Β-sheet Folding Research Articles

Barnacle-inspired and polyphenol-assisted modification of bacterial cellulose-based wound dressings for promoting infectious wound healing

Bacterial cellulose (BC) is an ideal candidate for wound dressings due to its natural origin, exceptional water-holding capacity, pliability, biocompatibility, and high absorption capability. However, the lack of essential antimicrobial activity limits its biomedical applications. This study reported BC-based wound dressings containing silk fibroin protein (SF), offering the potential for biomimetic properties, and (−)-epigallocatechin-3-gallate (EGCG) for polyphenol-assisted surface modification to promote infectious wound healing. Glycerol was used as the carbon source to promote the formation of an adhesive layer by facilitating the β-sheet folding of SF, and different concentrations of EGCG were employed to interact with SF through strong hydrogen bonding facilitated by the polyphenolic groups. The functionalized membrane exhibited outstanding water-holding capacity, swelling ratio, and degradation properties, along with enhanced hydrophilicity, adhesiveness, and a remarkable free radical scavenging ability. Both in vitro and in vivo experiments confirmed its potent bacteriostatic activity. The composite membrane displayed excellent biocompatibility, including cellular and hemocompatibility. Importantly, it effectively promoted wound healing in murine back infections. These findings suggest the significant feasibility of the innovative modification approach, and that functionalized membranes have great potential as wound-dressing materials for infection management in future clinical applications.

Design of Functional Globular β-Sheet Miniproteins.

The design of discrete β-sheet peptides is far less advanced than e. g. the design of α-helical peptides. The reputation of β-sheet peptides as being poorly soluble and aggregation-prone often hinders active design efforts. Here, we show that this reputation is unfounded. We demonstrate this by looking at the β-hairpin and WW domain. Their structure and folding have been extensively studied and they have long served as model systems to investigate protein folding and folding kinetics. The resulting fundamental understanding has led to the development of hyperstable β-sheet scaffolds that fold at temperatures of 100 °C or high concentrations of denaturants. These have been used to design functional miniproteins with protein or nucleic acid binding properties, in some cases with such success that medical applications are conceivable. The β-sheet scaffolds are not always completely rigid, but can be specifically designed to respond to changes in pH, redox potential or presence of metal ions. Some engineered β-sheet peptides also exhibit catalytic properties, although not comparable to those of natural proteins. Previous reviews have focused on the design of stably folded and non-aggregating β-sheet sequences. In our review, we now also address design strategies to obtain functional miniproteins from β-sheet folding motifs.

Glycine N-Thiocarboxyanhydride: A Key to Glycine-Rich Protein Mimics.

Glycine-rich proteins (GRPs) containing a high content of glycine residues (>30%) possess unique structural stability. However, the controllable synthesis of glycine-rich poly(amino acid)s (PAAs) to mimic GRPs has not been realized yet due to the poor solubility of polyglycine segments. We developed a novel method to synthesize glycine-rich PAAs via the controlled ring-opening copolymerization of glycine-N-thiocarboxyanhydrides (Gly-NTA) with sarcosine-N-carboxyanhydride and ε-Cbz-l-lysine-N-carboxyanhydride. The random copolymerization is evidenced by a kinetic study that shows that the propagation rate constant of Gly-NTA is close to those of comonomers. The copolymers exhibit predictable molecular weights between 4.5 and 24.6 kg/mol and tunable glycine incorporation, varying from 10.3 to 59.2%. Poly(Gly-r-Sar) samples with various glycine contents form nanoparticles or a hydrogel in water. Remarkably, the β-sheet folding of poly(Gly-r-Lys) remains intact in a neutral environment where the amine groups are protonated. Overall, the strategy paves the way to engineer glycine-rich PAAs and thereby expands their applications.

Three alginate lyases provide a new gut Bacteroides ovatus isolate with the ability to grow on alginate.

Humans consume alginate in the form of seaweed, food hydrocolloids, and encapsulations, making the digestion of this mannuronic acid (M) and guluronic acid (G) polymer of key interest for human health. To increase knowledge on alginate degradation in the gut, a gene catalog from human feces was mined for potential alginate lyases (ALs). The predicted ALs were present in nine species of the Bacteroidetes phylum, of which two required supplementation of an endo-acting AL, expected to mimic cross-feeding in the gut. However, only a new isolate grew on alginate. Whole-genome sequencing of this alginate-utilizing isolate suggested that it is a new Bacteroides ovatus strain harboring a polysaccharide utilization locus (PUL) containing three ALs of families: PL6, PL17, and PL38. The BoPL6 degraded polyG to oligosaccharides of DP 1-3, and BoPL17 released 4,5-unsaturated monouronate from polyM. BoPL38 degraded both alginates, polyM, polyG, and polyMG, in endo-mode; hence, it was assumed to deliver oligosaccharide substrates for BoPL6 and BoPL17, corresponding well with synergistic action on alginate. BoPL17 and BoPL38 crystal structures, determined at 1.61 and 2.11 Å, respectively, showed (α/α)6-barrel + anti-parallel β-sheet and (α/α)7-barrel folds, distinctive for these PL families. BoPL17 had a more open active site than the two homologous structures. BoPL38 was very similar to the structure of an uncharacterized PL38, albeit with a different triad of residues possibly interacting with substrate in the presumed active site tunnel. Altogether, the study provides unique functional and structural insights into alginate-degrading lyases of a PUL in a human gut bacterium. IMPORTANCE Human ingestion of sustainable biopolymers calls for insight into their utilization in our gut. Seaweed is one such resource with alginate, a major cell wall component, used as a food hydrocolloid and for encapsulation of pharmaceuticals and probiotics. Knowledge is sparse on the molecular basis for alginate utilization in the gut. We identified a new Bacteroides ovatus strain from human feces that grew on alginate and encoded three alginate lyases in a gene cluster. BoPL6 and BoPL17 show complementary specificity toward guluronate (G) and mannuronate (M) residues, releasing unsaturated oligosaccharides and monouronic acids. BoPL38 produces oligosaccharides degraded by BoPL6 and BoPL17 from both alginates, G-, M-, and MG-substrates. Enzymatic and structural characterization discloses the mode of action and synergistic degradation of alginate by these alginate lyases. Other bacteria were cross-feeding on alginate oligosaccharides produced by an endo-acting alginate lyase. Hence, there is an interdependent community in our guts that can utilize alginate.

Distorted aryl homochirality controlled by β-sheet folding

Distortion of planar aromatics occurs in the fused rings conjugated with bulky substituents, which generates racemic enantiomers with high transformation energy barriers. However, direct synthesis of homochiral distorted aryl compounds is a very challenging task. Here, we presented a molecular folding strategy to control distorted aryl homochirality. Amino acids and their derivatives conjugated on the polycyclic aromatic hydrocarbons including benzenes, naphthalenes and triphenylenes, which formed parallel β-sheet arrays through intramolecular hydrogen bonds. The folding behavior enabled distorted or twisted geometry of aromatics, of which the handedness was associated with the absolute chirality of amino acids. X-ray crystallography, theoretical calculations and circular dichroism spectroscopy verified the distorted homochirality in the solid and solution phase. The relatively small rotational barrier between the enantiomers made the molecule sensitive to the environment and thus realized the solvent-controlled chiral inversion. The β-sheet folding strategy can be widely used in polycyclic aromatic hydrocarbons with various functions, which provided a promising strategy to control inherent chirality of aromatics with adaptive chiroptical responses.

A bispecific inhibitor of complement and coagulation blocks activation in complementopathy models via a novel mechanism

AbstractInhibitors of complement and coagulation are present in the saliva of a variety of blood-feeding arthropods that transmit parasitic and viral pathogens. Here, we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits the formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all β-sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo–electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.

Identification and prediction of fold-switching proteins.

Folded proteins are assumed to be built upon fixed scaffolds of secondary structure, α-helices and β-sheets. Experimentally determined structures of >58,000 non-redundant proteins support this assumption, though it has recently been challenged by ∼100 fold-switching proteins. Though ostensibly rare, these proteins raise the question of how many uncharacterized proteins have shapeshifting, rather than fixed, secondary structures. We used a comparative sequence-based approach to predict fold switching in the universally conserved NusG transcription factor family, one member of which has a 50-residue regulatory subunit experimentally shown to switch between α-helical and β-sheet folds. Our approach predicts that 24% of sequences in this family undergo similar α-helix ⇌ β-sheet transitions. While these predictions cannot be reproduced by other state-of-the-art computational methods, they are confirmed by circular dichroism and nuclear magnetic resonance spectroscopy for 10 out of 10 sequence-diverse variants. Extending principles underlying our predictive approach, we identified coevolution of amino acid pairs uniquely corresponding to both conformations of 58 fold-switching proteins from distinct families. Using a set of these coevolved amino acid pairs, we successfully biased AlphaFold2 to predict two experimentally consistent conformations of a candidate protein with unsolved structure. These results indicate that fold-switching sequences have been preserved by natural selection, suggesting that their functionalities provide evolutionary advantage and paving the way for predictions of multiple distinct protein structures from single sequences.

Modulating amyloids’ formation path with sound energy

Protein folding is crucial for biological activity. Proteins' failure to fold correctly underlies various pathological processes, including amyloidosis, the aggregation of insoluble proteins (e.g., lysozymes) in organs. The exact conditions that trigger the structural transition of amyloids into β-sheet-rich aggregates are poorly understood, as is the case for the amyloidogenic self-assembly pathway. Ultrasound is routinely used to destabilize a protein's structure and enhance amyloid growth. Here, we report on an unexpected ultrasound effect on lysozyme amyloid species at different stages of aggregation: ultrasound-induced structural perturbation gives rise to nonamyloidogenic folds. Our infrared and X-ray analyses of the chemical, mechanical, and thermal effects of sound on lysozyme's structure found, in addition to the expected ultrasound-induced damage, evidence of irreversible disruption of the β-sheet fold of fibrillar lysozyme resulting in their structural transformation into monomers with no β-sheets. This structural transition is reflected in changes in the kinetics of protein self-assembly, namely, either prolonged nucleation or accelerated fibril growth. Using solution X-ray scattering, we determined the structure, the mass fraction of lysozyme monomer, and the morphology of its filamentous assemblies formed under different sound parameters. A nanomechanical analysis of ultrasound-modified protein assemblies revealed a correlation between the β-sheet content and elastic modulus of the protein material. Suppressing one of the ultrasound-derived effects allowed us to control the structural transformations of lysozyme. Overall, our comprehensive investigation establishes the boundary conditions under which ultrasound damages protein structure and fold. This knowledge can be utilized to impose medically desirable structural modifications on amyloid β-sheet-rich proteins.

Mutation-driven evolution of antibacterial function in an ancestral antifungal scaffold: Significance for peptide engineering.

Mutation-driven evolution of novel function on an old gene has been documented in many development- and adaptive immunity-related genes but is poorly understood in immune effector molecules. Drosomycin-type antifungal peptides (DTAFPs) are a family of defensin-type effectors found in plants and ecdysozoans. Their primitive function was to control fungal infection and then co-opted for fighting against bacterial infection in plants, insects, and nematodes. This provides a model to study the structural and evolutionary mechanisms behind such functional diversification. In the present study, we determined the solution structure of mehamycin, a DTAFP from the Northern root-knot nematode Meloidogyne hapla with antibacterial activity and an 18-mer insert, and studied the mutational effect through using a mutant with the insert deleted. Mehamycin adopts an expected cysteine-stabilized α-helix and β-sheet fold in its core scaffold and the inserted region, called single Disulfide Bridge-linked Domain (abbreviated as sDBD), forms an extended loop protruding from the scaffold. The latter folds into an amphipathic architecture stabilized by one disulfide bridge, which likely confers mehamycin a bacterial membrane permeability. Deletion of the sDBD remarkably decreased the ability but accompanying an increase in thermostability, indicative of a structure-function trade-off in the mehamycin evolution. Allosteric analysis revealed an interior interaction between the two domains, which might promote point mutations at some key sites of the core domain and ultimately give rise to the emergence of antibacterial function. Our work may be valuable in guiding protein engineering of mehamycin to improve its activity and stability.

Cross β Amyloid Nanotubes Demonstrate Promiscuous Catalysis in a Chemical Reaction Network via Co-option.

In Darwin's warm pond rich with nutrients, lesser number of early catalytic machineries with modest capabilities were able to demonstrate promiscuity by catalyzing diverse biochemical transformations important for protometabolism. Herein, we report catalytically promiscuous amyloid-based short peptide assemblies that could concomitantly catalyse three metabolically important yet orthogonal reactions. The surface exposed catalytic dyads featuring lysines and imidazoles were utilized for C=N condensation via dynamic covalent linkages and modulation of protonation events, respectively. Further, the peptide assemblies could promiscuously catalyse hydrolysis as well as retro-aldol reactions, that could be co-opted to facilitate C=N bond formation, either by a feedforward-driven reaction network or by replenishing depleted substrates. The catalytic diversity of short peptide based promiscuous β-sheet folds suggests their possible role in promoting the protometabolic network in early earth.

Instant and Tough Adhesives for Rapid Gastric Perforation and Traumatic Pneumothorax Sealing.

Hydrogel adhesives are hot spots due to their ubiquity and practical relevance. However, achieving a robust wet adhesion is still a challenge due to the preferential formation of hydrogen bonds between interfacial fluids and bulk hydrogel, as well as targeted substrates. Herein, a half-dry adhesive consisting of a silk fibroin (SF) semi-interpenetrating network and poly(acrylic acid) covalent network, which can allow a rapid liquid adsorption and repulsion process encountering a wet tissue, is reported. The remaining water enables excellent hydrogel flexibility to a dynamic surface, while the β-sheet fold endows its tough bulk strength under the peeling-off process. Notably, the wet adhesion energy versus porcine skin is 1440 J m-2 due to the combination of hydrogen bonds, electrostatic interactions, and chain entanglement derived from SF. In particular, both in vitro and in vivo outcomes indicate excellent hemostatic effects and result in incision closure of skin, artery, gastric perforation, and lung. After the first-stage closure, polyacrylic-silk fibroin adhesive (PSA) sealants can detach from the lung surface, fitting well to the healing period. By virtue of the reliable adhesion and good noncytotoxicity, PSA may be a prospective candidate for tissue sealant and drug carrier applications.

Many dissimilar NusG protein domains switch between α-helix and β-sheet folds

Folded proteins are assumed to be built upon fixed scaffolds of secondary structure, α-helices and β-sheets. Experimentally determined structures of >58,000 non-redundant proteins support this assumption, though it has recently been challenged by ~100 fold-switching proteins. Though ostensibly rare, these proteins raise the question of how many uncharacterized proteins have shapeshifting–rather than fixed–secondary structures. Here, we use a comparative sequence-based approach to predict fold switching in the universally conserved NusG transcription factor family, one member of which has a 50-residue regulatory subunit experimentally shown to switch between α-helical and β-sheet folds. Our approach predicts that 24% of sequences in this family undergo similar α-helix ⇌ β-sheet transitions. While these predictions cannot be reproduced by other state-of-the-art computational methods, they are confirmed by circular dichroism and nuclear magnetic resonance spectroscopy for 10 out of 10 sequence-diverse variants. This work suggests that fold switching may be a pervasive mechanism of transcriptional regulation in all kingdoms of life.

Crystal structure of thermally stable homodimeric cytochrome c'-β from Thermus thermophilus.

Cytochrome c'-β is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel β-sheet fold. Here, the crystal structure of cytochrome c'-β from the thermophilic Thermus thermophilus (TTCP-β) is reported at 1.74 Å resolution. TTCP-β has a typical antiparallel β-sheet fold similar to that of cytochrome c'-β from the moderately thermophilic Methylococcus capsulatus (MCCP-β). The phenylalanine cap structure around the distal side of the heme is also similar in TTCP-β and MCCP-β, indicating that both proteins similarly bind nitric oxide and carbon monoxide, as observed spectroscopically. Notably, TTCP-β exhibits a denaturation temperature of 117°C, which is higher than that of MCCP-β. Mutational analysis reveals that the increased homodimeric interface area of TTCP-β contributes to its high thermal stability. Furthermore, 14 proline residues, which are mostly located in the TTCP-β loop regions, possibly contribute to the rigid loop structure compared with MCCP-β, which has only six proline residues. These findings, together with those from phylogenetic analysis, suggest that the structures of Thermus cytochromes c'-β, including TTCP-β, are optimized for function under the high-temperature conditions in which the source organisms live.

Phylogenetic and protein prediction analysis reveals the taxonomically diverse distribution of virulence factors in Bacillus cereus strains.

Bacillus cereus is a food contaminant with widely varying enterotoxic potential due to its virulence proteins. In this article, phylogenetic analysis of the amino acid sequences from the whole-genomes of 41 strains, evolutionary distance calculation of the amino acid sequences of the virulence genes, and functional and structural predictions of the virulence proteins were performed to reveal the taxonomically diverse distribution of virulence factors. The genome evolution of the strains showed a clustering trend based on the protein-coding virulence genes. The strains of B. cereus have evolved into non-toxic risk and toxic risk clusters with medium-high- and medium-low-risk subclusters. The evolutionary transfer distances of incomplete virulence genes relative to housekeeping genes were greater than those of complete virulence genes, and the distance values of HblACD were higher than those of nheABC and CytK among the complete virulence genes. Cytoplasmic localization was impossible for all the virulence proteins, and NheB, NheC, Hbl-B, and Hbl-L1 were predicted to be extracellular. Nhe and Hbl proteins except CytK had similar spatial structures. The predicted structures of Nhe and Hbl mainly showed ‘head’ and ‘tail’ domains. The ‘head’ of NheA and Hbl-B, including two α-helices separated by β-tongue strands, might play a special role in the formation of Nhe trimers and Hbl trimers, respectively. The ‘cap’ of CytK, which includes two ‘latches’ with many β-sheets, formed a β-barrel structure with pores, and a ‘rim’ balanced the structure. The evolution of B. cereus strains showed a clustering tendency based on the protein-coding virulence genes, and the complete virulence-gene operon combination had higher relative genetic stability. The beta-tongue or latch associated with β-sheet folding might play an important role in the binding of virulence structures and pore-forming toxins in B. cereus.

A Fungal Defensin Inhibiting Bacterial Cell-Wall Biosynthesis with Non-Hemolysis and Serum Stability.

Defensins are a class of cationic disulfide-bridged antimicrobial peptides (AMPs) present in many eukaryotic organisms and even in bacteria. They primarily include two distinct but evolutionarily related superfamilies (cis and trans). Defensins in fungi belong to the members of the cis-superfamily with the cysteine-stabilized α-helical and β-sheet fold. To date, many fungal defensin-like peptides (fDLPs) have been found through gene mining of the genome resource, but only a few have been experimentally characterized. Here, we report the structural and functional characterization of Pyronesin4 (abbreviated as Py4), a fDLP previously identified by genomic sequencing of the basal filamentous ascomycete Pyronema confluens. Chemically, synthetic Py4 adopts a native-like structure and exhibits activity on an array of Gram-positive bacteria including some clinical isolates of Staphylococcus and Staphylococcus warneri, a conditioned pathogen inhabiting in human skin. Py4 markedly altered the bacterial morphology and caused cytoplasmic accumulation of the cell-wall synthesis precursor through binding to the membrane-bound Lipid II, indicating that it works as an inhibitor of cell-wall biosynthesis. Py4 showed no hemolysis and high mammalian serum stability. This work identified a new fungal defensin with properties relevant to drug exploration. Intramolecular epistasis between mutational sites of fDLPs is also discussed.

Many sequence-diverse domains switch between alpha-helix and beta-sheet folds

The protein folding paradigm asserts that the three-dimensional structure of a protein is determined by its amino acid sequence. Here we show that a substantial population of proteins from the NusG superfamily of transcription factors do not adhere to this paradigm. Previous work demonstrated that one member of this superfamily has a regulatory domain that completely switches between α-helical and β-sheet folds, but the pervasiveness of this fold-switching mechanism is uncertain. To address this question, we developed a sequence-based predictor, which revealed that thousands of proteins from this superfamily switch folds.

Explanation of Structure and Function of kv1.3 Potent Blocker from Mesobuthus eupeus Venom Gland: A New Promise in Drug Development

Background: Scorpions and other venomous animals are sought with great concern because venom is a source of novel peptides with exciting features. Some toxins of scorpion venom are effectors of potassium channels. Previous studies strongly support the importance of potassium channel toxins for use as pharmacological tools or potential drugs. Objectives: Here, a three-dimensional (3-D) structure and function of a potent acidic blocker of the human voltage-gated potassium ion channel, Kv1.3, previously identified in the scorpion Mesobuthus eupeus venom gland, were interpreted. Methods: The 3-D structure of meuK2-2 was generated using homology modeling. The interaction of meuK2-2 with the Kv1.3 channel was evaluated using a computational protocol employing peptide-protein docking experiments, pose clustering, and 100 ns molecular dynamic simulations to make the 3-D models of the meuK2-2/Kv1.3 complex trustworthy. Results: A CSα/β (cysteine-stabilized α-helical and β-sheet) fold was found for the 3-D structure of meuK2-2. In a different mechanism from what was identified so far, meuK2-2 binds to both turret and pore loop of Kv1.3 through two key residues (Ala28 and Ser11) and H-bonds. The binding of meuK2-2 induces some conformational changes to Kv1.3. Eventually, the side chain of a positively charged amino acid (His9) occupies the channel's pore. All together blocks the ion permeation pathway. Conclusions: MeuK2-2 could block Kv1.3 by a new mechanism. So, it could be a unique target for further investigations to develop a pharmacological tool and potential drug.

The desulfite mechanism exploration in a mode: Interaction between casein and sulfite by multi-spectrometry

Sulfite is a restricted food additive in various food preservatives. In our previous study, the sulfite was effectively removed by casein in shrimp samples. In this paper, the possible desulfite mechanism were explored by examining the interaction of casein with sulfite by fluorescence quenching spectroscopy, Ultraviolet (UV) adsorption, Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy. The binding constant (KS), binding thermodynamics, and the interaction effect on the casein's conformation were investigated. The results showed that casein bound with sulfite via hydrophilic and hydrophobic interactions, with Ks values of 1.07 × 105 at 293 K and 2.78 × 104 at 307 K respectively. The secondary structures and the conformation of casein were changed due to the interaction between casein and sulfite, with a decrease in positive Cotton effect, indicating the casein-sulfite interaction reduced the structural stability of β-sheet folding of casein. As a result, this work provides the theoretical basis for the desulfite mechanism for the sulfite removal by casein in food processing.

Behavior of Araujiain, a new cysteine phytoprotease, in organic media with low water content

In this paper we studied the effect of different organic solvents (1-octanol, trichloroethylene, ethanol, ethyl acetate, tetrahydrofuran, cyclohexane, propanone, acetonitrile, dichloromethane, chlorobenzene, N,N-dimethylformamide, acetophenone, diethyl ether, methanol, ethylene glycol and toluene) with low and constant water content on substrate preferences, thermostability and stability (caseinolytic activity retention after 4 h) of proteases of Araujia hortorum Fourn. ( Asclepiadaceae ). The stability of araujiain was high in N,N-dimethylformamide and ethanol at 40oC, but decreased at higher temperature. Araujiain substrates preferences in buffer Tris-HCl (pH 8), ethylene glycol and N,N-dimethylformamide exhibited different patterns, but the enzyme showed a high preference by glutamine derivative in all cases. According to FTIR spectroscopy studies, araujiain changed its secondary structure and as a consequence, it also changed its substrate preferences. This enzyme showed lower α-helical character and greater β-sheet folding in buffer than in organic media. A larger amount of antiparallel β-sheet residues indicates the formation of tighter intermolecular hydrogen bonds and enzymatic aggregates. These facts could explain the higher esterolytic activities, the greater stability and good hydrolytic potential of araujiain in some organic media such as N,N-dimethylformamide.

Factors Stabilizing β-Sheets in Protein Structures from a Quantum-Chemical Perspective.

Protein folds are determined by the interplay between various (de)stabilizing forces, which can be broadly divided into a local strain of the protein chain and intramolecular interactions. In contrast to the α-helix, the β-sheet secondary protein structure is significantly stabilized by long-range interactions between the individual β-strands. It has been observed that quite diverse amino acid sequences can form a very similar small β-sheet fold, such as in the three-β-strand WW domain. Employing "calibrated" quantum-chemical methods, we show herein on two sequentially diverse examples of the WW domain that the internal strain energy is higher in the β-strands and lower in the loops, while the interaction energy has an opposite trend. Low strain energy computed for peptide sequences in the loop 1 correlates with its postulated early formation in the folding process. The relatively high strain energy within the β-strands (up to 8 kcal mol-1 per amino acid residue) is compensated by even higher intramolecular interaction energy (up to 15 kcal mol-1 per residue). It is shown in a quantitative way that the most conserved residues across the structural family of WW domains have the highest contributions to the intramolecular interaction energy. On the other hand, the residues in the regions with the lowest strain are not conserved. We conclude that the internal interaction energy is the physical quantity tuned by evolution to define the β-sheet protein fold.