Intermolecular Hydrophobic Interactions Research Articles

Lactic acid bacteria fermentation-induced egg white protein structure deformation influencing gelling properties, with membrane concentration as a strategy to improve texture.

Egg white (EW), a rich protein source, holds promise for creating a high-protein, low-fat gel product. However, browning issues during heating and sterilization have hindered its wider application. In this study, lactic acid bacteria fermentation was employed to eliminate reducing sugar in EW, and its impact on the molecular structure and gelling properties was explored. The results revealed that fermentation would trigger protein structural unfolding and aggregation, evident from higher fluorescence intensity and enlarged protein particle diameters, resulting in the decrease in gelling hardness. In comparison, Streptococcus thermophilus-fermented EW (under 6×108CFU/mL incubation rate, fermented for 6h) exhibited the highest gel hardness, ascribed to the relatively weaker structure transformation, with high water holding capacity and stronger intermolecular hydrophobic interaction. To further enhance the gelling properties of fermented EW, membrane concentration treatment was applied, exhibiting superior characteristics in appearance, aroma, and taste. In summary, lactic acid bacteria fermentation and concentration are feasible solutions to improve appearance and texture of EW gels simultaneously. The research findings offer eco-friendly and practical strategies for enhancing the quality of EW gels, providing valuable theoretical insights for the development of innovative, texture-rich, and healthy nutritional foods.

Wheat gliadin/xanthan gum intermolecular complexes: Interaction mechanism and structural characterization

In this study, the interactions between wheat gliadin (GL) and xanthan gum (XG) were investigated to design new systems with potential applications as a gluten-free substitute product. Combining spectral with morphological and molecular docking methods allowed the establishment of the complexation mechanism between globular hydrophobic GL and the hydrophilic XG with an extended and partially disordered backbone. GL maintains intact its hydrophobic core even at high GL/XG ratios and organizes into small aggregate–type assemblies. The stable and uniform complexes have a low GL content, based on intermolecular hydrogen bonds and hydrophobic interactions. The GL/XG combining ratio influences the size, structure and interaction mechanism of the microparticles. The preferred sites of interaction and the binding affinities were determined by molecular docking on GL libraries and XG models. This research may provide significant knowledge for the development of low-GL wheat food products using a dietary fiber polysaccharide as a functional compound.

Designing multi-epitope vaccine against human cytomegalovirus integrating pan-genome and reverse vaccinology pipelines

Human cytomegalovirus (HCMV) is accountable for high morbidity in neonates and immunosuppressed individuals. Due to the high genetic variability of HCMV, current prophylactic measures are insufficient. In this study, we employed a pan-genome and reverse vaccinology approach to screen the target for efficient vaccine candidates. Four proteins, envelope glycoprotein M, UL41A, US23, and US28, were shortlisted based on cellular localization, high solubility, antigenicity, and immunogenicity. A total of 29 B-cell and 44 T-cell highly immunogenic and antigenic epitopes with high global population coverage were finalized using immunoinformatics tools and algorithms. Further, the epitopes that were overlapping among the finalized B-cell and T-cell epitopes were linked with suitable linkers to form various combinations of multi-epitopic vaccine constructs. Among 16 vaccine constructs, Vc12 was selected based on physicochemical and structural properties. The docking and molecular simulations of VC12 were performed, which showed its high binding affinity (−23.35 kcal/mol) towards TLR4 due to intermolecular hydrogen bonds, salt bridges, and hydrophobic interactions, and there were only minimal fluctuations. Furthermore, Vc12 eliciting a good response was checked for its expression in Escherichia coli through in silico cloning and codon optimization, suggesting it to be a potent vaccine candidate.

The pH dependent structural and viscoelastic behavior of Ovomucin-Complex isolated from egg white under alkaline conditions

Ovomucin-Complex (OVM) is responsible for providing the natural gel-like properties of egg white, especially in alkali-induced egg white gels. In this study, OVM was modified by alkaline to produce gel-type materials. The structure, physicochemical and viscoelastic properties of OVMs under different pH (7–12) were probed using FTIR, HPLC, SEM, CLSM and rheometer. The results of rheological measurements in linear viscoelastic region revealed that alkaline modified OVMs exhibited a soft viscoelastic gel in alkaline conditions, with the transition occurring near pH 8, compared with a viscoelastic solution at neutral pH. In addition to the pH-dependent gelation behavior of OVMs, further rheological studies under nonlinear deformations reveal shear thinning and an apparent yield stress, which were also highly affected by pH. The results from HPLC and molecular interactions suggest that the alkaline modification led to the depolymerization of OVMs in alkaline conditions, enhancing the formation of intermolecular disulfide bonds, electrostatic force and hydrophobic interactions. The differences in these mechanical properties were also reflected in the OVM structure. The SEM and CLSM images demonstrated that the arrangement of gel pores under pH 10 conditions is denser and well-distributed than those under other conditions. The FTIR results verified that alkaline modification leads to the decreased content of β-sheet. Nevertheless, prolonged exposure to pH 12.0 resulted in a gradual liquefaction of the gel due to increased electrostatic repulsion and surface hydrophobicity, as well as the reduced disulfide bond and an increase in the disordered structure. These findings provide a novel insight for the development of mucin gel foods.

Thermostable α-Amylases and Laccases: Paving the Way for Sustainable Industrial Applications

The growing demand in industrial and biotechnological settings for more efficient enzymes with enhanced biochemical features, particularly thermostability and thermotolerance, necessitates a timely response. Renowned for their versatility, thermostable enzymes offer significant promise across a range of applications, including agricultural, medicinal, and biotechnological domains. This comprehensive review summarizes the structural attributes, catalytic mechanisms, and connection between structural configuration and functional activity of two major classes of thermostable enzymes: α-amylases and laccases. These enzymes serve as valuable models for understanding the structural foundation behind the thermostability of proteins. By highlighting the commercial importance of thermostable enzymes and the interest these generate among researchers in further optimization and innovation, this article can greatly contribute to ongoing research on thermostable enzymes and aiding industries in optimizing production processes via immobilization, use of stabilizing additives, chemical modification, protein engineering (directed evolution and mutagenesis), and genetic engineering (through cloning and expression of thermostable genes). It also gives insights to the exploration of suitable strategies and factors for enhancing thermostability like increasing substrate affinity; introducing electrostatic, intramolecular, and intermolecular hydrophobic interactions; mitigating steric hindrance; increasing flexibility of an active site; and N- and C-terminal engineering, thus resulting in heightened multipronged stability and notable enhancements in the enzymes’ industrial applicability.

Role of Unimers to Polymersomes Transition in Pluronic Blends for Controlled and Designated Drug Conveyance.

This study investigates the nanoscale self-assembly from mixtures of two symmetrical poly(ethylene oxide)-poly(propylene oxide)-pol(ethylene oxide) (PEO-PPO-PEO) block copolymers (BCPs) with different lengths of PEO blocks and similar PPO blocks. The blended BCPs (commercially known as Pluronic F88 and L81, with 80 and 10% PEO, respectively) exhibited rich phase behavior in an aqueous solution. The relative viscosity (ηrel) indicated significant variations in the flow behavior, ranging from fluidic to viscous, thereby suggesting a possible micellar growth or morphological transition. The tensiometric experiments provided insight into the intermolecular hydrophobic interactions at the liquid-air interface favoring the surface activity of mixed-system micellization. Dynamic light scattering (DLS) and small-angle neutron scattering (SANS) revealed the varied structural morphologies of these core-shell mixed micelles and polymersomes formed under different conditions. At a concentration of ≤5% w/v, Pluronic F88 exists as molecularly dissolved unimers or Gaussian chains. However, the addition of the very hydrophobic Pluronic L81, even at a much lower (<0.2%) concentration, induced micellization and promoted micellar growth/transition. These results were further substantiated through molecular dynamics (MD) simulations, employing a readily transferable coarse-grained (CG) molecular model grounded in the MARTINI force field with density and solvent-accessible surface area (SASA) profiles. These findings proved that F88 underwent micellar growth/transition in the presence of L81. Furthermore, the potential use of these Pluronic mixed micelles as nanocarriers for the anticancer drug quercetin (QCT) was explored. The spectral analysis provided insight into the enhanced solubility of QCT through the assessment of the standard free energy of solubilization (ΔG°), drug-loading efficiency (DL%), encapsulation efficiency (EE%), and partition coefficient (P). A detailed optimization of the drug release kinetics was presented by employing various kinetic models. The [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay, a frequently used technique for assessing cytotoxicity in anticancer research, was used to gauge the effectiveness of these QCT-loaded mixed nanoaggregates.

Exploring promising natural compounds for breast cancer treatment: in silico molecular docking targeting WDR5-MYC protein interaction

Cancer is an aberrant differentiation of normal cells, characterized by uncontrolled growth and the potential to acquire invasive and aggressive properties that ultimately lead to metastasis. In the realm of scientific exploration, a multitude of pathways has been investigated and targeted by researchers, among which one specific pathway is recognized as WDR5-MYC. Continuous investigations and research show that WDR5-MYC is a therapeutic target protein. Hence, the discovery of naturally occurring compounds with anticancer properties has been suggested as a rapid and efficient alternative for the development of anticancerous therapeutics. A virtual screening approach was used to identify the most potent compounds from the NP-lib database at the MTiOpenScreen webserver against WDR5-MYC. This process yielded a total of 304 identified compounds. Subsequently, after screening, four potent compounds, namely Estrone (ZINC000003869899), Ethyl-1,2-benzanthracene (ZINC000003157052), Strychnine (ZINC000000119434) and 7H-DIBENZO [C, G] CARBAZOLE (ZINC000001562130), along with a cocrystallized 5-[4-(trifluoromethyl) phenyl]-1H-tetrazole inhibitor (QBP) as a reference ligand, were considered for stringent molecular docking. Thus, each compound exhibited significant docking energy between −8.2 and −7.7 kcal/mol and molecular contacts with essential residue Asn225, Lys250, Ser267 and Lys272 in the active pocket of WDR5-MYC against the QBP inhibitor (the native ligand QBP serves as a reference in the comparative analysis of docked complexes). The results support the potent compounds for drug-likeness and strong binding affinity with WDR5-MYC protein. Further, the stability of the selected compounds was predicted by molecular dynamics simulation (100 ns) contributed by intermolecular hydrogen bonds and hydrophobic interactions. This demonstrates the potential of the selected compounds to be used against breast cancer treatment. Communicated by Ramaswamy H. Sarma

Fabrication of water-in-oil-in-gel emulsion gel based on pH-shifting soybean lipophilic protein and carboxymethyl chitosan: Gel performance, physicochemical properties and digestive characteristics

In this study, Soybean lipophilic protein (LP) was modified by pH-shifting treatment and the modified LP was cross-linked with carboxymethyl chitosan (CMCS) to form an interpenetrating network structure to prepare water-in-oli-in-gel emulsion gels (W/O/G). The effects of the variation of phase ratio and addition of cross-linking agent (genipin) on the physicochemical properties and digestive characteristics of W/O/G were investigated. FT-IR and intermolecular force analysis showed that pH-shifting treatment exposed more hydrophobic groups in LP, and promoted the increase of intermolecular hydrophobic interaction and hydrogen bonding force. With the increase of GP addition and W1/O ratio, the swelling rate, water-holding capacity and UV stability of W/O/G gradually increased. While the porosity, encapsulation efficiency and in vitro degradation rate kept decreasing. W/O/G exhibited thixotropy in rheological tests and withstood a maximum compressive force of 1870 mN at a strain of 40%. Additionally, CMCS confered a certain pH response capability to W/O/G, which both maintained the gel network during pH changes and enabled the slow-release of the encapsulated bioactive in simulated in vitro digestion and improves bioaccessibility up to 80.63%. This study provided a theoretical basis for the cross-linking of modified LP and CMCS to form interpenetrating networks and enriched the gelation treatment of W/O/W emulsions.

Role of oil polarity on myofibrillar protein emulsions stability: A multi-scale research

The oil polarity was hypothesized to significantly influence the microscopic conformation of interfacial myofibrillar protein (MP) and thus the mesoscopic interfacial rheology of MP film, which ultimately tuned the macroscopic stability of MP-stabilized emulsions. In this research, the inner mechanism was revealed hierarchically based on emulsions multi-scale properties, by a combination of in vitro interfacial concentration measurement, dynamic interfacial tension measurement and interfacial dilatational rheology (highlighting Lissajous plots). Then, the above conclusion was verified through in silico molecular dynamic simulation. At macroscopic level, the light scattering results showed that emulsion prepared with higher polarity oil exhibited higher physical stability. This could be explained from the mesoscopic perspective that: (1) the adsorption dynamics of MP was faster (though lower diffusion rate, but higher penetration and rearrangement rate) on the higher polarity interface, which decreased interfacial tension more efficiently; (2) adsorption ratio, surface load and desorption ratio indicated that ‘multilayer interfacial film’ was formed by loosely-bound MP on the lower polarity interface, which decreased the effective interfacial coverage to impair emulsions stability; (3) Lissajous plots showed that interfacial structure with higher stability against large deformation was formed on the higher polarity oil interface. Finally, the microscopic molecular mechanism for explaining the above findings was revealed by in silico molecular dynamic simulation: though MP was less unfolded on the higher polarity interface leading to weaker intermolecular hydrophobic interactions, this compact molecular structure with stronger intramolecular rigidity predominantly contributed to steadier interfacial film structure. This multi-scale study provides a deep insight into the inner relationship between oil polarity and emulsions stability.

Crystal structure of rice APIP6 reveals a new dimerization mode of RING-type E3 ligases that facilities the construction of its working model

Ubiquitination is an important modification process in eukaryotic organisms, and ubiquitin ligase (E3) is the most diversified component of this system. APIP6 (AvrPiz-t interacting protein 6) is one of the E3s of rice, and is involved in the recognition of AvrPiz-t, one effector from the pathogen Magnaporthe oryzae, for the initiation of host defense against M. oryzae. However, the structural detail of how APIP6 performs its function remains elusive. Here, we present crystal structure of the RING domain (i.e., the E2-interaction domain) of APIP6 (APIP6-RING). APIP6-RING exists as a homodimer in crystal packing, in solution and in vivo. APIP6-RING consists of one β hairpin and one α helix, and β hairpins of two APIP6-RING molecules interact with each other in a novel ‘shoulder-to-shoulder’ mode to form a β sheet, and also rendering APIP6-RING to form a homodimer. Hydrogen bonds play a major role in the dimer formation of APIP6-RING, while hydrophobic-intermolecular interactions are inconspicuous. Due to the interaction mode between RING-type E3 and E2 is generally conserved, we constructed and verified a model of APIP6-RING/E2 complex, and proposed a working model of APIP6 with E2, ubiquitin, and the substrate AvrPiz-t. Taken together, our research presents the first structure of plant simple RING-type E3 ligase that exists in an unreported dimerization manner, as well as a working model of APIP6.

Effect of salinity on solution properties of a partially hydrolyzed polyacrylamide

The growing energy demand requires maximization of oil recovery from known reserves, while enhanced oil recovery (EOR) techniques are focused on sweeping residuals available in reservoirs previously flooded with water. Polymer additives are widely applied in EOR processes, as they can increase the viscosity and reduce the water permeability leading to mobility decrease of the injected solution. In this study, structural and rheological characterizations of a partially hydrolyzed polyacrylamide (FLOPAAM 3630S) were performed in a wide range of salinity relevant to conditions in existing oil reservoirs. Macromolecular solutions were identified at low polymer concentrations and/or at elevated salt levels, while gelation, i.e., formation of coherent structure, was observed at high FLOPAAM 3630S doses at low salinity. The transition points between these stages were determined and a comparison of scattering and rheology measurements revealed that the driving force of such a transformation is of non-electrostatic origin, but rather relies on the hydrophobic intermolecular interactions. The results bring new insights into the design of efficient EOR processes applied in oil reservoirs of different salt levels.

Fabrication of soy protein isolate - High methoxyl pectin composite emulsions for improving the stability and bioavailability of carotenoids

A handy and efficient emulsifying emulsion was developed based on self-assembled soy protein isolate (SPI) and high methoxy pectin (HMP) for entrapping and loading carotenoids. The complex, ca. 195 nm, showed a significant improvement in solubility, emulsification (32.42 m2/g) and stability (83.86%). Moreover, the physical stability of torularhodin was remarkably improved after entrapping into the SPI-HMP complex. The retention rate for entrapped torularhodin could be up to 66.03%, 95.14% and 84.31% under treatment with ultraviolet irradiation for 8 h, thermal stock at 25 and 50 °C for 28 days, respectively. Increase of two affinities, enhancement of intermolecular hydrogen bond and hydrophobic interaction and charge changes caused by the preparation process of the complex were the main reasons for improving the properties of emulsion. At the same time, composite lotion encapsulation significantly improves the bioavailability of torularhodin, including the absorption of carotenoids in the small intestine, the content in the systemic circulation and the storage capacity in the liver. Therefore, the preparation of low price, safer, green and efficient SPI-HMP complex emulsion has long-term application prospects on the development, transportation and application of antioxidants.

Single-Molecular Poly(propylene oxide) (PPO) Nucleus-Guided Assembly for Hydrophobicity-Dependent Molecular Transport in the Nanopore.

By combining DNA nanotechnology and solid-phase nanopore technology, the aggregation behavior of polymer guided by a single-molecular poly(propylene) (PPO) nucleus in a 3D DNA network has been studied. At low temperature, the PPO chain is evenly dispersed in the rigid 3D DNA network; at higher temperature, the PPO chain self-collapses to a single-molecular nucleus; and upon addition of amphiphilic block copolymers below the critical micelle concentration (CMC), the chains tend to aggregate on the isolated hydrophobic nucleus through intermolecular hydrophobic interactions. The process has been characterized by a rheological test and an electrochemical test. This study not only provides a preliminary understanding of the nucleation and growth process of block copolymers but also offers a theoretical basis for the study of protein self-folding and aggregation in the future. On this basis, utilizing this nucleation and growth event, a novel smart nanopore has been developed for hydrophobicity-dependent molecular transport.

Baking using oleogels

Baking using oleogels

Self‐Delivery O2‐Ecomonizer to Reverse Antiapoptosis of Hypoxic Tumor for Enhanced Photodynamic Therapy

AbstractOxygen‐dependent photodynamic therapy (PDT) holds unique advantages on tumor treatment by inducing cell apoptosis, but it is often limited owing to the unfavorable tumor microenvironments. In this study, a self‐delivery O2‐ecomonizer (designated as CeTam) is prepared to relieve hypoxia and reverse antiapoptotic pathway for enhanced photodynamic tumor therapy. Based on Chlorin e6 (Ce6) and Tamoxifen (Tam), CeTam is assembled through intermolecular hydrophobic interaction without any vehicles. Nanosized CeTam has an enhanced stability and improved drug delivery efficiency, which prefers to accumulate at tumor tissue for effective cellular uptake. More importantly, CeTam can effectively inhibit mitochondrial respiration to relieve hypoxia and reduce Bcl‐2 expression to reverse antiapoptotic pathway. As a result, this self‐delivery O2‐ecomonizer successfully regulates the unfavorable tumor microenvironments to enhance the PDT efficacy on tumor elimination. Taken together, this work reports a multifunctional nanoplatform by a simple self‐assembly strategy to remedy the deficiencies of PDT on hypoxic tumor treatment, which can advance the development of individualized biomedicine for precise tumor therapy.

Structural Significance of Hydrophobic and Hydrogen Bonding Interaction for Nanoscale Hybridization of Antiseptic Miramistin Molecules with Molybdenum Disulfide Monolayers

This paper reports an easy route to immobilize the antiseptic drug miramistin (MR) molecules between the sheets of molybdenum disulfide, known for excellent photothermal properties. Two hybrid layered compounds (LCs) with regularly alternating monolayers of MR and MoS2, differing in thickness of organic layer are prepared and studied by powder X-ray diffraction (PXRD), X-ray photoelectron spectroscopy (XPS), density functional theory (DFT) calculations and quantum theory of atoms in molecules (QTAIM) topological analysis. The obtained structural models elucidate the noncovalent interaction network of MR molecules confined in the two-dimensional spacing surrounded by sulfide sheets. It emerged that the characteristic folded geometry of MR molecule previously evidenced for pure miramistin is preserved in the hybrid structures. Quantification of the energetics of bonding interactions unveils that the most important contribution to structure stabilization of both compounds is provided by the weak but numerous CH…S bonding contacts. They are accompanied by the intra- and inter-molecular interactions within the MR layers, with dominating bonding effect of intermolecular hydrophobic interaction. The results obtained in the models provide a comprehensive understanding of the driving forces controlling the assembly of MR and MoS2 and may lead towards the development of novel promising MoS2-based photothermal therapeutic agents.

Hybrid Hydrogels for Neomycin Delivery: Synergistic Effects of Natural/Synthetic Polymers and Proteins.

This paper reports new physical hydrogels obtained by the freezing/thawing method. They include pullulan (PULL) and poly(vinyl alcohol) (PVA) as polymers, bovine serum albumin (BSA) as protein, and a tripeptide, reduced glutathione (GSH). In addition, a sample containing PULL/PVA and lysozyme was obtained in similar conditions. SEM analysis evidenced the formation of networks with porous structure. The average pore size was found to be between 15.7 μm and 24.5 μm. All samples exhibited viscoelastic behavior typical to networks, the hydrogel strength being influenced by the protein content. Infrared spectroscopy analysis revealed the presence of intermolecular hydrogen bonds and hydrophobic interactions (more pronounced for BSA content between 30% and 70%). The swelling kinetics investigated in buffer solution (pH = 7.4) at 37 °C evidenced a quasi-Fickian diffusion for all samples. The hydrogels were loaded with neomycin trisulfate salt hydrate (taken as a model drug), and the optimum formulations (samples containing 10-30% BSA or 2% lysozyme) proved a sustained drug release over 480 min in simulated physiological conditions. The experimental data were analyzed using different kinetic models in order to investigate the drug release mechanism. Among them, the semi-empirical Korsmeyer-Peppas and Peppas-Sahlin models were suitable to describe in vitro drug release mechanism of neomycin sulfate from the investigated hybrid hydrogels. The structural, viscoelastic, and swelling properties of PULL/PVA/protein hybrid hydrogels are influenced by their composition and preparation conditions, and they represent important factors for in vitro drug release behavior.

Structure-Based Identification of Natural-Product-Derived Compounds with Potential to Inhibit HIV-1 Entry.

Broadly neutralizing antibodies (bNAbs) are potent in neutralizing a wide range of HIV strains. VRC01 is a CD4-binding-site (CD4-bs) class of bNAbs that binds to the conserved CD4-binding region of HIV-1 envelope (env) protein. Natural products that mimic VRC01 bNAbs by interacting with the conserved CD4-binding regions may serve as a new generation of HIV-1 entry inhibitors by being broadly reactive and potently neutralizing. This study aimed to identify compounds that mimic VRC01 by interacting with the CD4-bs of HIV-1 gp120 and thereby inhibiting viral entry into target cells. Libraries of purchasable natural products were virtually screened against clade A/E recombinant 93TH057 (PDB: 3NGB) and clade B (PDB ID: 3J70) HIV-1 env protein. Protein-ligand interaction profiling from molecular docking and dynamics simulations showed that the compounds had intermolecular hydrogen and hydrophobic interactions with conserved amino acid residues on the CD4-binding site of recombinant clade A/E and clade B HIV-1 gp120. Four potential lead compounds, NP-005114, NP-008297, NP-007422, and NP-007382, were used for cell-based antiviral infectivity inhibition assay using clade B (HXB2) env pseudotype virus (PV). The four compounds inhibited the entry of HIV HXB2 pseudotype viruses into target cells at 50% inhibitory concentrations (IC50) of 15.2 µM (9.7 µg/mL), 10.1 µM (7.5 µg/mL), 16.2 µM (12.7 µg/mL), and 21.6 µM (12.9 µg/mL), respectively. The interaction of these compounds with critical residues of the CD4-binding site of more than one clade of HIV gp120 and inhibition of HIV-1 entry into the target cell demonstrate the possibility of a new class of HIV entry inhibitors.

Rational design of an N-terminal cysteine-containing tetrapeptide that inhibits tyrosinase and evaluation of its mechanism of action

There has been a resurgence of interest in bioactive peptides as therapeutic agents. This is particularly interesting for tyrosinase, which can be inhibited by thiol-containing peptides. This work demonstrates that an N-terminal cysteine-containing tetrapeptide can be rationally designed to inhibit tyrosinase activity in vitro and in cells. The tetrapeptide cysteine (C), arginine (R), asparagine (N) and leucine (L) or CRNL is a potent inhibitor of tyrosinase activity with an IC50 value of 39.62 ± 6.21 μM, which is comparable to currently used tyrosinase inhibitors. Through structure-activity studies and computational modeling, we demonstrate the peptide interacts with the enzyme via electrostatic (R with E322), hydrogen bonding (N with N260) and hydrophobic (L with V248) intermolecular interactions and that a combination of these is required for potent activity. Moreover, copper chelating activity might be one of the mechanisms of tyrosinase inhibition by CRNL. Kinetic studies show that tetrapeptide is a competitive inhibitor with two-step irreversible inhibition. In addition, CRNL had no toxicity and could reduce melanin levels in the murine melanoma cell line (B16F1). Overall, CRNL is a very promising candidate for hyperpigmentation treatment.

Crystal structure analysis of pyrrolidone carboxyl peptidase from Thermus thermophilus

Pyrrolidone carboxyl peptidase (PCP) hydrolytically removes the L-pyroglutamic acid from the amino terminal region of pyroglutamyl proteins or peptides. So far, only a limited number of structures of PCP have been solved. Here we report the crystal structure of pyrrolidone carboxyl peptidase from Thermus thermophilus (TtPCP) which has been solved using the molecular replacement method and refined at 1.9 Å resolution. TtPCP follows the α/β/α architecture in which the central β-sheets are surrounded by α-helices on both sides. The inter subunit contact between two monomers consists of two short antiparallel β-strands and part of a long protrusion loop. By comparing the TtPCP with its structural homologs, we identified the putative catalytic triad residues as Glu76, Cys139 and His160. A unique disulfide link found in some homologs of TtPCP, formed between two monomers that provide thermal stability to the protein, is not observed in TtPCP. Hence, being a thermophilic protein, the putative thermal stability of TtPCP could be due to more intra and inter-molecular hydrogen bonds, hydrophobic and ion pair interactions when compared with its mesophilic counterpart. The structural details of TtPCP will be helpful to understand the basis of the intrinsic stability of thermophilic proteins. Also, it could be useful for protein engineering.