Aromatic-aromatic Interactions Research Articles

Developing Isomeric Peptides for Mimicking the Sequence-Activity Landscapes of Enzyme Evolution.

Enzymes catalyze almost all material conversion processes within living organisms, yet their natural evolution remains unobserved. Short peptides, derived from proteins and featuring active sites, have emerged as promising building blocks for constructing bioactive supramolecular materials that mimic native proteins through self-assembly. Herein, we employ histidine-containing isomeric tetrapeptides KHFF, HKFF, KFHF, HFKF, FKHF, and FHKF to craft supramolecular self-assemblies, aiming to explore the sequence-activity landscapes of enzyme evolution. Our investigations reveal the profound impact of peptide sequence variations on both assembly behavior and catalytic activity as hydrolytic simulation enzymes. During self-assembly, a delicate balance of multiple intermolecular interactions, particularly hydrogen bonding and aromatic-aromatic interactions, influences nanostructure formation, yielding various morphologies (e.g., nanofibers, nanospheres, and nanodiscs). Furthermore, the analysis of the structure-activity relationship demonstrates a strong correlation between the distribution of the His active site on the nanostructures and the formation of the catalytic microenvironment. This investigation of the sequence-structure-activity paradigm reflects how natural enzymes enhance catalytic activity by adjusting the primary structure during evolution, promoting fundamental research related to enzyme evolutionary processes.

Thr-to-Ala Mutation Leads to a Larger Aromatic Pair and Reduced Packing Density in α1,α3-Helices during Thioredoxin Cold Adaptation.

This study investigates the impact of aromatic-aromatic interactions on the cold adaptation of thioredoxin (Trx), a small redox protein with a conserved Trx-fold structure. Two Trx orthologs, one from the psychrophilic Arctic bacterium Sphingomonas sp. (SpTrx) and the other from the mesophilic Escherichia coli (EcTrx), display distinct aromatic interactions in their α1,α3-helices. SpTrx features a larger Trp11-Phe69 pair, while EcTrx employs a smaller Phe12-Tyr70 pair along with an additional Asp9-Thr66 hydrogen bond. Smaller aromatic residues in SpTrx (Phe-Phe or Phe-Tyr pair) lead to decreased thermal and thermodynamic stabilities, increased conformational flexibility, and reduced enzyme activity. In contrast, EcTrx's thermal stability is primarily influenced by the larger Trp residue, especially in the more hydrophobic Trp-Phe pair compared to the Trp-Tyr pair. Both SpTrx and EcTrx exhibit a strengthening of the Asp-Thr hydrogen bond by a Phe-Tyr pair and a weakening by a Trp-Phe pair. Additionally, the Asp8-Thr65 hydrogen bond in SpTrx contributes to the destabilization of the Phe-Phe pair. Molecular dynamics simulations of SpTrx indicate that a smaller aromatic pair or the Asp-Thr hydrogen bond in the α1,α3-helices further destabilizes the α2-helix across the central β-sheet. Our results suggest that the Thr-to-Ala mutation destabilizes the α1,α3-helices, resulting in a larger aromatic pair and reduced packing density in psychrophilic Trxs during cold adaptation. These findings enhance our understanding of Trx's adaptation to colder temperatures.

Molecular Characterization of Alpha-Amylase of Aspergillus flavus and Aspergillus oryzae: An In Silico Study

Alpha amylases (EC 3.2.1.1.) hydrolyze polysaccharides like starch and glycogen to create oligosaccharides of different sizes by random cleavage of internal 1, 4-glucosidic linkages. These enzymes are produced by many different bacteria and fungi. In this investigation, α-amylase from Aspergillus flavus and Aspergillus oryzae was taken into consideration. Amino acid sequences and protein structure were retrieved from databases. Sequences of Aspergillus oryzae possess a high number of charged residues whereas Aspergillus flavus have a higher abundance of uncharged polar and hydrophobic amino acid residues. 3 common superfamilies were observed between both species. The alignment of sequences showed their similar conservative nature. Non-covalent intra-protein interactions like the salt bridge, aromatic-aromatic interactions, aromatic-sulfur interactions and cation-pi interactions helped to increase the stability of α-amylase of Aspergillus oryzae. The tunnels and cavities also help to increase the functionality and catalytic activity of α-amylase. This study will also be helpful for protein engineering.

Tuning Phase Transition of Molecular Self-Assembly by Artificial Chaperones through Aromatic-Aromatic Interactions.

The molecular chaperones are essential and play significant roles in controlling the protein phase transition and maintaining physiological homeostasis. However, manipulating phase transformation in biomimetic peptide self-assembly is still challenging. This work shows that an artificial chaperone modulates the energy landscape of supramolecular polymerization, thus controlling the phase transition of amyloid-like assemblies from crystals to hydrogels to solution. The absence of a chaperone allows the NapP to form crystals, while the presence of the chaperone biases the pathway to form nanofibrous hydrogels to soluble oligomers by adjusting the chaperone ratios. Mechanistic studies reveal that the aromatic-aromatic interaction is the key to trapping the molecules in a higher energy fold. Adding the chaperone relieves this restriction, lowers the energy barrier, and transforms the crystal into a hydrogel. This phase transformation can also be achieved in the macromolecular crowding environment, thus providing new insights into understanding molecular self-assembly in multiple component systems.

MPA-Pred: A machine learning approach for predicting the binding affinity of membrane protein-protein complexes.

Membrane protein-protein interactions are essential for several functions including cell signaling, ion transport, and enzymatic activity. These interactions are mainly dictated by their binding affinities. Although several methods are available for predicting the binding affinity of protein-protein complexes, there exists no specific method for membrane protein-protein complexes. In this work, we collected the experimental binding affinity data for a set of 114 membrane protein-protein complexes and derived several structure and sequence-based features. Our analysis on the relationship between binding affinity and the features revealed that the important factors mainly depend on the type of membrane protein and the functional class of the protein. Specifically, aromatic and charged residues at the interface, and aromatic-aromatic and electrostatic interactions are found to be important to understand the binding affinity. Further, we developed a method, MPA-Pred, for predicting the binding affinity of membrane protein-protein complexes using a machine learning approach. It showed an average correlation and mean absolute error of 0.83 and 0.91 kcal/mol, respectively, using the jack-knife test on a set of 114 complexes. We have also developed a web server and it is available at https://web.iitm.ac.in/bioinfo2/MPA-Pred/. This method can be used for predicting the affinity of membrane protein-protein complexes at a large scale and aid to improve drug design strategies.

Analyzing the aromatic-aromatic interactions in proteins: A2ID 2.0

The Aromatic-Aromatic Interactions Database (A2ID) is a comprehensive repository dedicated to documenting aromatic-aromatic (π-π) networks observed in experimentally determined protein structures. The first version of A2ID was reported in 2011 [Int J Biol Macromol, 2011, 48, 540]. It has undergone a series of significant updates, leading to its current version, which focuses on the identification and analysis of 3,444,619 π-π networks from proteins. The geometrical parameters such as centroid-centroid distances (r) and interplanar angles (ϕ) were used to identify and characterize π-π networks. It was observed that among the 84,500 proteins with at least one aromatic π-π network, about 92.50 % of the instances are found to be either 2π (77.34 %) or 3π (15.23 %) networks. The analysis of interacting amino acid pairs in 2π networks indicated a dominance of PHE residues followed by TYR. The updated version of A2ID incorporates analysis of π-π networks based on SCOP2 and ECOD classifiers, in addition to the existing SCOP, CATH, and EC classifications. This expanded scope allows researchers to explore the characteristics and functional implications of π-π networks in protein structures from multiple perspectives. The current version of A2ID along with its extensive dataset and detailed geometric information is publicly accessible using https://acds.neist.res.in/a2idv2.

Understanding the Stabilization Mechanism of a Thermostable Mutant of Hygromycin B Phosphotransferase by Protein Sector-Guided Dynamic Analysis.

Point mutations can exert beneficial effects on proteins, including stabilization. The stabilizing effects of mutations are typically attributed to changes in free energy and residue interactions. However, these explanations lack detail and physical insights, which hinder the mechanistic study of protein stabilization and prevent accurate computational prediction of stabilizing mutations. Here, we investigate the physical mechanism underlying the enhanced thermostability of a Hygromycin B phosphotransferase mutant, Hph5. We find that the unpredictable mutation A118V induces rotation of F199, allowing it to establish an aromatic-aromatic interaction with W235. In contrast, the predictable mutation T246A acts through static hydrophobic interactions within the protein core. These discoveries were accelerated by a residue-coevolution-based theory, which links mutational effects to stability-associated local structures, providing valuable guidance for mechanistic exploration. The established workflow will benefit the development of accurate stability prediction programs and can be used to mine a protein stability database for undiscovered physical mechanisms.

Reversible Light-Induced Dimerization of Secondary Face Azobenzene-Functionalized β-Cyclodextrin Derivatives.

β-cyclodextrin (βCyD) derivatives equipped with aromatic appendages at the secondary face exhibit tailorable self-assembling capabilities. The aromatic modules can participate in inclusion phenomena and/or aromatic-aromatic interactions. Supramolecular species can thus form that, at their turn, can engage in further co-assembling with third components in a highly regulated manner; the design of nonviral gene delivery systems is an illustrative example. Endowing such systems with stimuli responsiveness while keeping diastereomeric purity and a low synthetic effort is a highly wanted advancement. Here, we show that an azobenzene moiety can be "clicked" to a single secondary O-2 position of βCyD affording 1,2,3-triazole-linked βCyD-azobenzene derivatives that undergo reversible light-controlled self-organization into dimers where the monomer components face their secondary rims. Their photoswitching and supramolecular properties have been thoroughly characterized by UV-vis absorption, induced circular dichroism, nuclear magnetic resonance, and computational techniques. As model processes, the formation of inclusion complexes between a water-soluble triazolylazobenzene derivative and βCyD as well as the assembly of native βCyD/βCyD-azobenzene derivative heterodimers have been investigated in parallel. The stability of the host-guest supramolecules has been challenged against the competitor guest adamantylamine and the decrease of the medium polarity using methanol-water mixtures. The collective data support that the E-configured βCyD-azobenzene derivatives, in aqueous solution, form dimers stabilized by the interplay of aromatic-aromatic and aromatic-βCyD cavity interactions after partial reciprocal inclusion. Photoswitching to the Z-isomer disrupts the dimers into monomeric species, offering opportunity for the spatiotemporal control of the organizational status by light.

Aromatic amino acids in the finger domain of the FMRFamide-gated Na[Formula: see text] channel are involved in the FMRFamide recognition and the activation.

FMRFamide-gated Na[Formula: see text] channel (FaNaC) is a member of the DEG/ENaC family and activated by a neuropeptide, FMRFamide. Structural information about the FMRFamide-dependent gating is, however, still elusive. Because two phenylalanines of FMRFamide are essential for the activation of FaNaC, we hypothesized that aromatic-aromatic interaction between FaNaC and FMRFamide is critical for FMRFamide recognition and/or the activation gating. Here, we focused on eight conserved aromatic residues in the finger domain of FaNaCs and tested our hypothesis by mutagenic analysis and in silico docking simulations. The mutation of conserved aromatic residues in the finger domain reduced the FMRFamide potency, suggesting that the conserved aromatic residues are involved in the FMRFamide-dependent activation. The kinetics of the FMRFamide-gated currents were also modified substantially in some mutants. Some results of docking simulations were consistent with a hypothesis that the aromatic-aromatic interaction between the aromatic residues in FaNaC and FMRFamide is involved in the FMRFamide recognition. Collectively, our results suggest that the conserved aromatic residues in the finger domain of FaNaC are important determinants of the ligand recognition and/or the activation gating in FaNaC.

Macrocyclic β-Sheets Stabilized by Hydrogen Bond Surrogates.

Mimics of protein secondary and tertiary structure offer rationally-designed inhibitors of biomolecular interactions. β-Sheet mimics have a storied history in bioorganic chemistry and are typically designed with synthetic or natural turn segments. We hypothesized that replacement of terminal inter-β-strand hydrogen bonds with hydrogen bond surrogates (HBS) may lead to conformationally-defined macrocyclic β-sheets without the requirement for natural or synthetic β-turns, thereby providing a minimal mimic of a protein β-sheet. To access turn-less antiparallel β-sheet mimics, we developed a facile solid phase synthesis protocol. We surveyed a dataset of protein β-sheets for naturally observed interstrand side chain interactions. This bioinformatics survey highlighted an over-abundance of aromatic-aromatic, cation-π and ionic interactions in β-sheets. In correspondence with natural β-sheets, we find that minimal HBS mimics show robust β-sheet formation when specific amino acid residue pairings are incorporated. In isolated β-sheets, aromatic interactions endow superior conformational stability over ionic or cation-π interactions. Circular dichroism and NMR spectroscopies, along with high-resolution X-ray crystallography, support our design principles.

Tim4, a macrophage receptor for apoptotic cells, binds polystyrene microplastics via aromatic-aromatic interactions

Understanding the interface between microplastics and biological systems will provide new insights into the impacts of microplastics on living organisms. When microplastics enter the body, they are engulfed preferentially by phagocytes such as macrophages. However, it is not fully understood how phagocytes recognize microplastics and how microplastics impact phagocyte functions. In this study, we demonstrate that T cell immunoglobulin mucin 4 (Tim4), a macrophage receptor for phosphatidylserine (PtdSer) on apoptotic cells, binds polystyrene (PS) microparticles as well as multi-walled carbon nanotubes (MWCNTs) through the extracellular aromatic cluster, revealing a novel interface between microplastics and biological systems via aromatic-aromatic interactions. Genetic deletion of Tim4 demonstrated that Tim4 is involved in macrophage engulfment of PS microplastics as well as of MWCNTs. While Tim4-mediated engulfment of MWCNTs causes NLRP3-dependent IL-1β secretion, that of PS microparticles does not. PS microparticles neither induce TNF-α, reactive oxygen species, nor nitric oxide production. These data indicate that PS microparticles are not inflammatory. The PtdSer-binding site of Tim4 contains an aromatic cluster that binds PS, and Tim4-mediated macrophage engulfment of apoptotic cells, a process called efferocytosis, was competitively blocked by PS microparticles. These data suggest that PS microplastics do not directly cause acute inflammation but perturb efferocytosis, raising concerns that chronic exposure to large amounts of PS microplastics may cause chronic inflammation leading to autoimmune diseases.

Novel reusable catalytic poly(4-styrenesulfonate-co-glycidylmethacrylate) foams for adsorption and photodegradation of the model pollutant dye methylene blue based on aromatic-aromatic interactions

With the aim of achieving new highly porous, water-insoluble solid adsorbents for pollutants in water whose adsorption mechanism is based on aromatic-aromatic interactions, solid materials bearing charged aromatic groups have been developed from a family of copolymers of sodium styrene sulfonate and glycidyl methacrylate synthesized by free radical copolymerization both in DMF and in DMF/H2O, with comonomer molar ratio ranging between 0.2 and 0.8. The copolymers have been rationally designed to furnish hydrophilic functional negatively charged aromatic rings to allow aromatic-aromatic interaction with cationic aromatic pollutants, and hydrophobic functional monomers able to trigger crosslinking reactions through ring opening of epoxy groups by thermal treatment. All these copolymers form hydrogels in water. However, foams with closed cell structure were formed with the copolymer synthesized in DMF at a 0.2/0.8 NaSS/GMA molar ratio in the feed upon thermal treatment for 5–15 min at 270 °C, that resulted in non-soluble, elastic, hard, and fragile solid materials. Volume expansion stood around 30-fold the original volume, and porosity of the resulting expanded objects reached values higher than 73 %. Due to the large surface area of these foams and the presence of the benzene sulfonate moieties, the materials showed maximum adsorption capacity of around 200 mg of the model dye methylene blue per g of material, with a high adsorption rate. Interestingly, the absorbent foams showed catalytic activity towards photodegradation of the dye and allowed reusing the materials for at least six cycles.

Energy landscapes and heat capacity signatures for peptides correlate with phase separation propensity.

Phase separation plays an important role in the formation of membraneless compartments within the cell and intrinsically disordered proteins with low-complexity sequences can drive this compartmentalisation. Various intermolecular forces, such as aromatic-aromatic and cation-aromatic interactions, promote phase separation. However, little is known about how the ability of proteins to phase separate under physiological conditions is encoded in their energy landscapes and this is the focus of the present investigation. Our results provide a first glimpse into how the energy landscapes of minimal peptides that contain - and cation- interactions differ from the peptides that lack amino acids with such interactions. The peaks in the heat capacity () as a function of temperature report on alternative low-lying conformations that differ significantly in terms of their enthalpic and entropic contributions. The analysis and subsequent quantification of frustration of the energy landscape suggest that the interactions that promote phase separation lead to features (peaks or inflection points) at low temperatures in . More features may occur for peptides containing residues with better phase separation propensity and the energy landscape is more frustrated for such peptides. Overall, this work links the features in the underlying single-molecule potential energy landscapes to their collective phase separation behaviour and identifies quantities ( and frustration metric) that can be utilised in soft material design.

Design of nanobody-based bispecific constructs by in silico affinity maturation and umbrella sampling simulations

Random mutagenesis is the natural opportunity for proteins to evolve and biotechnologically it has been exploited to create diversity and identify variants with improved characteristics in the mutant pools. Rational mutagenesis based on biophysical assumptions and supported by computational power has been proposed as a faster and more predictable strategy to reach the same aim. In this work we confirm that substantial improvements in terms of both affinity and stability of nanobodies can be obtained by using combinations of algorithms, even for binders with already high affinity and elevated thermal stability. Furthermore, in silico approaches allowed the development of an optimized bispecific construct able to bind simultaneously the two clinically relevant antigens TNF-α and IL-23 and, by means of its enhanced avidity, to inhibit effectively the apoptosis of TNF-α-sensitive L929 cells. The results revealed that salt bridges, hydrogen bonds, aromatic-aromatic and cation-pi interactions had a critical role in increasing affinity. We provided a platform for the construction of high-affinity bispecific constructs based on nanobodies that can have relevant applications for the control of all those biological mechanisms in which more than a single antigen must be targeted to increase the treatment effectiveness and avoid resistance mechanisms.

PIF - A Java library for finding atomic interactions and extracting geometric features supporting the analysis of protein structures.

Proteins play an essential role in the functioning of living organisms. The enormity of the atomic interactions in proteins is essential in controlling their spatial structures and dynamics. It can also provide scientists with valuable information that help to determine the native structures of proteins. This paper presents the PIF (Protein Interaction Finder) library for the Java language, enabling the identification of selected atomic interactions (hydrogen and disulfide bonds, ionic, hydrophobic, aromatic-aromatic, sulfur-aromatic, and amino-aromatic interactions) based on the three-dimensional structure of proteins. The interaction calculation rules applied in PIF rely on documented theoretical foundations gathered from experimental studies of interactions in native protein structures. The library has a universal purpose, supporting drug discovery and development processes and protein structure modeling. Finding the atomic interactions can also deliver numerical features for various Artificial Intelligence (AI) models built for protein analysis. The conducted research comparing the results obtained with the use of the PIF library and competing tools has shown that our solution can effectively determine the interactions occurring in protein structures for entire collections of proteins. Moreover, as a solution that provides a programming interface, the PIF library can be used in any Java project, making it a universal tool.

Significance of D- tryptophan in Contryphan-Ar1131 Conus peptide: Oxidative folding, trypsin binding, and photostabilization activity

Distinct differences have been observed between L-tryptophan and D-tryptophan containing contryphan-Ar1131 in oxidative folding, trypsin binding, and photostabilization activity on avobenzone. [W5] contryphan-Ar1131 and [w5] contryphan-Ar1131 were chemically synthesized and characterized using RP-HPLC and mass spectrometry. Structural differences due to the change of configuration of tryptophan were evident from the optimized structures of contryphan-Ar1131 using density functional theory (DFT). The comparison of early events of oxidative folding has revealed the role of D-tryptophan in accelerating the formation of a disulfide bond. The optimized structures of the reduced form of peptides revealed the occurrence of aromatic-aromatic and aromatic-proline interactions in [w5] contryphan-Ar1131 which may be critical in aiding the oxidative folding reaction. The presence of the Lys6-Pro7 peptide bond indicates that contryphan-Ar1131 is resistant but may bind to trypsin allowing to assign the binding affinity of peptides to the protein surface. Competitive binding studies and molecular docking along with molecular dynamic (MD) simulations have revealed that [w5] contryphan-Ar1131 has more affinity for the active site of trypsin. Given tryptophan is a photostabilizer of FDA-approved chemical UV-A filter avobenzone, the report has compared the photostabilization activity of [W5]/ [w5] contryphan-Ar1131 on avobenzone under natural sunlight. [w5] contryphan-Ar1131 has better photostabilization activity than that of [W5] contryphan-Ar1131 and also individual D-tryptophan and L-tryptophan amino acids. These biochemical studies have highlighted the significance of D-tryptophan in contryphan-Ar1131 and its photostabilization activity on avobenzone may find applications in cosmetics.

Thermostability engineering of industrial enzymes through structure modification.

Thermostability is an essential requirement of enzymes in the industrial processes to catalyze the reactions at high temperatures; thus, enzyme engineering through directed evolution, semi-rational design and rational design are commonly employed to construct desired thermostable mutants. Several strategies are implemented to fulfill enzymes' thermostability demand including decreasing the entropy of the unfolded state through substitutions Gly → Xxx or Xxx → Pro, hydrogen bond, salt bridge, introducing two different simultaneous interactions through single mutant, hydrophobic interaction, filling the hydrophobic cavity core, decreasing surface hydrophobicity, truncating loop, aromatic-aromatic interaction and introducing positively charged residues to enzyme surface. In the current review, horizons about compatibility between secondary structures and substitutions at preferable structural positions to generate the most desirable thermostability in industrial enzymes are broadened. KEY POINTS: • Protein engineering is a powerful tool for generating thermostable industrial enzymes. • Directed evolution and rational design are practical approaches in enzyme engineering. • Substitutions in preferable structural positions can increase thermostability.

Adsorptive Structure and Mobility on Carbon Nanotube Exteriors Using Benzoic Acid as a Molecular Probe of Amphiphilic Water Contaminants.

Benzoic acid is the simplest aromatic carboxylic acid that is also a common water contaminant. Its structural and amphiphilic properties are shared by many other contaminants of concern. Based on a molecular dynamics study, this work reports the competitive adsorption of benzoic acid with water on the curved exteriors of carbon nanotubes of varying oxygen content. With the help of cylindrically approximated pair correlation functions, carboxyl-carboxyl associations were found to serve as an additional mechanism providing stability to the adsorbed benzoic acid on tube exteriors. These associations are secondary to the main aromatic-aromatic interactions during the adsorption process and therefore were not sufficient to establish the energy hierarchy at the adsorbed state with increase in surface oxygen content. The same mechanism was previously ascribed to the adsorption of the structurally similar but bulkier tannic acid. Both water and benzoic acid were organized into numerous mobility groups and a correspondence was established between species residence time and the average translation time taken to escape the tube vicinity. Vigorous exchange of water molecules among the first adsorption shell, the second adsorption shell, and the immediate vicinity radially outside was estimated to take place within a short time of about 10 ps.

Structural insights into the binding of nanobody Rh57 to active RhoA-GTP

RhoA protein is a small GTPase that acts as a molecular switch. When bound to guanosine triphosphate (GTP), RhoA can activate several key signal pathways. Recently, nanobody Rh57 specific binding with GTP bound active RhoA was discovered and developed as a BRET biosensor without cytotoxicity. To further clarify the nanobody Rh57's mechanism of action, we co-expressed, purified, and crystallized the RhoA-Rh57 nanobody complex and solved the structure by X-ray diffraction with a resolution of 2.76 Å. The structure showed that the interaction is mainly through hydrogen bonds, salt bridges, aromatic-aromatic interactions, and hydrophobic interactions. The involved regions include CDR3 and non-hypervariable loop of Rh57, and the SWI switch loops of RhoA, respectively. The different SWI conformation of inactivated RhoA-GDP prevented the Rh57's binding. The possible explanation of Rh57 as a non-cytotoxic BRET intracellular tracer is that Rh57's binding did not overlap with downstream PRK1 and thus did not interfere with the downstream signaling pathway. Our research provides an in-depth understanding of how nanobodies recognize activated RhoA-GTP while not binding inactivated RhoA-GDP. This structural information may also provide critical information for further optimization of relevant nanobodies.

Methionine‐aromatic Interaction as a Tool to Enhance Protein Stability

Nonbonded aromatic interactions play a significant role in stabilizing a folded protein structure(1, 2). Apart from well‐studied aromatic‐aromatic interactions in proteins, preferential proximity of sulfur containing cysteine and methionine sidechains and aromatic rings is also observed in protein hydrophobic cores and at the interfaces of protein‐protein complexes. Methionine‐aromatic motifs are examples of such S‐aromatic motifs that provide additional stabilization at a longer distance range over pure hydrophobic interactions(3). Although the idea of preferential association of aromatic amino acids has been successfully implemented for de novo design of peptides and proteins, the additional stability provided by met‐aromatic motif over pure hydrophobic interaction and its potential significance in designing a stabler protein fold has not been examined before. The present work shows the relevance of S‐aromatic interaction in increasing thermodynamic stability of a protein fold using Small Ubiquitin‐Like Modifier (SUMO) protein. We found that a highly conserved methionine‐phenylalanine motif in the core is a unique signature of SUMO but absent in other homologous Ubiquitin‐like (Ubl) protein folds. Using biophysical measurements and solution NMR based experiments, we found that the specific ‘up’ conformation between the methionine‐phenylalanine pair is critical to SUMO’s β‐grasp fold stability and its function(4). Given the additional stability provided by met‐aromatic motif over classical hydrophobic interaction, we engineered new S‐aromatic motifs in SUMO by replacing phenylalanine with other naturally occurring aromatic amino acids. Combining all atom MD simulations and solution NMR studies, we identify the energetically preferred interaction geometry of different met‐aromatic motifs inside the core. We found that the engineered SUMOs with increasing S‐aromatic interaction strength enhances the overall protein stability. Overall, our results reveal the potential role of S‐aromatic interactions as a design tool in enhancing thermodynamic stability of a protein fold.