Solvent-exposed Residues Research Articles

Structural insight into the poly(3-hydroxybutyrate) hydrolysis by intracellular PHB depolymerase from Bacillus thuringiensis.

Poly((R)-3-hydroxybutyrate) (PHB) is a microbial biopolymer widely used in commercial biodegradable plastics. PHB degradation in cell is catalyzed by PHB depolymerase (PhaZ), which hydrolyzes the polyester into mono- and/or oligomeric (R)-3-hydroxylbutyrates (3HB). A novel intracellular PhaZ from Bacillus thuringiensis (BtPhaZ) was identified for potential applications in polymer biodegradation and 3HB production. Herein, we present the crystal structure of BtPhaZ at 1.42-Å resolution, making the first crystal structure for an intracellular PhaZ. BtPhaZ comprises a canonical α/β hydrolase catalytic domain and a unique α-helical cap domain. Despite lacking sequence similarity, BtPhaZ shares high structural homology with many α/β hydrolase members, exhibiting a similar active-site architecture. Alongside the most conserved superfamily signature, several new conserved signatures have been identified, contributing not only to the formations of the Ser-His-Asp catalytic triad and the oxyanion hole but also to the active-site conformation. The putative P-1 subsite appears to have limited space for accommodating only one 3HB-monomer, which may provide an explanation why the major hydrolytic product for BtPhaZ is monomeric form. Furthermore, a cluster of solvent-exposed hydrophobic residues in the helical cap domain forms an adsorption site for polymer-binding. Detailed structural comparisons reveal that various PhaZs employ distinct residues for the biopolymer-binding and hydrolysis.

Structural basis of nucleic acid recognition by the N-terminal cold shock domain of the plant glycine-rich protein AtGRP2

AtGRP2 is a glycine-rich, RNA-binding protein that plays pivotal roles in abiotic stress response and flowering time regulation in Arabidopsis thaliana. AtGRP2 consists of an N-terminal cold shock domain (CSD) and two C-terminal CCHC-type zinc knuckles interspersed with glycine-rich regions. Here, we investigated the structure, dynamics, and nucleic acid-binding properties of AtGRP2-CSD. The 2D [1H,15N] heteronuclear single quantum coherence spectrum of AtGRP2-CSD1-79 revealed the presence of a partially folded intermediate in equilibrium with the folded state. The addition of 11 residues at the C terminus stabilized the folded conformation. The three-dimensional structure of AtGRP2-CSD1-90 unveiled a β-barrel composed of five antiparallel β-strands and a 310 helical turn, along with an ordered C-terminal extension, a conserved feature in eukaryotic CSDs. Direct contacts between the C-terminal extension and the β3-β4 loop further stabilized the CSD fold. AtGRP2-CSD1-90 exhibited nucleic acid binding via solvent-exposed residues on strands β2 and β3, as well as the β3-β4 loop, with higher affinity for DNA over RNA, particularly favoring pyrimidine-rich sequences. Furthermore, DNA binding induced rigidity in the β3-β4 loop, evidenced by 15N-{1H} NOE values. Mutation of residues W17, F26, and F37, in the central β-sheet, completely abolished DNA binding, highlighting the significance of π-stacking interactions in the binding mechanism. These results shed light on the mechanism of nucleic acid recognition employed by AtGRP2, creating a framework for the development of biotechnological strategies aimed at enhancing plant resistance to abiotic stresses.

Morphology evolution of supramolecular aggregates from C3-symmetric peptide amphiphiles

During the aging of supramolecular systems, new stable materials with different morphologies, molecular packing, and hierarchical organization may form from the initial assembly. Understanding the mechanisms and pathways accompanying age-related changes may allow for the controlled synthesis of multiple materials from a single molecule in a nature-inspired manner. Herein, we demonstrate the multi-stage evolution of a single solution of C3-symmetric peptide amphiphile containing diphenylalanine fragment and benzyl-1,3,5-tricarboxamide core. Transmission electron microscopy reveals the rapid formation of 20–40 nm wide nanoribbons in an aqueous solution within 60 s after preparation. The lateral edges of nanoribbons contain solvent-exposed aromatic residues that tend to merge via an unconventional edge-to-edge zipping mechanism, leading to the nanoribbon’s growth in width. After reaching a critical width and flexibility, “sticky edges” on the same nanoribbon may merge via a self-zipping mechanism, forming rigid nanotubes. Eventually, mechanical perturbations cause buckling and fracture of nanotubes into shorter nanotubes via flexural or tensile failure. This work reports new evolution pathways in soft 2D assemblies and introduces solution ageing as a promising method to control morphology of peptide amphiphiles.

Selection Across the Three-Dimensional Structure of Venom Proteins from North American Scolopendromorph Centipedes.

Gene duplication followed by nucleotide differentiation is one of the simplest mechanisms to develop new functions for genes. However, the evolutionary processes underlying the divergence of multigene families remain controversial. We used multigene families found within the diversity of toxic proteins in centipede venom to test two hypotheses related to venom evolution: the two-speed mode of venom evolution and the rapid accumulation of variation in exposed residues (RAVER) model. The two-speed mode of venom evolution proposes that different types of selection impact ancient and younger venomous lineages with negative selection being the predominant form in ancient lineages and positive selection being the dominant form in younger lineages. The RAVER hypothesis proposes that, instead of different types of selection acting on different ages of venomous lineages, the different types of selection will selectively contribute to amino acid variation based on whether the residue is exposed to the solvent where it can potentially interact directly with toxin targets. This hypothesis parallels the longstanding understanding of protein evolution that suggests that residues found within the structural or active regions of the protein will be under negative or purifying selection, and residues that do not form part of these areas will be more prone to positive selection. To test these two hypotheses, we compared the venom of 26 centipedes from the order Scolopendromorpha from six currently recognized species from across North America using both transcriptomics and proteomics. We first estimated their phylogenetic relationships and uncovered paraphyly among the genus Scolopendra and evidence for cryptic diversity among currently recognized species. Using our phylogeny, we then characterized the diverse venom components from across the identified clades using a combination of transcriptomics and proteomics. We conducted selection-based analyses in the context of predicted three-dimensional properties of the venom proteins and found support for both hypotheses. Consistent with the two-speed hypothesis, we found a prevalence of negative selection across all proteins. Consistent with the RAVER hypothesis, we found evidence of positive selection on solvent-exposed residues, with structural and less-exposed residues showing stronger signal for negative selection. Through the use of phylogenetics, transcriptomics, proteomics, and selection-based analyses, we were able to describe the evolution of venom from an ancient venomous lineage and support principles of protein evolution that directly relate to multigene family evolution.

Investigation of F508del CFTR unfolding and a search for stabilizing small molecules

Mutation of phenylalanine at position 508 in the cystic fibrosis transmembrane conductance regulator (F508del CFTR) yields a protein unstable at physiological temperatures that is rapidly degraded in the cell. This mutation is present in about 90% of cystic fibrosis patients, hence there is great interest in compounds reversing its instability. We have previously reported the expression of the mutated protein at low temperature and its purification in detergent. Here we describe the use of the protein to screen compounds present in a library of Federal Drug Administration (FDA) – approved drugs and also in a small natural product library. The kinetics of unfolding of F508del CFTR at 37 °C were probed by the increase in solvent-exposed cysteine residues accessible to a fluorescent reporter molecule. This occurred in a bi-exponential manner with a major (≈60%) component of half-life around 5 min and a minor component of around 60 min. The faster kinetics match those observed for loss of channel activity of F508del CFTR in cells at 37 °C. Most compounds tested had no effect on the fluorescence increase, but some were identified that significantly slowed the kinetics. The general properties of these compounds, and any likely mechanisms for inducing stability in purified CFTR are discussed. These experimental data may be useful for artificial intelligence – aided design of CFTR-specific drugs and in the identification of stabilizing additives for membrane proteins (in general).

De novo missense variants in exon 9 of SEPHS1 cause a neurodevelopmental condition with developmental delay, poor growth, hypotonia, and dysmorphic features

Selenophosphate synthetase (SEPHS) plays an essential role in selenium metabolism. Two mammalian SEPHS paralogues, SEPHS1 and SEPHS2, share high sequence identity and structural homology with SEPHS. Here, we report nine individuals from eight families with developmental delay, growth and feeding problems, hypotonia, and dysmorphic features, all with heterozygous missense variants in SEPHS1. Eight of these individuals had a recurrent variant at amino acid position 371 of SEPHS1 (p.Arg371Trp, p.Arg371Gln, and p.Arg371Gly); seven of these variants were known to be de novo. Structural modeling and biochemical assays were used to understand the effect of these variants on SEPHS1 function. We found that a variant at residue Trp352 results in local structural changes of the C-terminal region of SEPHS1 that decrease the overall thermal stability of the enzyme. In contrast, variants of a solvent-exposed residue Arg371 do not impact enzyme stability and folding but could modulate direct protein-protein interactions of SEPSH1 with cellular factors in promoting cell proliferation and development. In neuronal SH-SY5Y cells, we assessed the impact of SEPHS1 variants on cell proliferation and ROS production and investigated the mRNA expression levels of genes encoding stress-related selenoproteins. Our findings provided evidence that the identified SEPHS1 variants enhance cell proliferation by modulating ROS homeostasis. Our study supports the hypothesis that SEPHS1 plays a critical role during human development and provides a basis for further investigation into the molecular mechanisms employed by SEPHS1. Furthermore, our data suggest that variants in SEPHS1 are associated with a neurodevelopmental disorder.

LC-Photo-CIDNP hyperpolarization of biomolecules bearing a quasi-isolated spin pair: Magnetic-Field dependence via a rapid-shuttling device

Liquid-state low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) is an emerging technology tailored to enhance the sensitivity of NMR spectroscopy via LED- or laser-mediated optical irradiation. LC-photo-CIDNP is particularly useful to detect solvent-exposed aromatic residues (Trp, Tyr), either in isolation or within polypeptides and proteins. This study investigates the magnetic-field dependence of the LC-photo-CIDNP of Trp-α-13C-β,β,2,4,5,6,7-d7, a Trp isotopolog bearing a quasi-isolated 1Hα-13Cαspin pair (QISP). We employed a new rapid-shuttling side-illumination field-cycling device that enables ultra-fast (90–120 ms) vertical movements of NMR samples within the bore of a superconducting magnet. Thus, LC-photo-CIDNP hyperpolarization occurs at low field, while hyperpolarized signals are detected at high field (700 MHz). Resonance lineshapes were excellent, and the effect of several fields (1.18–7.08 T range) on hyperpolarization efficiency could be readily explored. Remarkably, unprecedented LC-photo-CIDNP enhancements ε ≅ 1,200 were obtained at 50 MHz (1.18 T), suggesting exciting avenues to hypersensitive LED-enhanced NMR in liquids at low field.

A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins.

The online version contains supplementary material available at 10.1007/s00723-023-01618-8.

Mutation Space of Spatially Conserved Amino Acid Sites in Proteins.

The mutation space of spatially conserved (MSSC) amino acid residues is a protein structural quantity developed and described in this work. The MSSC quantifies how many mutations and which different mutations, i.e., the mutation space, occur in each amino acid site in a protein. The MSSC calculates the mutation space of amino acids in a target protein from the spatially conserved residues in a group of multiple protein structures. Spatially conserved amino acid residues are identified based on their relative positions in the protein structure. The MSSC examines each residue in a target protein, compares it to the residues present in the same relative position in other protein structures, and uses physicochemical criteria of mutations found in each conserved spatial site to quantify the mutation space of each amino acid in the target protein. The MSSC is analogous to scoring each site in a multiple sequence alignment but in three-dimensional space considering the spatial location of residues instead of solely the order in which they appear in a protein sequence. MSSC analysis was performed on example cases, and it reproduces the well-known observation that, regardless of secondary structure, solvent-exposed residues are more likely to be mutated than internal ones. The MSSC code is available on GitHub: "https://github.com/Cantu-Research-Group/Mutation_Space".

Differences in the organization of interface residues tunes the stability of the SARS-CoV-2 spike-ACE2 complex.

The continuous emergence of novel variants represents one of the major problems in dealing with the SARS-CoV-2 virus. Indeed, also due to its prolonged circulation, more than ten variants of concern emerged, each time rapidly overgrowing the current viral version due to improved spreading features. As, up to now, all variants carry at least one mutation on the spike Receptor Binding Domain, the stability of the binding between the SARS-CoV-2 spike protein and the human ACE2 receptor seems one of the molecular determinants behind the viral spreading potential. In this framework, a better understanding of the interplay between spike mutations and complex stability can help to assess the impact of novel variants. Here, we characterize the peculiarities of the most representative variants of concern in terms of the molecular interactions taking place between the residues of the spike RBD and those of the ACE2 receptor. To do so, we performed molecular dynamics simulations of the RBD-ACE2 complexes of the seven variants of concern in comparison with a large set of complexes with different single mutations taking place on the RBD solvent-exposed residues and for which the experimental binding affinity was available. Analyzing the strength and spatial organization of the intermolecular interactions of the binding region residues, we found that (i) mutations producing an increase of the complex stability mainly rely on instaurating more favorable van der Waals optimization at the cost of Coulombic ones. In particular, (ii) an anti-correlation is observed between the shape and electrostatic complementarities of the binding regions. Finally, (iii) we showed that combining a set of dynamical descriptors is possible to estimate the outcome of point mutations on the complex binding region with a performance of 0.7. Overall, our results introduce a set of dynamical observables that can be rapidly evaluated to probe the effects of novel isolated variants or different molecular systems.

Chitinase is a Potent Insecticidal Molecular Target of Camptothecin and Its Derivatives

Camptothecin (CPT) is a prominent molecule in natural product research because of its application prospects in medicine and agriculture. In this study, CPT and its derivatives were discovered to be competitive inhibitors of group II and group h insect chitinases, both of which are key components of insect chitinolytic systems. CPT and 7-ethyl-10-hydroxycamptothecin (SN-38) inhibited group II chitinase from Ostrinia furnacalis (OfChtII) with Ki values of 5.1 and 2.0 μM, respectively. Results from tryptophan fluorescence spectroscopy, molecular docking analysis, and molecular dynamics simulations revealed that both CPT and SN-38 inhibit OfChtII-C1 by interacting with solvent-exposed tryptophan residues in a substrate-binding cleft. CPT exhibited high insecticidal activity toward the orthopteran pest Locusta migratoria, possibly because of the midgut metabolism of CPT, with only moderate activities toward lepidopteran pests. Even though SN-38 exhibited much lower insecticidal activities than CPT, it still showed higher inhibitory activity toward chitinase. This study reports a new molecular target of CPT and provides insights into molecular design of CPT-based insecticides against different kinds of pests.

Stable binding to phosphatidylserine-containing membranes requires conserved arginine residues in tandem C domains of blood coagulation factor VIII.

At sites of vascular damage, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated platelet surfaces with activated factor IX (fIXa) to form the intrinsic "tenase" complex. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through β-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex. In this study, we devised a novel platform for generating recombinant C1, C2, and C1C2 domain constructs and performed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Binding measurements between phosphatidylserine (PS)-containing lipid membrane surfaces and fVIII C domains demonstrated an ionic strength dependence on membrane binding affinity. Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. Lastly, we determined the X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of PS, which binds to a basic cleft and makes charge-charge contact with R2320. We conclude that basic clefts in the fVIII C domains bind to PS-containing membranes through conserved arginine residues via a C domain modularity, where each C domain possesses modest electrostatic-dependent affinity and tandem C domains are required for high affinity binding.

Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine.

The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag is assembled on the protein in three steps, comprising (i)reaction with 4-fluoro-2,6-dicyanopyridine (FDCP); (ii)reaction of the cyano groups with -cysteine, penicillamine or -cysteine to complete the lanthanoid chelating moiety; and (iii)titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with and ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron-electron resonance experiments after titration with ions.

Structure-selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern.

Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to affect the quality of the antibody response focusing it to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and three variants of concern (VOC), B.1.351, B.1.617.2, and BA.1. We demonstrate that immunogens built on structure selection can be used to influence the quality of the antibody response by focusing it to a conserved site of vulnerability shared between wildtype virus and VOCs, resulting in neutralizing antibodies across variants.

π-Clamp-Mediated Homo- and Heterodimerization of Single-Domain Antibodies via Site-Specific Homobifunctional Conjugation.

Post-translational protein–protein conjugation produces bioconjugates that are unavailable via genetic fusion approaches. A method for preparing protein–protein conjugates using π-clamp-mediated cysteine arylation with pentafluorophenyl sulfonamide functional groups is described. Two computationally designed antibodies targeting the SARS-CoV-2 receptor binding domain were produced (KD = 146, 581 nM) with a π-clamp sequence near the C-terminus and dimerized using this method to provide a 10–60-fold increase in binding (KD = 8–15 nM). When two solvent-exposed cysteine residues were present on the second protein domain, the π-clamp cysteine residue was selectively modified over an Asp-Cys-Glu cysteine residue, allowing for subsequent small-molecule conjugation. With this strategy, we build molecule–protein–protein conjugates with complete chemical control over the sites of modification.

Structural and biochemical analysis reveals how ferulic acid improves catalytic efficiency of Humicolagrisea xylanase

Humicolagrisea var. thermoidea is an aerobic and thermophilic fungus that secretes the GH11 xylanase HXYN2 in the presence of sugarcane bagasse. In this study, HXYN2 was expressed in Pichiapastoris and characterized biochemically and structurally in the presence of beechwood xylan substrate and ferulic acid (FA). HXYN2 is a thermally stable protein, as indicated by circular dichroism, with greater activity in the range of 40–50 °C and pH 5.0–9.0, with optimal temperature and pH of 50 °C and 6.0, respectively. FA resulted in a 75% increase in enzyme activity and a 2.5-fold increase in catalytic velocity, catalytic efficiency, and catalytic rate constant (kcat), with no alteration in enzyme affinity for the substrate. Fluorescence quenching indicated that FA forms a complex with HXYN2 interacting with solvent-exposed tryptophan residues. The binding constants ranged from moderate (pH 7.0 and 9.0) to strong (pH 4.0) affinity. Isothermal titration calorimetry, structural models and molecular docking suggested that hydrogen bonds and hydrophobic interactions occur in the aglycone region inducing conformational changes in the active site driven by initial and final enthalpy- and entropy processes, respectively. These results indicate a potential for biotechnological application for HXYN2, such as in the bioconversion of plant residues rich in ferulic acid.

Spontaneous Refolding of Amyloid Fibrils from One Polymorph to Another Caused by Changes in Environmental Hydrophobicity.

Here, we report a new phenomenon in which lysozyme fibrils formed in a solution of acetic acid spontaneously refold to a different polymorph through a disassembled intermediate upon the removal of acetic acid. The structural changes were revealed and characterized by deep-UV resonance Raman spectroscopy, nonresonance Raman spectroscopy, intrinsic tryptophan fluorescence spectroscopy, and atomic force microscopy. A PPII-like structure with highly solvent-exposed tryptophan residues predominates the intermediate aggregates before refolding to polymorph II fibrils. Furthermore, the disulfide (SS) bonds undergo significant rearrangements upon the removal of acetic acid from the lysozyme fibril environment. The main SS bond conformation changes from gauche-gauche-trans in polymorph I to gauche-gauche-gauche in polymorph II. Changing the hydrophobicity of the fibril environment was concluded to be the decisive factor causing the spontaneous refolding of lysozyme fibrils from one polymorph to another upon the removal of acetic acid. Potential biological implications of the discovered phenomenon are discussed.

Substrate binding in the mitochondrial ADP/ATP carrier is a step-wise process guiding the structural changes in the transport cycle

Mitochondrial ADP/ATP carriers import ADP into the mitochondrial matrix and export ATP to the cytosol to fuel cellular processes. Structures of the inhibited cytoplasmic- and matrix-open states have confirmed an alternating access transport mechanism, but the molecular details of substrate binding remain unresolved. Here, we evaluate the role of the solvent-exposed residues of the translocation pathway in the process of substrate binding. We identify the main binding site, comprising three positively charged and a set of aliphatic and aromatic residues, which bind ADP and ATP in both states. Additionally, there are two pairs of asparagine/arginine residues on opposite sides of this site that are involved in substrate binding in a state-dependent manner. Thus, the substrates are directed through a series of binding poses, inducing the conformational changes of the carrier that lead to their translocation. The properties of this site explain the electrogenic and reversible nature of adenine nucleotide transport.

Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents.

Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody–drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the β-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.

Evolution of tunnels in α/β-hydrolase fold proteins-What can we learn from studying epoxide hydrolases?

The evolutionary variability of a protein’s residues is highly dependent on protein region and function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues whereas active site residues are considered to be conserved. The abovementioned rules apply also to α/β-hydrolase fold proteins—one of the oldest and the biggest superfamily of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We selected soluble epoxide hydrolases as representative of this family to conduct the first systematic study on the evolution of tunnels. We hypothesised that tunnels are lined by mostly conserved residues, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. The hypothesis was confirmed, and we suggested a general and detailed way of the tunnels’ evolution analysis based on entropy values calculated for tunnels’ residues. We also found three different cases of entropy distribution among tunnel-lining residues. These observations can be applied for protein reengineering mimicking the natural evolution process. We propose a ‘perforation’ mechanism for new tunnels design via the merging of internal cavities or protein surface perforation. Based on the literature data, such a strategy of new tunnel design could significantly improve the enzyme’s performance and can be applied widely for enzymes with buried active sites.