 3JFC coupling measurements difficult” is not fully clear. Could you elaborate on how the absence of a CH3 group specifically complicates the 3JFC coupling measurements?
7. Potential applications to protein–ligand interactions:
   
   It would be valuable to expand the discussion on how these fluorinated labels might be applied to studying protein–ligand interactions. Would such interactions be detectable as perturbations in the 19F chemical shifts? Do you expect these effects to be site-specific and sensitive to local changes, or rather global, given the apparent sensitivity of the 19F shifts to the overall protein structure?
8. Estimating energy differences between rotamers:
   
   Based on your experimental data and the DFT calculations of 3JFC couplings, would it be possible to estimate the energy differences between the various rotamers?