Protein-substrate Interactions Research Articles

Computational investigation of cis-1,4-polyisoprene binding to the latex-clearing protein LcpK30.

Latex clearing proteins (Lcps) catalyze the oxidative cleavage of the C = C bonds in cis-1,4-polyisoprene (natural rubber), producing oligomeric compounds that can be repurposed to other materials. The active catalytic site of Lcps is buried inside the protein structure, thus raising the question of how the large hydrophobic rubber chains can access the catalytic center. To improve our understanding of hydrophobic polymeric substrate binding to Lcps and subsequent catalysis, we investigated the interaction of a substrate model containing ten carbon-carbon double bonds with the structurally characterized LcpK30, using multiple computational tools. Prediction of the putative tunnels and cavities in the LcpK30 structure, using CAVER-Pymol plugin 3.0.3, fpocket and Molecular Dynamic (MD) simulations provided valuable insights on how substrate enters from the surface to the buried active site. Two dominant tunnels were discovered that provided feasible routes for substrate binding, and the presence of two hydrophobic pockets was predicted near the heme cofactor. The larger of these pockets is likely to accommodate the substrate and to determine the size distribution of the oligomers. Protein-ligand docking was carried out using GOLD software to predict the conformations and interactions of the substrate within the protein active site. Deeper insight into the protein-substrate interactions, including close-contacts, binding energies and potential cleavage sites in the cis-1,4-polyisoprene, were obtained from MD simulations. Our findings provide further justification that the protein-substrate complexation in LcpK30 is mainly driven by the hydrophobic interactions accompanied by mutual conformational changes of both molecules. Two potential binding modes were identified, with the substrate in either extended or folded conformations. Whilst binding in the extended conformation was most favorable, the folded conformation suggested a preference for cleavage of a central double bond, leading to a preference for oligomers with 5 to 6 C = C bonds. The results provide insight into further enzyme engineering studies to improve catalytic activity and diversify the substrate and product scope of Lcps.

Effect of quercetin on the protein-substrate interactions in SIRT6: Insight from MD simulations

SIRT6 is of interest for its promising effect in the treatment of aging-related diseases. Studies have shown quercetin (QUE) and its derivatives have varying degrees of effect on the catalytic effect of SIRT6. In the research, the effect of QUE on the protein-substrate interaction in the SIRT6-mediated mono-ADP ribosylation system was investigated by conventional molecular dynamics (MD) simulations combined with MM/PBSA binding free energy calculations. The results show that QUE can bind stably to SIRT6 with the binding energy of −22.8 kcal/mol and further affect the atomic interaction between SIRT6 and NAD+ (or H3K9), resulting in an increased affinity between SIRT6-NAD+ and decreased SIRT6-H3K9 binding capacity. At the same time, the binding of QUE can also alter some structural characteristics of the protein, with large shifts occurring in the residue regions involving the N-terminal (residues 1–27), Rossmann fold regions (residues 55–92), and ZBD (residues 164–179). Thus, QUE shows great potential as a scaffold for the design of novel potent SIRT6 modulators.

In silico anticancer activity of isoxazolidine and isoxazolines derivatives: DFT study, ADMET prediction, and molecular docking

This study presents a comprehensive analysis of six isoxazolidine and isoxazoline derivatives, employing a multifaceted approach that integrates Density Functional Theory (DFT), AdmetSAR analysis, and molecular docking simulations to explore their electronic, pharmacokinetic, and anticancer properties. Utilizing DFT analysis with the B3LYP-D3BJ functional and the 6-311++G(d,p) basis set, molecular geometries were optimized, and vibrational frequencies in the IR spectrum were evaluated, offering insights into the molecular structure and stability of the pharmaceutical compounds. Electrostatic potential maps were analyzed to predict functional group reactivity and protein-substrate interactions. Frontier Molecular Orbital (FMO) analysis and Density of States (DOS) plots revealed varying stability levels among the compounds, with 1b, 2b, and 3b exhibiting slightly higher stability. Chemical potential and hardness analyses highlighted stronger binding affinity for compounds 1b and 2b, suggesting stronger potential interactions. AdmetSAR analysis predicted favorable human intestinal absorption (HIA) rates for all compounds, with compound 3b showing superior oral effectiveness. Molecular docking and dynamics simulations were conducted targeting the receptor (PDB: 1JU6). Molecular docking simulations confirmed the high affinity of these compounds towards the target protein 1JU6, particularly compound 3b, which exhibited the most favorable binding energy of -8.50 kcal/mol. Molecular dynamics simulations demonstrated the superior stability of ligand 3b over 1b and 5-FU over 100 ns, suggesting its potential for further study. The 3b-protein complex exhibited stability through hydrophobic and hydrogen bond interactions, with 3b demonstrating reduced solvent exposure compared to 1b and 5-FU. This study underscores the promising role of compound 3b in anticancer treatments, providing a solid foundation for future drug development and optimization efforts.

Loop Evolutionary Patterns Shape Catalytic Efficiency of TRI101/201 for Trichothecenes: Insights into Protein-Substrate Interactions.

Trichothecenes are highly toxic mycotoxins produced by Fusarium fungi, while TRI101/201 family enzymes play a crucial role in detoxification through acetylation. Studies on the substrate specificity and catalytic kinetics of TRI101/201 have revealed distinct kinetic characteristics, with significant differences observed in catalytic efficiency toward deoxynivalenol, while the catalytic efficiency for T-2 toxin remains relatively consistent. In this study, we used structural bioinformatics analysis and a molecular dynamics simulation workflow to investigate the mechanism underlying the differential catalytic activity of TRI101/201. The findings revealed that the binding stability between trichothecenes and TRI101/201 hinges primarily on a hydrophobic cage structure within the binding site. An intrinsic disordered loop, termed loop cover, defined the evolutionary patterns of the TRI101/201 protein family that are categorized into four subfamilies (V1/V2/V3/M). Furthermore, the unique loop displayed different conformations among these subfamilies' structures, which served to disrupt (V1/V2/V3) or reinforce (M) the hydrophobic cages. The disrupted cages enhanced the water exposure of the hydrophilic moieties of substrates like deoxynivalenol and thereby hindered their binding to the catalytic sites of V-type enzymes. In contrast, this water exposure does not affect substrates like T-2 toxin, which have more hydrophobic substituents, resulting in a comparable catalytic efficiency of both V- and M-type enzymes. Overall, our studies provide theoretical support for understanding the catalytic mechanism of TRI101/201, which shows how an intrinsic disordered loop could impact the protein-ligand binding and suggests a direction for rational protein design in the future.

Deciphering the Allosteric Activation Mechanism of SIRT6 Using Molecular Dynamics Simulations.

As a member of the histone deacetylase protein family, the NAD+-dependent SIRT6 plays an important role in maintaining genomic stability and regulating cell metabolism. Interestingly, SIRT6 has been found to have a preference for hydrolyzing long-chain fatty acyls relative to deacetylation, and it can be activated by fatty acids. However, the mechanisms by which SIRT6 recognizes different substrates and can be activated by small molecular activators are still not well understood. In this study, we carried out extensive molecular dynamic simulations to shed light on these mechanisms. Our results revealed that the binding of the myristoylated substrate stabilizes the catalytically favorable conformation of NAD+, while the binding of the acetyl-lysine substrate leads to a loose binding of NAD+ in SIRT6. Based on these observations, we proposed a reasonable allosteric binding mode for myristic acid, which can enhance the catalytic activity of SIRT6 by stabilizing the binding of NAD+ with His131 as well as the acetylated substrate. Furthermore, our molecular dynamics simulations demonstrated that synthetic SIRT6 activators, such as UBCS039, MDL-801, and 12q, block the flipping of ribose in NAD+ and therefore can stabilize substrate-NAD+-His131 interactions in a manner similar to fatty acids. In summary, our newly proposed activation mechanism of SIRT6 highlights the importance of protein-substrate interactions, which would facilitate the rational design of new SIRT6 activators.

Synthesis, Characterization, Antimicrobial, Density Functional Theory, and Molecular Docking Studies of Novel Mn(II), Fe(III), and Cr(III) Complexes Incorporating 4-(2-Hydroxyphenyl azo)-1-naphthol (Az).

This work synthesized three new CrAz2, MnAz2, and FeAz2 complexes and investigated them using IR, mass, UV spectroscopy, elemental analysis, conductivity and magnetic tests, and thermogravimetric analysis. The azo-ligand, 4-(2-hydroxyphenylAzo)-1-naphthol (Az), couples with metal ions via its nitrogen (in -N=N- bonds) and oxygen (in hydroxyl group) atoms, according to the IR spectra of these complexes. Through thermal examination (TG/TGA), the number and location of water in the complexes were also determined. Density functional theory (DFT) theory is applied to ameliorate the structures of the ligand (Az) and metal complexes and analyze the quantum chemical characteristics of these complexes. The antifungal and antibacterial activity of the ligand and its complexes opposed to several hazardous bacteria and fungi was investigated in vitro. Metal complexes were discovered to have a higher inhibitory impact on some organisms than the free ligand. The MnAz2 complex exhibited the best activity among the studied materials, whereas the CrAz2 complex had the lowest. The compounds' binding affinity to the E. coli (PDB ID: 1hnj) structure was predicted using molecular docking. Binding energies were calculated by analyzing protein-substrate interactions. These encouraging findings imply that these chemicals may have physiological effects and may be valuable for a variety of medical uses in the future.

Cellular Response to Bone Morphogenetic Proteins-2 and -7 Covalently Bound to Photocrosslinked Heparin-Diazoresin Multilayer.

Despite the plethora of research that exists on recombinant human bone morphogenetic protein-2 and -7 (rhBMP-2 and rhBMP-7) and has been clinically approved, there is still a need to gain information that would allow for their more rational use in bone implantology. The clinical application of supra-physiological dosages of these superactive molecules causes many serious adverse effects. At the cellular level, they play a role in osteogenesis and cellular adhesion, migration, and proliferation around the implant. Therefore, in this work, we investigated the role of the covalent binding of rhBMP-2 and rhBMP-7 separately and in combination with ultrathin multilayers composed of heparin and diazoresin in stem cells. In the first step, we optimized the protein deposition conditions via quartz crystal microbalance (QCM). Then, atomic force microscopy (AFM) and enzyme-linked immunosorbent assay (ELISA) were used to analyze protein-substrate interactions. The effect of the protein binding on the initial cell adhesion, migration, and short-term expression of osteogenesis markers was tested. In the presence of both proteins, cell flattening and adhesion became more prominent, resulting in limited motility. However, the early osteogenic marker expression significantly increased compared to the single protein systems. The presence of single proteins resulted in the elongation of cells, which promoted their migration activity.

Finding inhibitors and new activators for the biliverdin transporter ABCB10.

The absence of the mitochondrial ATP binding cassette (ABC)-transporter ABCB10 in mice is embryonic lethal, with fetuses presenting severe anemia and oxidative damage. Initially, ABCB10 was thought to be a heme transporter, but recent evidence from our group and collaborators suggests instead that ABCB10 transports biliverdin, a heme degradation product with antioxidant properties. Since some ABC transporters are promiscuous, interacting and/or transporting a large variety of drugs, it is important to determine the specificity of ABCB10 for its substrate. Commonly, ABC transporters present a basal ATPase activity that is increased by substrate binding. As expected, the substrate biliverdin significantly increases the basal ATPase activity of the transporter. Now, we are studying the effect of related compounds on the ATPase activity of purified ABCB10 reconstituted in lipid nanodiscs. We have found inhibition with pyrroles closely related to biliverdin, suggesting that specific substrate-protein interactions are required for the biliverdin-induced activation. We have also found additional compounds (pyrroles and porphyrins) that stimulate the ATPase activity of the transporter, although at a lower level than biliverdin does. Competition assays are underway to determine if these compounds bind to a single site or if there is evidence of the coexistence of multiple drug binding sites on the transporter. Finding compounds that can modulate or inhibit the activity of this essential transporter brings additional tools for needed biochemical and structural studies and opens the door for possible therapeutic purposes. Funding: 1R01GM145938-01 to MEZ.

Hydrolysis Mechanism of Carbamate Methomyl by a Novel Esterase PestE: A QM/MM Approach.

Methomyl is one of the most important carbamates that has caused potential hazardous effects on both human beings and the environment. Here, we systematically investigated the hydrolysis mechanism of methomyl catalyzed by esterase PestE using molecular dynamics simulations (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. The hydrolysis mechanism involves two elementary steps: (Ⅰ) serine-initiated nucleophilic attack and (Ⅱ) C-O bond cleavage. Our work elicits the atomic level details of the hydrolysis mechanism and free energy profiles along the reaction pathway. The Boltzmann-weighted average potential barriers are 19.1 kcal/mol and 7.5 kcal/mol for steps Ⅰ and Ⅱ, respectively. We identified serine-initiated nucleophilic attack as the rate determining-step. The deep learning-based kcat prediction model indicated that the barrier of the rate-determining step is 15.4 kcal/mol, which is in good agreement with the calculated results using Boltzmann-weighted average method. We have elucidated the importance of the protein-substrate interactions and the roles of the key active site residues during the hydrolysis process through noncovalent interactions analysis and electrostatic potential (ESP) analysis. The results provide practical value for achieving efficient degradation of carbamates by hydrolases.

Synthesis, DFT, Biological and Molecular Docking Analysis of Novel Manganese(II), Iron(III), Cobalt(II), Nickel(II), and Copper(II) Chelate Complexes Ligated by 1-(4-Nitrophenylazo)-2-naphthol

Novelmanganese(II), iron(III), cobalt(II), nickel(II), and copper(II) chelates were synthesized and studied using elemental analysis (EA), infrared spectroscopy, mass spectrometry, ultraviolet-visible spectroscopy, and conductivity, as well as magnetic measurements and thermogravimetric analysis (TG). The azo-ligand 1-[(4-nitrophenyl)diazenyl]-2-naphthol (HL) chelates to the metal ions via the nitrogen and oxygen centers of the azo group and the hydroxyl, respectively. The amounts of H2O present and its precise position were identified by thermal analysis. Density functional theory (DFT) was employed to theoretically elucidate the molecular structures of the ligand and the metal complexes. Furthermore, the quantum chemical parameters were also evaluated. The antimicrobial properties were evaluated against a group of fungal and bacterial microbes. Interestingly, the bioactivity of the complexes is enhanced compared to free ligands. Within this context, the CuL complex manifested the lowest activity, whereas the FeL complex had the greatest. Molecular docking was used to foretell the drugs' binding affinity for the structure of Escherichia coli (PDB ID: 1hnj). Protein-substrate interactions were resolved, and binding energies were accordingly calculated.

Synthesis, structural, DFT, antibacterial, antifungal, anti-inflammatory, and molecular docking analysis of new VO(II), Fe(III), Mn(II), Zn(II), and Ag(I) complexes based on 4-((2-hydroxy-1-naphthyl)azo) benzenesulfonamide

In this work, we synthesized and studied novel VO(II), Mn(II), Fe(III), Zn(II), and Ag(I) − 4-((2-hydroxy-1-naphthyl)azo) benzenesulfonamide complexes. Characterization techniques such as thermogravimetry (T.G.A), magnetic susceptibility testing, electrical conductivity studies, infrared spectroscopy, and mass spectroscopy all played crucial roles. Metal ions are coordinated by the azo Nitrogen (NN) and the hydroxyl Oxygen (–OH), as seen by the infrared spectra (IR). Our investigation of water's distribution and thermal behavior, in addition to its overall number, was made possible by techniques from thermogravimetry (T.G.A). The theoretical optimization of ligand (HL) and its metal complexes’ molecular structures was accomplished by the use of density functional theory (DFT). Other quantum chemical properties might potentially be derived by using the Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) energy levels. In vitro testing was performed on both the ligand and its complexes to see whether or not they were capable of inhibiting the growth of harmful bacteria and fungi. The metal complexes have enhanced biological activity against the target microorganisms compared to the unbound ligand. In vitro research was also conducted on the anti-inflammatory effects of the aforementioned substances. Surprisingly, the AgL complex was the least active of the compounds tested, while the FeL complex was the most active. Based on molecular docking studies, we hypothesized that the synthesized compounds would bind to the cyclooxygenase-2 (COX-2) enzyme (Protein Data Base (PDB) ID: 5IKT) and the E. coli DNA gyrase B receptor (PDB ID: 1KNZ). Binding energies were calculated after studying protein-substrate interactions in depth. The molecular docking indications are that these compounds have physiological effects and might have broad use in the pharmaceutical industry.

Atypical Substrates of the Organic Cation Transporter 1.

The human organic cation transporter 1 (OCT1) is expressed in the liver and mediates hepatocellular uptake of organic cations. However, some studies have indicated that OCT1 could transport neutral or even anionic substrates. This capability is interesting concerning protein-substrate interactions and the clinical relevance of OCT1. To better understand the transport of neutral, anionic, or zwitterionic substrates, we used HEK293 cells overexpressing wild-type OCT1 and a variant in which we changed the putative substrate binding site (aspartate474) to a neutral amino acid. The uncharged drugs trimethoprim, lamivudine, and emtricitabine were good substrates of hOCT1. However, the uncharged drugs zalcitabine and lamotrigine, and the anionic levofloxacin, and prostaglandins E2 and F2α, were transported with lower activity. Finally, we could detect only extremely weak transport rates of acyclovir, ganciclovir, and stachydrine. Deleting aspartate474 had a similar transport-lowering effect on anionic substrates as on cationic substrates, indicating that aspartate474 might be relevant for intra-protein, rather than substrate-protein, interactions. Cellular uptake of the atypical substrates by the naturally occurring frequent variants OCT1*2 (methionine420del) and OCT1*3 (arginine61cysteine) was similarly reduced, as it is known for typical organic cations. Thus, to comprehensively understand the substrate spectrum and transport mechanisms of OCT1, one should also look at organic anions.

The Effect of the Topmost Layer and the Type of Bone Morphogenetic Protein-2 Immobilization on the Mesenchymal Stem Cell Response.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) plays a key role in the stem cell response, not only via its influence on osteogenesis, but also on cellular adhesion, migration, and proliferation. However, when applied clinically, its supra-physiological levels cause many adverse effects. Therefore, there is a need to concomitantly retain the biological activity of BMP-2 and reduce its doses. Currently, the most promising strategies involve site-specific and site-directed immobilization of rhBMP-2. This work investigated the covalent and electrostatic binding of rhBMP-2 to ultrathin-multilayers with chondroitin sulfate (CS) or diazoresin (DR) as the topmost layer. Angle-resolved X-ray photoelectron spectroscopy was used to study the exposed chemical groups. The rhBMP-2 binding efficiency and protein state were studied with time-of-flight secondary ion mass spectrometry. Quartz crystal microbalance, atomic force microscopy, and enzyme-linked immunosorbent assay were used to analyze protein–substrate interactions. The effect of the topmost layer was tested on initial cell adhesion and short-term osteogenesis marker expression. The results show the highest expression of selected osteomarkers in cells cultured on the DR-ended layer, while the cellular flattening was rather poor compared to the CS-ended system. rhBMP-2 adhesion was observed only on negatively charged layers. Cell flattening became more prominent in the presence of the protein, even though the osteogenic gene expression decreased.

In silico Analysis of Renilla Muelleri, Photinus Pyralis and Metridia Longa Luciferase

Luciferase derived from different organism is widely used for various biological process in the cells or tissues. Current in silico study was done to investigate the protein-substrate interactions of Renilla muelleri, Metridia longa and Photinus pyralis luciferase with their respective substrates coelenterazine, Luciferin, and ATP. RaptorX which is an online tool was used for luciferase modeling. PyRx v9.0 was used for protein-substrate binding. Online server Cluspro was used for protein-protein docking. CASTp was used for protein active site pocket prediction. Photinus pyralis luciferase was bonded with ATP molecules through Glu83, Asp153, Asp44, Ser85, Gln87 and His171, while Photinus Pyralis luciferase was bonded with luciferin molecules through five different residues i.e. His171, Arg62, Met90, Leu63 and Val168. Photinus pyralis residues that were docked with ATP and luciferin molecules were present in N terminal domain of Photinus pyralis luciferase. In case of Renilla muelleri, catalytic residues, His285 was present in its all the docking complex. Renilla muelleri and Metridia longa luciferase were also docked with different substrate and found that efficiency of Renilla muelleri and Metridia longa luciferase was lower towards Photinus pyralis substrates as compared to their own substrate coelentereazine. According to the findings, it has been concluded that luciferase of every light emitting organism required specific (its own) substrate for proper light reaction. Keywords: Renilla muelleri; Metridia longa; Photinus pyralis; luciferase

Seven metal-based bi-dentate NO azocoumarine complexes: Synthesis, physicochemical properties, DFT calculations, drug-likeness, in vitro antimicrobial screening and molecular docking analysis

Herein, new Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) complexes based on azocoumarin ligand, 2-[(E)-(2-oxo-2H-chromen-6-yl)diazenyl]-1-phenylbutane-1,3-dione (L), were synthesized and characterized via elemental analyses, IR, mass, UV–Vis spectra, magnetic susceptibility, conductivity measurements, and thermogravimetric analysis (TG). From IR spectra, it was elucidated that the azocoumarin ligand behaves as a neutral bi-dentate ligand and coordinates to the metal ions through the azo Nitrogen and carbonyl Oxygen. Number and position of the water molecules were investigated through thermal analysis. The molecular structures of the ligand (L) and its metal complexes were optimized theoretically using density functional theory (DFT) for various possible orientations of the H2O and Cl moieties around the metal-center, to find out the most reliable coordination modes. Moreover, the quantum chemical parameters were evaluated.The bioactivity of the ligand and its complexes were screened for their in vitro anti-bacterial and anti-fungal activity against series of pathogenic bacterial and fungal strains. The metal complexes possess high biological activity with low minimum inhibition concentration (MIC) against different organisms. In order to ascertain the bio-effect of the present compounds, their MIC was compared with previously reported MIC showing lower MIC values. Molecular docking was used to predict the binding efficiency between the prepared compounds and the receptor of E. coli(1HNJ), the target enzyme for the antimicrobial reagents. The protein-substrate interactions were investigated and the binding energies were calculated. The drug-likeness and ADMET prediction showed compliance with the Lipinski rule, and the compounds were found to have good absorption, distribution, metabolism, and excretion generally. The results were very encouraging, so we can say that these products are biologically active and can be used later in other applications towards drug development.

Substrate Protein Interactions and Methylglyoxal Modifications Reduce the Aggregation Propensity of Human Alpha-A-Crystallin G98R Mutant.

The G98R mutation in αA-crystallin is associated with presenile cataract development in humans. Previous studies have indicated that mutant proteins altered structure, decreased stability, increased oligomeric size, loss of chaperone-like activity, and susceptibility to proteolysis could be contributing factors to cataract formation. To evaluate the effect of substrate protein interactions with the mutant protein on cataract formation, we have performed chaperone assays with alcohol dehydrogenase (ADH), citrate synthase (CS), and βB2-crystallin (βB2), and analyzed the reaction mixtures by multi-angle light scattering (MALS) analysis. It appears that αAG98R protein initially gets stabilized upon interaction with substrate proteins. Analysis of the chaperone-client protein complexes revealed that wild-type αA-crystallin interacts with substrate proteins to form compact complexes leading to a slight increase in oligomeric mass, whereas αAG98R forms less compact and high molecular weight complexes with the substrate, and the resulting complexes continue to increase in size over time. As a result, the soluble complexes formed initially by the mutant protein begin to scatter light and precipitate. We found that the stability and chaperone activity of the αAG98R can be improved by modifying the protein with low concentrations (50 µM) of methylglyoxal (MGO). Incubation of αAG98R protein (1 mg/ml) under aseptic conditions for 30 days at 37°C resulted in precipitation of the mutant protein. In contrast, mutant protein incubations carried out with 50 µM MGO remained soluble and transparent. SDS-PAGE analysis showed gradual autolysis of the mutant protein in the absence of MGO. The average molar mass of the mutant protein oligomers changed from 7,258 ± 12 kDa to 3,950 ± 08 kDa within 60 min of incubation with MGO. There was no further significant change in the molar mass of mutant protein when tested on day 7 of MGO treatment. Our data suggest that the initial stabilization of αAG98R by substrate proteins could delay congenital cataracts’ appearance, and the uncontrolled long-term interaction amongst mutant subunits and substrate proteins could be the rationale behind presenile cataracts formation. The results also demonstrate the potential benefit of low concentrations of MGO in stabilizing mutant chaperone protein(s).

Engineering a heterologously expressed fructosyltransferase from Aspergillus oryzae N74 in Komagataella phaffii (Pichia pastoris) for kestose production

Fructo-oligosaccharides (FOS) are one of the most well-studied and commercialized prebiotics. FOS can be obtained either by controlled hydrolysis of inulin or by sucrose transfructosylation. FOS produced from sucrose are typically classified as short-chain FOS (scFOS), of which the best known are 1-kestotriose (GF2), 1,1-kestotetraose (GF3), and 1,1,1-kestopentaose (GF4), produced by fructosyltransferases (FTases) or β-fructofuranosidases. In previous work, FOS production was studied using the Aspergillus oryzae N74 strain, its ftase gene was heterologously expressed in Komagataella phaffii (Pichia pastoris), and the enzyme’s tertiary structure modeled. More recently, residues that may be involved in protein–substrate interactions were predicted. In this study, the aim was to experimentally validate previous in silico results by independently producing recombinant wild-type A. oryzae N74 FTase and three single-point mutations in Komagataella phaffii (Pichia pastoris). The R163A mutation virtually abolished the transfructosylating activity, indicating a requirement for the positively charged arginine residue in the catalytic domain D. In contrast, transfructosylating activity was improved by introducing the mutations V242E or F254H, with V242E resulting in higher production of GF2 without affecting that of GF3. Interestingly, initial sucrose concentration, reaction temperature and the presence of metal cofactors did not affect the enhanced activity of mutant V242E. Overall, these results shed light on the mechanism of transfructosylation of the FTase from A. oryzae and expand considerations regarding the design of biotechnological processes for specific FOS production.

Time-resolved DEER EPR and solid-state NMR afford kinetic and structural elucidation of substrate binding to Ca2+-ligated calmodulin

Recent advances in rapid mixing and freeze quenching have opened the path for time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state NMR of protein-substrate interactions. DEER, in conjunction with phase memory time filtering to quantitatively extract species populations, permits monitoring time-dependent probability distance distributions between pairs of spin labels, while solid-state NMR provides quantitative residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein. Here, we demonstrate the power of these combined approaches to unravel the kinetic and structural pathways in the binding of the intrinsically disordered peptide substrate (M13) derived from myosin light-chain kinase to the universal eukaryotic calcium regulator, calmodulin. Global kinetic analysis of the data reveals coupled folding and binding of the peptide associated with large spatial rearrangements of the two domains of calmodulin. The initial binding events involve a bifurcating pathway in which the M13 peptide associates via either its N- or C-terminal regions with the C- or N-terminal domains, respectively, of calmodulin/4Ca2+ to yield two extended "encounter" complexes, states A and A*, without conformational ordering of M13. State A is immediately converted to the final compact complex, state C, on a timescale τ ≤ 600 μs. State A*, however, only reaches the final complex via a collapsed intermediate B (τ ∼ 1.5 to 2.5 ms), in which the peptide is only partially ordered and not all intermolecular contacts are formed. State B then undergoes a relatively slow (τ ∼ 7 to 18 ms) conformational rearrangement to state C.

Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking.

Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4-methylthiobutyl (4MTB) and indol-3-ylmethyl (I3M) glucosinolates to study how GTR1 from Arabidopsis thaliana transports these substrates in computational simulation approaches. The designed pipeline reported here includes massive docking of 4MTB and I3M in an ensemble of GTR1 conformations (in both inward and outward conformations) extracted from molecular dynamics simulations, followed by clustered and substrate–protein interactions profiling. The identified key residues were mutated, and their role in substrate transport was tested. We were able to identify key residues that integrate a major binding site of these substrates, which is critical for transport activity. In silico approaches employed here represent a breakthrough in the plant transportomics field, as the identification of key residues usually takes a long time if performed from a purely wet-lab experimental perspective. The inclusion of structural bioinformatics in the analyses of plant transporters significantly speeds up the knowledge-gaining process and optimizes valuable time and resources.

Label-free Surface Enhanced Raman Scattering (SERS) on Centrifugal Silver Plasmonic Paper (CSPP): A Novel Methodology for Unprocessed Biofluids Sampling and Analysis.

Label-free SERS is a powerful bio-analytical technique in which molecular fingerprinting is combined with localized surface plasmons (LSPs) on metal surfaces to achieve high sensitivity. Silver and gold colloids are among the most common nanostructured substrates used in SERS, but since protein-rich samples such as serum or plasma can hinder the SERS effect due to protein–substrate interactions, they often require a deproteinization step. Moreover, SERS methods based on metal colloids often suffer from a poor reproducibility. Here, we propose a paper-based SERS sampling method in which unprocessed human serum samples are first soaked on paper strips (0.4 × 2 cm2), and then mixed with colloidal silver nanoparticles by centrifugation to obtain a Centrifugal Silver Plasmonic Paper (CSPP). The CSPP methodology has the potential to become a promising tool in bioanalytical SERS applications: it uses common colloidal substrates but without the need for sample deproteinization, while having a good reproducibility both in terms of overall spectral shape (r > 0.96) and absolute intensity (RSD < 10%). Moreover, this methodology allows SERS analysis more than one month after serum collection on the paper strip, facilitating storage and handling of clinical samples (including shipping from clinical sites to labs).