F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.176780.1 Review Articles Understanding Protein–Protein Interactions in Biology: Applications of NMR Spectroscopy and Isothermal Titration Calorimetry

[version 1; peer review: 1 not approved]

 Ammous-Boukhris Nihel Conceptualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-5744-0328 1 Gargouri Ali Conceptualization Writing – Review & Editing 1 Mokdad-Gargouri Raja Conceptualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0003-1319-0061 a 1 Center of Biotechnology of Sfax, Laboratory of Eukaryotes Molecular Biotechnology, University of Sfax, Sfax, Tunisia raja.gargouri@cbs.rnrt.tn

No competing interests were disclosed.

 16 1 2026 2026 15 66 8 1 2026 Copyright: © 2026 Ammous-Boukhris N et al. 2026 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Protein–protein interactions play a central role in cellular signaling, immune recognition, and therapeutic development, yet they are often characterized by weak affinities, transient binding, and pronounced conformational flexibility. These features present significant challenges for conventional structural biology techniques. Biophysical approaches, particularly nuclear magnetic resonance (NMR) spectroscopy, and isothermal titration calorimetry (ITC) have emerged as powerful tools for elucidating peptide interactions under near-physiological conditions. NMR offers residue-level information on interaction interfaces, conformational changes, and protein dynamics in solution, making it uniquely suited for the analysis of weak and transient interactions. In contrast, ITC provides a direct and label-free measurement of binding thermodynamics, yielding quantitative parameters such as affinity, stoichiometry, and the enthalpic and entropic contributions to binding. This review highlights the principles, applications, and limitations of NMR and ITC in protein–protein interaction research, emphasizing how their combined use enables an integrated understanding of structure, dynamics, and energetics. Representative examples from the literature are discussed, including viral peptide–host protein interactions such as those involving Epstein–Barr virus latent membrane protein 1 (LMP1). Together, these studies illustrate the unique ability of NMR and ITC to capture structural and dynamic features of peptide recognition that are critical for understanding biological function and guiding peptide-based therapeutic design.

 Nuclear Magnetic Resonance Isothermal Titration Calorimetry Peptide Interaction Biophysical approaches Tunisian Ministry of Higher Education and Scientific Research Grant:LR19/CBS02 This research is funded by the Tunisian Ministry of Higher Education and Scientific Research (Grant: LR19/CBS02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

Protein–protein interactions (PPIs) are central to all biological processes, including signal transduction, enzymatic regulation, transcriptional control, and cellular architecture. 1 Rather than acting as isolated entities, proteins function within complex and dynamic interaction networks, where the specificity, strength, and regulation of PPIs determine biological outcomes. 2, 3 Understanding these interactions at the molecular level is therefore essential for elucidating biological mechanisms and for developing therapeutic strategies targeting dysregulated protein interactions.

Traditional approaches to studying PPIs, such as yeast two-hybrid assays 4 or coimmunoprecipitation, 5 are powerful for identifying interaction partners but provide limited quantitative or mechanistic information. In contrast, biophysical techniques offer direct insight into the molecular basis of PPIs by characterizing binding affinities, thermodynamic driving forces, structural interfaces, and conformational dynamics. 6, 7 Among these techniques, nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) have emerged as particularly valuable tools.

NMR spectroscopy enables the study of PPIs at atomic resolution in solution, allowing researchers to map interaction interfaces, detect conformational changes, and characterize weak or transient interactions that are often inaccessible to crystallographic methods. 8 Importantly, NMR can capture the dynamic nature of protein interactions, which is increasingly recognized as a key determinant of biological function. 8 Complementing this structural and dynamic information, ITC provides a direct and label-free measurement of the thermodynamic parameters governing protein binding, including binding affinity, enthalpy, entropy, and stoichiometry. 9, 10

This review focuses on the application of NMR spectroscopy and ITC to the study of proteinprotein interactions in biological research. By highlighting the unique and complementary insights provided by these techniques, we aim to illustrate how biophysical approaches contribute to a deeper mechanistic understanding of PPIs and their roles in complex biological systems.

 1. Nuclear magnetic resonance spectroscopy in the study of protein–protein interactions principles of NMR applied to PPI

NMR spectroscopy exploits the magnetic properties of atomic nuclei to obtain detailed information about the structure and environment of biomolecules in solution. In the context of PPIs, NMR is particularly powerful because it allows proteins to be studied under nearphysiological conditions without the need for crystallization or immobilization. Most biomolecular NMR experiments rely on isotopically labeled proteins, typically incorporating 15N and/or 13C, which enables the resolution of individual residues within a protein sequence. 9, 11 Unlike many structural techniques that provide static snapshots, NMR is inherently sensitive to molecular motions across a wide range of timescales. 12 This feature makes it especially suitable for investigating PPIs that are dynamic, weak, or transient-characteristics that are common in biological signaling and regulatory networks.

Mapping PPIs Interfaces

One of the most common applications of NMR in PPI research is the identification of interaction interfaces. 12 Chemical shift perturbation (CSP) experiments are widely used for this purpose. In these experiments, changes in NMR resonance frequencies are monitored as one protein is titrated with its binding partner. Residues located at or near the binding interface experience changes in their local chemical environment, resulting in measurable chemical shift changes. 12, 13 By mapping these perturbations onto the protein’s three-dimensional structure or sequence, researchers can identify interaction surfaces with residue-level resolution. This approach is particularly valuable for guiding mutagenesis experiments, validating interaction models, and distinguishing direct binding interfaces from allosteric effects. Importantly, CSP analysis can be applied even when only one of the interacting proteins is isotopically labeled, making it experimentally efficient. 12– 14

Characterization of Weak and Transient Interactions

Many biologically relevant PPIs are characterized by low affinities and short lifetimes, such as those involved in signaling cascades or regulatory processes. These interactions are often difficult to capture using crystallography or cryo-electron microscopy. NMR, however, is well suited to detect and characterize such weak interactions, as it can observe binding events across a broad range of dissociation constants. 15

Techniques such as line broadening analysis, relaxation measurements, and exchange spectroscopy allow NMR to probe binding kinetics and exchange processes between free and bound states. This capability is particularly important for studying encounter complexes and transient binding modes that may play critical roles in molecular recognition and specificity. 15

Insights into Conformational Dynamics and Allostery

Beyond identifying binding interfaces, NMR provides unique insights into how PPIs influence conformational dynamics. 16 Changes in backbone and side-chain motions upon complex formation can be monitored using relaxation experiments, revealing how binding affects protein flexibility and internal motions. These dynamic effects are often central to biological function. 17 For example, PPIs may induce conformational selection or allosteric regulation, where binding at one site influences the structure or activity at a distant site. NMR is uniquely capable of detecting such long-range effects, highlighting the role of dynamics as an integral component of protein interaction mechanisms rather than a secondary feature. 18

Advantages and Limitations of NMR in PPI Studies

NMR offers several key advantages for the study of PPIs, including residue-specific resolution, sensitivity to dynamics, and the ability to study proteins in solution under physiologically relevant conditions. It is particularly powerful for analyzing weak, transient, and disordered interactions that are difficult to access using other techniques.

However, NMR also has limitations. The size of protein complexes that can be studied is constrained by spectral complexity and sensitivity, although advances such as transverse relaxation-optimized spectroscopy (TROSY) and higher magnetic field strengths have significantly extended these limits. 19 In addition, NMR experiments often require isotopic labeling and substantial experimental expertise, which can limit throughput. 20

2. Isothermal Titration Calorimetry (ITC) in the study of PPIs

Principles of ITC Applied to Protein Interactions

Isothermal titration calorimetry (ITC) is a label-free biophysical technique that directly measures the heat exchanged during molecular binding events. 21 In ITC experiments, one protein is titrated into a solution containing its binding partner, and the resulting heat changes are recorded as a function of molar ratio. From a single experiment, ITC provides a complete thermodynamic profile of the interaction, including the binding affinity (Kd), enthalpy change (ΔH), entropy change (ΔS), and binding stoichiometry (n). 21

In the context of PPIs, ITC is particularly valuable because it measures binding energetics directly, without relying on fluorescent labels, surface immobilization, or indirect readouts. This directness makes ITC a gold-standard method for quantifying PPIs and for validating interactions identified by other biochemical or biophysical techniques. 22

Quantitative Characterization of Binding Affinity and Stoichiometry

A key strength of ITC in PPI research is its ability to accurately determine binding affinities over a broad range, from weak micromolar interactions to tight nanomolar complexes, provided that appropriate experimental conditions are used. 23 The determination of binding stoichiometry is especially important for protein complexes that may form higher-order assemblies or oligomeric states, as ITC can distinguish between different binding models. This quantitative information is critical for understanding biological function, as the strength and stoichiometry of protein interactions often dictate signaling thresholds, complex formation, and regulatory mechanisms within the cell. 24

Thermodynamic Insights into Protein-Protein Recognition

Beyond affinity measurements, ITC uniquely provides insight into the thermodynamic forces that drive PPIs. The relative contributions of enthalpy and entropy to binding can reveal the underlying molecular mechanisms of recognition. 25 Enthalpy-driven interactions are often associated with the formation of specific non-covalent contacts such as hydrogen bonds and electrostatic interactions, whereas entropy-driven interactions may reflect hydrophobic effects, solvent reorganization, or conformational changes. In PPI studies, comparing thermodynamic signatures across related protein complexes or mutant variants can identify residues or regions critical for binding. Such analyses are particularly valuable for dissecting allosteric effects and for understanding how mutations alter interaction energetics without necessarily disrupting binding interfaces. 26

Applications in Mutational and Comparative Studies

ITC is widely used in mutational analyses of protein-protein interfaces, where point mutations are introduced to assess their impact on binding energetics. 27, 28 By comparing thermodynamic parameters between wild-type and mutant proteins, researchers can distinguish between residues that contribute directly to binding affinity and those that influence interaction stability indirectly. Additionally, ITC is frequently employed in comparative studies of homologous proteins or interaction partners, enabling the identification of evolutionary or functional differences in binding mechanisms. 29 These applications highlight ITC’s role as a powerful tool for linking molecular energetics to biological function.

Advantages and Limitations of ITC in PPI Research

The principal advantage of ITC is its ability to provide a complete thermodynamic description of PPIs in a single experiment, using native proteins and solution conditions that closely mimic the cellular environment. This makes ITC an essential complement to structural techniques such as NMR and crystallography. However, ITC also has limitations. The technique typically requires relatively large amounts of highly purified protein, which can be challenging for unstable or low-yield systems. 30 In addition, ITC provides limited structural information and is less sensitive to very weak interactions without careful experimental optimization. 30 As a result, ITC is most powerful when used in combination with structural and spectroscopic methods.

 3. Applications of NMR and ITC: Representative examples from the literature 3.1 Peptide–Protein interaction: Epitope mapping

The pioneering work by Mayer and Meyer demonstrated the application of STD NMR to map ligand epitopes in protein–ligand complexes. In studies involving peptide ligands binding to large proteins, STD NMR identified specific peptide residues that exhibited the strongest saturation transfer, indicating close proximity to the protein surface. Hydrophobic and aromatic residues typically showed the highest STD intensities, revealing their key role in anchoring the peptide within the binding site. 31

3.2 Immune recognition: Peptide–MHC interactions

STD NMR has been applied to study peptide binding to major histocompatibility complex (MHC) molecules. In these studies, STD spectra revealed strong signals for peptide anchor residues occupying deep pockets within the MHC binding groove, while solvent-exposed residues showed weaker or no STD effects. These experiments provided direct experimental evidence for the differential contribution of peptide residues to MHC recognition under nearphysiological conditions.

 3.3 Peptide–Lectin interactions

In more recent work, STD NMR was used to characterize peptide interactions with lectins involved in immune recognition. STD epitope mapping revealed that specific amino acid side chains dominated the interaction, and competition experiments with known ligands confirmed binding site specificity. These studies highlighted the utility of STD NMR in analyzing multivalent and low-affinity peptide interactions. 32

3.4 Peptide–LMP1 interactions

In our recent work, we reported the identification and biophysical characterization of a novel peptide, termed B1.12, that specifically interacts with the extracellular loop of the Epstein–Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1). Unlike most previous studies that have focused on the cytoplasmic C-terminal activation regions (CTARs) of LMP1, our study targeted the extracellular domain, which remains comparatively underexplored despite its potential relevance for therapeutic intervention. Using a peptide selection strategy, B1.12 was identified as a specific binder of the LMP1 extracellular loop. Biophysical analyses demonstrated that the interaction is specific, weak-to-moderate in affinity, and dynamic, consistent with peptide–protein recognition events involving flexible binding surfaces. The interaction between the peptide B1.12 and the extracellular loop of the EBV oncoprotein LMP1 is intrinsically weak, dynamic, and involves a membrane-associated target, making it poorly accessible to conventional biochemical assays. NMR spectroscopy was therefore critical for detecting this interaction under solution conditions without immobilization or labeling of LMP1. Ligand-based NMR methods enabled sensitive detection of binding and provided residue-level epitope mapping of B1.12, identifying the peptide residues directly involved in recognition.

Isothermal titration calorimetry (ITC) complemented these findings by providing a direct and quantitative thermodynamic characterization of the interaction. ITC confirmed specific binding and yielded the affinity and stoichiometry of the B1.12-LMP1 complex, while also revealing the energetic balance between enthalpic and entropic contributions. Together, NMR and ITC provided a coherent mechanistic description of the interaction, combining sensitive detection, molecular insight, and quantitative validation, and established a robust framework for peptide optimization and therapeutic targeting of LMP1. 33

3.5 Peptide drug discovery and screening

Saturation Transfer Difference (STD) NMR has been widely used to identify binding epitopes of peptides interacting with large protein receptors. For example, STD NMR studies of peptide ligands binding to lectins and immune receptors revealed which peptide residues were in closest contact with the protein surface. These experiments provided rapid identification of pharmacophore regions without the need for isotopic labeling of the receptor, making STD NMR particularly valuable in peptide screening and optimization. 34 STD NMR has been also used in peptide-based drug discovery. For example, peptide inhibitors targeting enzyme active sites were screened using STD NMR to rapidly distinguish binders from non-binders. 34 Epitope maps derived from STD intensities guided rational peptide optimization by identifying residues critical for binding while eliminating non-essential regions.

 3.6 Peptide–Protein interaction: p53 transactivation domain–MDM2

One of the most cited examples of NMR in peptide interaction studies is the interaction between the intrinsically disordered p53 transactivation domain (TAD) and its regulator MDM2. Using 1H- 15N HSQC titration experiments, researchers demonstrated that p53 TAD undergoes folding upon binding, adopting an α-helical conformation when interacting with MDM2. Chemical shift perturbation analysis identified key hydrophobic residues involved in binding, while NOE data confirmed helix formation in the bound state. This work established NMR as a powerful tool for studying coupled folding and binding of disordered peptides. 34 NMR has been extensively applied to characterize low-affinity peptide–protein interactions, such as prolinerich peptides binding to SH3 domains. In these systems, CSP analysis combined with fastexchange behavior in HSQC spectra allowed mapping of the binding interface despite dissociation constants in the micromolar-millimolar range. Relaxation measurements further revealed dynamic exchange between multiple bound conformations, highlighting the dynamic nature of peptide recognition. 35

3.7 Peptide–Membrane interactions: Antimicrobial peptides

NMR has played a central role in elucidating the mechanism of action of antimicrobial peptides (AMPs). Solution NMR studies using micelles and bicelles showed that peptides such as magainin and LL-37 adopt amphipathic α-helical conformations upon membrane binding. Solid-state NMR further revealed peptide orientation and depth of insertion within lipid bilayers, clarifying how AMPs disrupt membrane integrity while maintaining selectivity for bacterial membranes. 36

3.8 Dynamics-Driven recognition: Calcineurin–NFAT peptide

The interaction between calcineurin and the NFAT regulatory peptide is a classical example where NMR revealed the importance of conformational dynamics. Relaxation dispersion experiments showed that the peptide samples bound-like conformations in the free state, supporting a conformational selection mechanism. This work demonstrated how NMR uniquely links peptide dynamics to biological function. 37

3.9 Recent hybrid NMR–Computational study

More recent studies have combined sparse NMR data with molecular dynamics simulations to characterize highly flexible peptide–protein complexes. For instance, NMR chemical shifts and PRE restraints were integrated with simulations to determine structural ensembles of signaling peptides bound to regulatory proteins. These approaches overcome limitations of traditional structure determination and represent a modern trend in peptide interaction studies. 38

4. Future perspectives

Advances in biophysical instrumentation and methodology continue to expand the applicability of NMR spectroscopy and ITC in PPI research. In the case of NMR, developments such as higher magnetic field strengths, improved probe technology, and enhanced pulse sequences are steadily increasing sensitivity and extending the size limits of protein complexes that can be studied. Emerging approaches, including in-cell NMR and solid-state NMR, further broaden the scope of PPI analysis by enabling the investigation of protein interactions in more native or heterogeneous biological environments. At the same time, computational integration is becoming increasingly important. The combination of NMR-derived structural and dynamic data with molecular dynamics simulations and integrative modeling approaches allows for more comprehensive descriptions of protein interaction landscapes. These hybrid methods are particularly promising for studying conformational ensembles and transient complexes that are difficult to capture using a single technique.

For ITC, ongoing improvements in instrument sensitivity and experimental design are reducing sample requirements and increasing throughput, making calorimetric measurements more accessible for challenging protein systems. Microcalorimetry and automated platforms are expected to further enhance the use of ITC in comparative and mutational studies of PPIs. In addition, the integration of ITC data with structural and spectroscopic methods is likely to become more systematic, enabling more detailed correlations between binding energetics and molecular mechanisms.

Looking forward, the increasing emphasis on systems-level and quantitative biology underscores the continued relevance of biophysical techniques. As PPIs are studied in increasingly complex networks, NMR and ITC will remain essential for grounding large-scale interaction data in detailed molecular and thermodynamic understanding.

 Conclusion

Protein–protein interactions are fundamental to biological function, governing processes ranging from signal transduction to gene regulation. A detailed understanding of these interactions requires not only the identification of interaction partners but also insight into the structural, dynamic, and energetic principles that underlie molecular recognition. Biophysical techniques play a central role in meeting this challenge. NMR spectroscopy provides unparalleled access to the structural and dynamic features of PPIs, enabling residue-specific mapping of interfaces and characterization of conformational changes and transient binding events. Complementing this, ITC offers a direct and quantitative assessment of binding energetics, revealing the thermodynamic forces that drive and regulate protein interactions. Together, these techniques form a powerful and complementary toolkit for the mechanistic analysis of PPIs. By integrating the strengths of NMR and ITC, researchers can construct cohesive models that link structure, dynamics, and energetics to biological function. As methodological advances continue to expand the capabilities of these tools, their combined application will remain essential for advancing our understanding of protein–protein interactions and their roles in complex biological systems.

 Data availability

No data are associated with this article.

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Competing interests: No competing interests were disclosed.

 18 3 2026 Copyright: © 2026 Zambelli B 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

In the manuscript of Ammous-Boukhris et al., the authors review the application of nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to the study of protein–protein interactions (PPI). The manuscript first introduces the biological relevance of PPI and then summarizes the basic principles of NMR and ITC as tools to characterize these systems. The review concludes with some examples from the literature and a short section discussing future perspectives.

The topic is clearly relevant for the structural biology and biophysics communities, and the manuscript follows a logical structure. However, in my opinion, the current version of the review remains quite general and introductory in scope. Much of the discussion stays at a general level, and the literature analysis does not capture recent developments in the field. In addition, the examples presented do not convincingly illustrate how NMR and ITC together provide mechanistic insight into PPIs. Therefore, the manuscript currently reads less like a structured review of the field and more like a series of general statements on the usefulness of NMR and ITC for studying protein–protein interactions, rather than a critical synthesis of the literature.

Here below, I report a point-by-point list of the issues that I found.

Major points:

1) The first important limitation of the manuscript is that much of the discussion remains at a general and descriptive level. Most sections essentially restate well-known properties of NMR spectroscopy and ITC without developing the methodological aspects in more detail. For example, the manuscript repeatedly notes that NMR provides information on binding interfaces and conformational dynamics, while ITC provides thermodynamic parameters such as binding affinity, enthalpy, entropy, and stoichiometry. These statements are correct, but they appear several times throughout the manuscript in very similar wording and, more importantly, are not explored further.

For a review article, it would be useful to go beyond these general descriptions and discuss more explicitly how the techniques are applied in practice, what their main limitations are, and in which contexts they are particularly informative. For instance, in the case of NMR the authors could briefly discuss how CSP data are interpreted in different exchange regimes, or how relaxation measurements can provide information on binding dynamics. Likewise, the section on ITC could address some of the experimental considerations involved in calorimetric measurements and the interpretation of thermodynamic signatures. Adding this type of discussion is needed to substantially increase the scientific depth of the review.

2) A second important issue concerns the references reported throughout the manuscript. Most (almost all) of the cited papers correspond to classic studies from the 1990s or early 2000s. These references might be important and could certainly be included, but the review would benefit from incorporating more recent literature reflecting methodological developments over the past decade. There have been significant advances in biomolecular NMR methods, such as those for studying larger macromolecular complexes or intrinsically disordered proteins, as well as the increasing use of integrative approaches combining NMR data with other biophysical methods such as ITC. Similarly, developments in calorimetric analysis and in the interpretation of binding energetics, such as global fitting approaches or interpretation of binding signatures, have expanded the scope of ITC in studies of biomolecular recognition. Including more recent examples and reviews would help position the manuscript within the current state of the art.

3) The manuscript contains a certain amount of repetition. In several sections the text reiterates the idea that NMR provides structural and dynamic information while ITC provides thermodynamic characterization. While this is a key concept and certainly worth emphasizing, it appears multiple times in similar formulations throughout the manuscript. As a result, some sections give the impression of repeating earlier points rather than advancing the discussion. The manuscript would likely benefit from consolidating some of these statements and focusing more on specific examples or methodological insights.

4) Section 3 is intended to present examples from the literature, but in practice the examples are described briefly, without any kind of in depth-analysis and with a short description of the biological system followed by a general statement that NMR was used to characterize the interaction. The reader is not given any detail about how the experiments were performed, what specific information was obtained, which experimental approach was used, or how the different techniques contributed to the overall interpretation of the system. This reviewer thinks that this section should be rethought and rewritten to be considerably deeper: the authors should focus on a smaller number of examples and discuss them in greater depth. For instance, three-four well-chosen case studies could be analyzed in more detail, explaining how NMR data and ITC measurements together helped clarify the mechanism of molecular recognition. It is important that the additive or synergic integration of these two techniques is at the center of the discussion, to better illustrate the central message of the review.

5) Although the manuscript declares the purpose to explore the complementary nature of NMR and ITC, almost all the examples discussed in Section 3 mainly focus only on NMR experiments. This creates imbalance in a review that is explicitly presented as focusing on the combined use of these two techniques. If the goal is to demonstrate how NMR and ITC together provide a more complete understanding of PPI, which is an interesting topic, the examples should illustrate how the structural or dynamic information obtained from NMR is complemented by thermodynamic measurements from ITC. At present this integration is not present.

6) While the authors explore different biological systems in Section 3, they fail to explain why these systems were chosen or what specific aspect of NMR/ITC methodology they are meant to illustrate. As it stands, the section reads more as a sequence of unconnected case studies than as a structured discussion of representative problems in PPI biophysics. Because of this, it is difficult for the reader to extract broader methodological lessons from the examples. It would therefore be more effective to reorganize this section around clearer themes—for instance interface mapping, dynamics-driven recognition, or the thermodynamic dissection of binding energetics — and then use a representative and well-chosen case study to illustrate each of these points in detail. This would make the overall message of the section much clearer.

7) The discussion of the authors’ work on peptide interactions with the Epstein–Barr virus LMP1 protein is interesting and relevant. However, this section is presented in greater detail than many of the other examples in the review, which are only briefly summarized. As a result, the emphasis on this system feels disproportionate in the context of a general review article. I suggest exploring all the examples in much greater details (including the one coming from the authors’ work, which, although explored deeper, still remains quite superficial for this type of review) and with the same level of details.

8) The acronym STD is used before being defined in the text. It is also important that the technique is briefly explained in its principles before presenting literature examples. The manuscript refers to STD intensities and epitope mapping, but the underlying concepts is not described. Since this technique is repeatedly mentioned in the examples, a short explanation of the experimental principles is important for readers who are not already familiar with it. Moreover, STD-NMR is a ligand-observed NMR technique that is typically applied to interactions between a protein and a small molecule. This point is not clearly explained in the current text, which may give the impression that STD-NMR is a general method for characterizing protein–protein interactions involving two folded and relatively large partners. The authors need to clarify the typical experimental context in which STD-NMR is applied and to explain that the technique refers to ligand-based interaction studies rather than for conventional protein–protein complexes.

Minor points

1) The review would benefit from the inclusion of one or two schematic figures illustrating the basic principles of the techniques discussed. For example, diagrams showing how chemical shift perturbation experiments are used to map binding interfaces in NMR, or a schematic representation of a typical ITC thermogram and binding isotherm, would make the manuscript more accessible to readers.

2) The reference list should be checked to ensure that all citations correspond accurately to the statements made in the text and that the bibliography reflects the current literature. For example, I could not find reference 34. Also, reference 32 dates 1996 and is defined “more recent work”, apparently being unrelated to the paragraph 3.3 in which it was cited (this reference describes the structure of a T cell receptor in complex with MHC and does not appear to be related to lectin–peptide interactions or to STD-NMR experiments).

3) At page 3 the authors state: NMR “allows proteins to be studied under near physiological conditions”. It is certainly true that NMR does not require crystallization or immobilization, which can introduce artefacts, but it requires protein concentrations that are outside of the physiological range. Thus, I would not state that NMR uses conditions that are almost physiological, rather I would stress the importance of working in solution compared to other techniques that work in the solid state.

4) In the same page, the authors state: NMR “enables resolution of individual residues”. It actually provides atomic-scale resolution.

5) Authors also mention that “NMR is sensitive to molecular motions across of a wide range of timescales”. This is an interest concept, and one of the real strengths of NMR in my opinion. Therefore, I advise the authors to explore this topic further, explaining how we can correlate NMR data on protein dynamics, also touching the theme of flexible regions/proteins and of intermediate exchange regimes.

6) The authors state that NMR “can observe binding events across a broad range of dissociation constants”. This is not fully true: due to the high concentration requirements for NMR experiments (in the hundreds of micromolar ranges), the dissociation constants that NMR can explore are actually quite high. That means that NMR can detect low affinity interactions, but for the same reason it is also prone to identify non-specific interactions, which are typically characterized by low affinity (in the millimolar range). This might be a drawback.

7) Paragraphs 3.3 and 3.5 report the same example of peptide-lectin interactions. Can they be unified?

8) Solid-state NMR is only briefly mentioned in the Future perspective section, while I think it should be explained in the review, at least when the authors report NMR limitations for higher MW, as solid-state NMR allows to overcome this limitation.

9) There are several minor formatting inconsistencies throughout the manuscript, such as missing spaces between words (for example “proteinprotein”, “nearphysiological”, or “Akey strength”). These appear to be formatting artifacts but should be corrected to improve readability.

Is the review written in accessible language?

Yes

Are all factual statements correct and adequately supported by citations?

No

Are the conclusions drawn appropriate in the context of the current research literature?

No

Is the topic of the review discussed comprehensively in the context of the current literature?

No

Reviewer Expertise:

Protein biophysics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.