Isothermal Titration Calorimetry Research Articles

Installation of an Indole on the BRCA1 Disordered Domain Using Triazine Chemistry

The functionalization of protein sidechains with highly water-soluble chlorotriazines (or derivatives thereof) using nucleophilic aromatic substitution reactions has been commonly employed to install various functional groups, including poly(ethylene glycol) tags or fluorogenic labels. Here, a poorly soluble dichlorotriazine with an appended indole is shown to react with a construct containing the disordered domain of BRCA1. Subsequently, this construct can undergo proteolytic cleavage to remove the SUMO-tag: the N-terminal poly(His) tag is still effective for purification. Steady-state fluorescence, circular dichroism spectroscopy, and isothermal titration calorimetry with the binding partner of BRCA1, PALB2, are used to characterize the indole-labeled BRCA1. Neither the reaction conditions nor the indole-tag appreciably alter the structure of the BRCA1. Mass spectrometry confirms that the target is modified once, although the location of modification cannot be determined by tandem mass spectrometry with collision-induced dissociation due to disadvantageous fragmentation patterns.

Experimental approaches to investigate biophysical interactions between homeodomain transcription factors and DNA.

Homeodomain transcription factors (TFs) bind to specific DNA sequences to regulate the expression of target genes. Structural work has provided insight into molecular identities and aided in unraveling structural features of these TFs. However, the detailed affinity and specificity by which these TFs bind to DNA sequences is still largely unknown. Qualitative methods, such as DNA footprinting, Electrophoretic Mobility Shift Assays (EMSAs), Systematic Evolution of Ligands by Exponential Enrichment (SELEX), Bacterial One Hybrid (B1H) systems, Surface Plasmon Resonance (SPR), and Protein Binding Microarrays (PBMs) have been widely used to investigate the biochemical characteristics of TF-DNA binding events. In addition to these qualitative methods, bioinformatic approaches have also assisted in TF binding site discovery. Here we discuss the advantages and limitations of these different approaches, as well as the benefits of utilizing more quantitative approaches, such as Mechanically Induced Trapping of Molecular Interactions (MITOMI), Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC), in determining the biophysical basis of binding specificity of TF-DNA complexes and improving upon existing computational approaches aimed at affinity predictions.

3D‐Printable Polymer Filters for the Selective Complexation of Silver Ions

ABSTRACTThis study presents the fabrication of polymeric filters, which are able to selectively bind silver ions from aqueous solution. Those filters are fabricated within an easy approach via 3D‐digital light processing (DLP)‐printing enabling a tailor‐made design. In first order, an acrylamide containing a benzo‐trithiacrown‐ether (BTCE) functionality is synthesized. This monomer is further 3D‐printed via layer by layer photo‐polymerization together with commercially available comonomers, resulting in polymer networks containing the BTCE‐groups in their side chains as selective binding moiety for silver ions. Within isothermal‐titration calorimetry (ITC) measurements, the complexation abilities of the BTCE to bind the Ag+ ions are determined. Furthermore, the resulting 3D‐printed filters are investigated regarding their complexation behavior, in a detailed fashion applying inductively coupled plasma optical emission spectroscopy (ICP‐OES).

PH‐Directed Capture‐SELEX for Nanomolar Affinity Aptamers for Kanamycin Detection

Kanamycin A is a widely used antibiotic, although it has a narrow therapeutic window demanding highly accurate monitoring. The sensing of kanamycin A using aptamers is of great interest since aptamers can be used for continuous monitoring with a rapid response. While kanamycin has been the target for at least four previous aptamer selections, the binding affinities of the reported DNA aptamers are still sub‐optimal. All the previous aptamer selections were performed at pH 7.5 or higher. Given that kanamycin A has four amino groups with pKa values close to 7, we herein selected DNA aptamers for kanamycin A at both pH 6 and pH 8. The selection at pH 6 enriched aptamers although the pH 8 selection library remained highly diverse in the end. The best aptamer named KAN6‐1 showed a dissociation constant of around 320 nM measured using isothermal titration calorimetry in the selection buffer. In buffers without salt, binding can happen from pH 6 to 8. Specific binding was confirmed using mutation studies. A strand displacement assay was developed with a limit of detection (LOD) of 100 nM in buffer. Similar LOD values were also obtained in lake water and in 10% human serum. Comparisons were also made with some previously reported DNA aptamers. This study shows the importance of pH value on the selection of aptamers and provides a new aptamer for kanamycin A detection.

Lapachol, a natural food component, interacts with human serum albumin: Insights of its impact on the pharmacokinetics of clinically used drugs

Lapachol (LAP), a natural 1,4-naphthoquinone used in popular medicine of South American, is an antioxidant and antimicrobial compound used as a food additive; however, its interactive profile with the main protein carrier of compounds in the human bloodstream (human serum albumin, HSA) was not still characterized. Additionally, the impact of LAP in binding clinically drugs to albumin is still unknown. Thus, the present work describes the interaction HSA:LAP using different biophysical techniques, i.e., 1H saturation-transfer difference nuclear magnetic resonance (1H STD-NMR), isothermal titration calorimetry (ITC), steady-state and time-resolved fluorescence measurements combined with molecular docking calculations. LAP interacts with subdomain region IIA (site I), mainly driven by enthalpy effects, while subdomain region IB (site III) was identified as the second binding site, mainly driven by entropy effects. The binding is spontaneous, strong (binding constant average, Kaverage ≈ 4.45 × 105 M−1), and there is a positive cooperativity in the presence of ibuprofen, with the LAP structure fully buried into the protein cavities. Overall, LAP might impact the residence time (pharmacokinetic profile) of drugs that bind to subdomains regions IIA and IB of albumin, e.g., warfarin, phenylbutazone, diflunisal, naproxen, camptothecin, doxorubicin, daunorubicin, suramin, and tyrosine kinase inhibitors.

Temperature dependent human serum albumin Corona formation: A case study on gold nanorods and nanospheres.

Protein molecules interact with nanoparticles to form a protein layer on the surface called the protein corona. Corona formation can be affected by the temperature and shape of the nanoparticles thereby impacting the fate of the nanoparticles inside physiological systems. We have investigated the human serum albumin (HSA) corona formation and its interactions with gold nanospheres and nanorods at different temperatures (18-42 °C). UV-Vis, fluorescence, isothermal titration calorimetry (ITC), x-ray photoelectron spectroscopy (XPS) and circular dichroism (CD) experiments have been performed to determine the changes due to the shape and temperature. UV-Vis spectra show a greater propensity of corona induced aggregation in the nanorods compared to the nanospheres. The Stern-Volmer plot indicates that static quenching is predominant in the HSA-AuNP interactions with higher quenching efficiency for nanorods than nanospheres. ITC analyses show that HSA-nanorods are involved in more favorable interactions compared to HSA-nanospheres. XPS studies indicate a more electron rich environment on the AuNRs surface and higher AuS interactions in AuNSs. CD spectra show higher secondary structural changes with temperature in case of HSA-AuNR interactions compared to HSA-AuNS interactions. The changes in the protein corona due to nanoparticle shape and variable temperature have been investigated through protein adsorption and composition, types of interactions and protein conformation.

The formation of protein coronas and the interaction of soy protein isolate and whey protein isolate with starch nanoparticles

The influence of food components on the physicochemical properties and biological responses of nanoparticles is pivotal. Since soy protein isolate (SPI) and whey protein isolate (WPI) are commonly used as plant and animal proteins in food, it is imperative to gain comprehensive knowledge of the interaction between SPI/WPI and nanoparticles. Accordingly, we examined the adsorption of SPI and WPI onto starch nanoparticles (SNPs) and their associated interactions. SNP aggregation was observed in the SPI/WPI, and coronas formed around the surfaces of the SNPs. The binding of the protein isolates reduced the zeta potential of the SNPs from −10.34 to −26.5 mV for the SPI and to −21.37 mV for the WPI. However, mixing the SNPs with the protein isolates did not alter the microenvironment of aromatic amino acids and had no substantial impact on the secondary structure of the proteins. Thermogravimetric analysis revealed that the formation of SPI/WPI coronas enhanced the thermal stability of the SNPs. Isothermal titration calorimetry indicated that the adsorption of the SPI and WPI onto the SNPs was driven primarily by weak van der Waals forces and hydrogen bonds. This study facilitates the predicting of organic nanoparticle behaviours in complex food matrix and human gastrointestinal environments.

PROTAC Beyond Cancer- Exploring the New Therapeutic Potential of Proteolysis Targeting Chimeras.

In the realm of oncology, the transformative impact of PROTAC (PROteolysis TAgeting Chimeras) technology has been particularly pronounced since its introduction in the 21st century. Initially conceived for cancer treatment, PROTACs have evolved beyond their primary scope, attracting increasing interest in addressing a diverse array of medical conditions. This expanded focus includes not only oncological disorders but also viral infections, bacterial ailments, immune dysregulation, neurodegenerative conditions, and metabolic disorders. This comprehensive review explores the broadening landscape of PROTAC application, highlighting ongoing developments and innovations aimed at deploying these molecules across a spectrum of diseases. Careful consideration of the design challenges associated with PROTACs reveals that, when appropriately addressed, these compounds present significant advantages over traditional therapeutic approaches, positioning them as promising alternatives. To evaluate the efficacy of PROTAC molecules, a diverse array of assays is employed, ranging from High-Throughput Imaging (HTI) assays to Cell Painting assays, CRBN engagement assays, Fluorescence Polarization assays, amplified luminescent proximity homogeneous assays, Timeresolved fluorescence energy transfer assays, and Isothermal Titration Calorimetry assays. These assessments collectively contribute to a nuanced understanding of PROTAC performance. Looking ahead, the trajectory of PROTAC technology suggests its potential recognition as a versatile therapeutic strategy for an expansive range of medical conditions. Ongoing progress in this field sets the stage for PROTACs to emerge as valuable tools in the multifaceted landscape of medical treatments.

The simultaneous application of fulvic acid and protein hydrolysate biostimulants enhances cucumber responses to Fe deficiency

Iron (Fe) is widely recognized as a critical factor in limiting crop production; however, eco-friendly strategies to address its deficiency are still required. The use of biostimulants has displayed promising results in mitigating Fe deficiency. Our hypothesis was that the combined application of two biostimulants with distinct molecular structures - fulvic acid (FA) and protein hydrolysate (PH) - could be more effective than the use of a single compound. The simultaneous presence of FA and PH (MIX) in a Fe-free nutrient solution led to a redistribution of endogenous Fe, resulting in a higher leaf SPAD index. Furthermore, the addition of FeCl3 as a Fe source (resupply) in MIX-treated plants enhanced the biostimulant effect, as evidenced by increased dry root and shoot weight and a more developed root system. In addition, the expression of Strategy-I-related genes, CsFRO1 and CsIRT1, remained elevated. These effects can be attributed to improved interaction between the roots and biostimulants through the formation of the FA-PH complex, as demonstrated by circular dichroism and isothermal titration calorimetry analyses.

Landscaping DNA binding potential of piperine derivatives by computational and biophysical methods

Piperine, the alkaloid from Black pepper, is known for its wide range of pharmacological effects. The DNA binding activity of piperine was reported earlier. In this work, we explore the DNA duplex binding properties of four piperine derivatives, piperonal, piperonyl alcohol, piperonylic acid, and piperic acid using biophysical and computational techniques. Various spectroscopic and calorimetric techniques were employed for the experimental analysis. We employed UV − vis absorption and fluorescence spectroscopy to verify the binding of piperine to calf thymus DNA (ctDNA). The energetics of this interaction were analysed using isothermal titration calorimetry (ITC). Conformational changes in DNA resulting from ligand interactions were investigated using circular dichroism spectroscopy. All these experimental results consistently demonstrate that the piperine derivatives exhibit stronger binding affinity to DNA than piperine. Computational analyses, utilizing molecular docking and dynamics, revealed similar results to experimental studies, indicating that the compounds bind to the minor groove of DNA like piperine, primarily through van der Waals interactions. Both in vitro and in silico investigations demonstrated the strong DNA binding potential of the examined piperine derivatives. This study is the first to report on the comparative interaction between these piperine derivatives and a DNA duplex.

Thermodynamics of Polyethylene Glycol Intrusion in Microporous Water.

Polymers can be used to augment the properties of microporous materials, affording enhanced processability, stability, and compatibility. Manipulating polymers to target specific properties, however, requires detailed knowledge of how different polymers and microporous materials interact. Here, we report a study of the thermodynamics of polyethylene glycol (PEG) intrusion into a representative hydrophobic zeolite (silicalite-1) and metal-organic framework [ZIF-67; Co(2-methylimidazolate)2] in water, both of which can be formed into colloidally stable aqueous dispersions─termed "microporous water"─with dry, guest-accessible pore networks. Through a combination of O2 capacity measurements and isothermal titration calorimetry (ITC), we establish relationships between PEG intrusion behavior, polymer length, polymer end groups, and the structure of the microporous framework. In particular, we find that PEG intrusion is exothermic for silicalite-1 but endothermic for ZIF-67. Our results provide fundamental insights into polymer intrusion in microporous materials that should inform efforts to design composite solids and fluids with enhanced functionality.

Crystallographic, kinetic, and calorimetric investigation of PKA interactions with L-type calcium channels and Rad GTPase

β-adrenergic signalling activates cAMP-dependent protein kinase (PKA), which regulates the activity of L-type voltage-gated calcium channels such as CaV1.2. Several PKA target sites in the C-terminal tail of CaV1.2 have been identified, and their phosphorylation has been suggested to increase currents in specific tissues or heterologous expression systems. However, augmentation of CaV1.2 currents in the heart is instead mediated by phosphorylation of Rad, a small GTPase that can inhibit CaV1.2. It is unclear how each of the proposed target sites in CaV1.2 and Rad rank towards their recognition by PKA, which could reveal a preferential phosphorylation. Here we used quantitative assays on three CaV1.2 and four Rad sites. Isothermal titration calorimetry (ITC) and enzyme kinetics show that there are two Tiers of targets, with CaV1.2 residue Ser1981 and Rad residues Ser25 and Ser272 forming Tier 1 substrates for PKA. These share a common feature with two Arginine residues at specific positions that can anchor the peptide into the substrate binding cleft of PKA. In contrast, PKA shows minimal activity for the other, Tier 2 substrates, characterized by low kcat values and undetectable binding via ITC. The existence of two Tiers suggests that PKA regulation of the CaV1.2 complex may occur in a graded fashion. We report crystal structures of the PKA catalytic subunit with and without a CaV1.2 and test the importance of several anchoring residues via mutagenesis. Different target sites utilize different anchors, highlighting the plasticity of PKAc to recognize substrates.

Enhancing Biocatalysis: Metal-Organic Frameworks as Multifunctional Enzyme Hosts.

ConspectusEnzymes are highly efficient and selective catalysts that operate under mild conditions, making them invaluable for various chemical transformations. However, their limitations, such as instability and high cost, call for advancements in enzyme immobilization and the development of suitable host materials. Metal-organic frameworks (MOFs), characterized by high porosity, crystallinity, and tunability, are promising candidates for enzyme encapsulation. Among these, zirconium-based MOFs (Zr-MOFs) stand out due to their exceptional structural diversity and chemical stability. The physical and chemical properties of Zr-MOFs can be tuned and characterized with atomic precision, and their interactions with enzymes can be analyzed through a range of techniques spanning from chemistry and materials science to biochemistry. This tunable platform provides opportunities to systematically investigate the impact of encapsulation on the stability and activity of enzymes in order to develop design rules for enzyme hosts.In this Account, we discuss experimentally validated concepts for designing MOF hosts based on their structural properties and enzyme encapsulation mechanisms. We present methods to enhance enzyme catalytic performance through encapsulation and strategies for creating multifunctional enzyme@MOF systems via host modifications. We start by highlighting the importance of host structural design that maximizes substrate diffusion and enzyme availability, with particular focus on MOFs containing hierarchical mesoporous structures such as those in the csq topology. We then delve into the encapsulation process and host-guest interactions examined through techniques such as microscopy, calorimetry, and computational methods, which provide guidelines to fine-tune the local pore chemical environment to enhance enzyme stability and catalytic activity. Techniques found in biochemistry, such as isothermal titration calorimetry (ITC) and confocal laser scanning microscopy (CLSM), were developed to investigate enzyme encapsulation mechanisms, revealing high-entropy-driven host-guest affinity. Additionally, we discuss cases in which enzyme@MOF systems demonstrated enhanced catalytic activities and multifunctional capabilities. Encapsulated enzymes have demonstrated improved thermal and chemical stabilities compared to their free counterparts, maintaining activity under conditions that typically lead to denaturation. Additionally, the highly tunable nature of the MOF platforms allows them to support more complex systems such as tandem reactions, enabling applications in biophotocatalysis, bioelectrocatalysis, and targeted therapeutic protein delivery.The versatility of enzyme@MOFs promises extensive applications in both research and industrial processes across fields including biotechnology, pharmaceutical development, and environmental science. We provide an outlook for promising directions for enzyme@MOF research, with the aim of continuing innovation and exploration. We hope that this Account can benefit chemists, biologists, and material scientists toward designing efficient and adaptable next-generation biocatalytic composite materials.

Nanostructured Hydrogels of Carboxylated Cellulose Nanocrystals Crosslinked by Calcium Ions

Bio-based eco-friendly cellulose nanocrystals (CNCs) gain an increasing interest for diverse applications. We report the results of an investigation of hydrogels spontaneously formed by the self-assembly of carboxylated CNCs in the presence of CaCl2 using several complementary techniques: rheometry, isothermal titration calorimetry, FTIR-spectroscopy, cryo-electron microscopy, cryo-electron tomography, and polarized optical microscopy. Increasing CaCl2 concentration was shown to induce a strong increase in the storage modulus of CNC hydrogels accompanied by the growth of CNC aggregates included in the network. Comparison of the rheological data at the same ionic strength provided by NaCl and CaCl2 shows much higher dynamic moduli in the presence of CaCl2, which implies that calcium cations not only screen the repulsion between similarly charged nanocrystals favoring their self-assembly, but also crosslink the polyanionic nanocrystals. Crosslinking is endothermic and driven by increasing entropy, which is most likely due to the release of water molecules surrounding the interacting COO− and Ca2+ ions. The hydrogels can be easily destroyed by increasing the shear rate because of the alignment of rodlike nanocrystals along the direction of flow and then quickly recover up to 90% of their viscosity in 15 s, when the shear rate is decreased.

Repurposed Anti-Multiple Sclerosis Drug Fty720 Targets Carbapenem-Resistant Acinetobacter baumannii via Multiple Pathways.

Bacterial antimicrobial resistance (AMR), particularly multidrug resistance (MDR) in gram-negative bacterial strains, has emerged as a formidable challenge of substantial consequence, necessitating an urgent pursuit of a sustainable and efficacious strategic response. Repurposing nonantibiotic drugs as potential antibiotics or antibiotic adjuvants is a valuable approach to targeting MDR bacteria. A total of 1,750 FDA-approved drugs (APExBIO, USA) were screened to test their antimicrobial activities against MDR bacteria using the broth microdilution method according to the standard of the Clinical and Laboratory Standards Institute (CLSI). Microscale thermophoresis (MST) analysis was performed to detect the Fty720-LPS interactions. Fty720-indcued lipid changes were measured by untargeted lipidomic analysis. Isothermal titration calorimetry (ITC) analysis was used to determine the Fty720-lipid binding affinities. DNA degradation was assessed via agarose gel electrophoresis with ethidium bromide (EB) staining and visualized using a gel imaging system. Galleria mellonella larvae infection model and Mouse peritonitis infection models were used to evaluated the antibacterial ability of Fty720 in vivo. In this study, we identified Fty720, a pharmaceutical agent for treating multiple sclerosis, as a potent inhibitor of carbapenem-resistant Acinetobacter baumannii (CRAB). We demonstrated that Fty720 exerts antibacterial effects through multiple strategies, including disruption of the structural integrity of the membranes by interacting with LPS and glycerophospholipids, as well as degradation of bacterial DNA. Furthermore, through judicious structural modification, the pivotal role of the positively charged moiety (NH2) in Fty720's antibacterial activity was substantiated. Intriguingly, the translation of Fty720's antibacterial efficacy was demonstrated in vivo, substantiating its pronounced influence on elevating survival rates among models afflicted with MDR gram-negative bacterial infections. Fty720 targets CRAB via multiple pathways, including disruption of outer and inner membrane integrity and DNA degradation. This investigation unveils the multifaceted antibacterial mechanisms of Fty720 while concurrently delineating a prospective therapeutic avenue to counteract MDR gram-negative bacterial strains.

Computational and in vitro binding studies of the redesigned pentoxifylline analogue against phosphodiesterases function in sperm

Pentoxifylline (PTX) is a widely used pharmacological agent for the selection of motile sperm in both normozoospermic and asthenozoospermic ejaculates prior to their use in assisted reproductive technologies (ARTs), e.g. intracytoplasmic sperm injection (ICSI). Since cAMP signalling pathways underlie motility, PTX stimulates motility through the inhibition of cAMP-hydrolysing phosphodiesterase (PDE) isoforms in sperm, viz. PDE4A, PDE4D, and PDE10A. However, it has been noted that PTX causes an early acrosome reaction (AR) and has deleterious effects on oocyte development and early embryogenesis. Previous in vitro and in silico binding studies on the aforementioned PDEs with PTX and a modified PTX called PTXm-1 suggested room for improvement in terms of binding affinity. Indeed, using a structure-guided in silico approach we designed a novel modified PTX (mPTX), which outperformed both PTX and PTXm-1 in binding to PDEs in our in silico binding studies based on docking and molecular dynamics (MD) as well as in vitro binding studies employing isothermal titration calorimetry (ITC). Our findings warrant comparative ex vivo evaluation of mPTX to know whether mPTX is more effective than PTX and PTXm-1 vis-à-vis functional competence for assisted reproductive technology.

Structural dynamics of calcium and integrin-binding protein 2 (CIB2) reveal uncommon flexibility and heterogeneous calcium and magnesium loading

Calcium- and Integrin-Binding protein 2 (CIB2) is a widely expressed protein with an uncertain biological role. Two of its four EF-hand motifs bind Mg(II) and/or Ca(II), thus triggering conformational changes. Although previous studies suggested that CIB2 preferentially binds Mg(II) over Ca(II) under physiological conditions, an atomic level characterization of CIB2 in the presence of both cations was lacking. Based on a combination of solution NMR, exhaustive molecular dynamics simulations and isothermal titration and differential scanning calorimetry, we characterized the interaction of CIB2 with both Ca(II) and Mg(II) ions and elucidated the protein regions involved in the interaction with the α7B integrin target. Analysis of experimental amide nitrogen relaxation rates shows that the EF4 motif exhibits high mobility regardless of the specific bound metal ion and demonstrates that the Ca(II)- and Mg(II)-bound state of CIB2 is relatively floppy, with pico-nanosecond motions induced in a region involved in target recognition. Overall, our data indicate a preferential, thermodynamically stable but structurally flexible state for CIB2, in which an Mg(II) ion is bound to EF3 and a Ca(II) ion to EF4. These results unveil the role of metal binding events in CIB2 and offer new insights into the dynamic regulation of target recognition.

Effect of Water Networks On Ligand Binding: Computational Predictions vs Experiments.

Rational drug design focuses on the explanation and prediction of complex formation between therapeutic targets and small-molecule ligands. As a third and often overlooked interacting partner, water molecules play a critical role in the thermodynamics of protein-ligand binding, impacting both the entropy and enthalpy components of the binding free energy and by extension, on-target affinity and bioactivity. The community has realized the importance of binding site waters, as evidenced by the number of computational tools to predict the structure and thermodynamics of their networks. However, quantitative experimental characterization of relevant protein-ligand-water systems, and consequently the validation of these modeling methods, remains challenging. Here, we investigated the impact of solvent exchange from light (H2O) to heavy water (D2O) to provide complete thermodynamic profiling of these ternary systems. Utilizing the solvent isotope effects, we gain a deeper understanding of the energetic contributions of various components. Specifically, we conducted isothermal titration calorimetry experiments on trypsin with a series of p-substituted benzamidines, as well as carbonic anhydrase II (CAII) with a series of aromatic sulfonamides. Significant differences in binding enthalpies found between light vs heavy water indicate a substantial role of the binding site water network in protein-ligand binding. Next, we challenged two conceptually distinct modeling methods, the grid-based WaterFLAP and the molecular dynamics-based MobyWat, by predicting and scoring relevant water networks. The predicted water positions accurately reproduce those in available high-resolution X-ray and neutron diffraction structures of the relevant protein-ligand complexes. Estimated energetic contributions of the identified water networks were corroborated by the experimental thermodynamics data. Besides providing a direct validation for the predictive power of these methods, our findings confirmed the importance of considering binding site water networks in computational ligand design.

Effects of amino acids on the release of acidic odor compounds in Langjiu and its potential mechanisms

Non-volatile amino acids could influence the overall aroma volatility and perception of Chinese liquor (Baijiu), yet the underlying mechanisms remain unclear. In this study, stir bar sorptive extraction-gas chromatography-mass spectrometry (SBSE-GC-MS) and amino acid analyzer were used to identify the odor compounds and free amino acids in Langjiu. Six acidic odor compounds with an odor activity value (OAV) ≥ 1 and the flavor amino acid L-phenylalanine (L-phe) were selected for analysis. Partition coefficient and olfactory threshold analyses were conducted to investigate the effect of L-phe on the release of these odor compounds. The results indicated that L-phe promoted the release of 3-methylbutanoic acid and pentanoic acid, while it inhibited the release of 2-methylpropanoic acid, butanoic acid, hexanoic acid, and octanoic acid. However, at high concentrations (257 mg/L), L-phe attenuated both the promotive and inhibitory effects. Fluorescence spectroscopy and isothermal titration calorimetry analyses demonstrated that the six acidic odor compounds effectively quenched the intrinsic fluorescence of L-phe through a static quenching mechanism, forming ground-state complexes. The primary interactions between L-phe and 3-methylbutanoic acid and pentanoic acid were attributed to hydrophobic interactions, while the interactions with 2-methylpropanoic acid, butanoic acid, hexanoic acid, and octanoic acid were mainly due to electrostatic interactions. This study provides new insights into the flavor regulation of Baijiu.

On the function of TRAP substrate-binding proteins: the isethionate-specific binding protein IseP.

Bacteria evolve mechanisms to compete for limited resources and survive in new niches. Here we study the mechanism of isethionate import from the sulfate-reducing bacterium Oleidesulfovibrio alaskensis. The catabolism of isethionate by Desulfovibrio species has been implicated in human disease, due to hydrogen sulfide production, and has potential for industrial applications. O.alaskensis employs a tripartite ATP-independent periplasmic (TRAP) transporter (OaIsePQM) to import isethionate, which relies on the substrate-binding protein (OaIseP) to scavenge isethionate and deliver it to the membrane transporter component (OaIseQM) for import into the cell. We determined the binding affinity of isethionate to OaIseP by isothermal titration calorimetry (ITC), KD = 0.95 µM (68% CI = 0.6-1.4 µM), which is weaker compared to other TRAP substrate-binding proteins. The X-ray crystal structures of OaIseP in the ligand-free and isethionate-bound forms were obtained and showed that in the presence of isethionate, OaIseP adopts a closed conformation whereby two domains of the protein fold over the substrate. We serendipitously discovered two crystal forms with sulfonate-containing buffers (HEPES and MES) bound in the isethionate-binding site. However, these do not evoke domain closure, presumably because of the larger ligand size. Together, our data elucidate the molecular details of how a TRAP substrate-binding protein binds a sulfonate-containing substrate, rather than a typical carboxylate-containing substrate. These results may inform future antibiotic development to target TRAP transporters and provide insights into protein engineering of TRAP transporter substrate-binding proteins.