Isothermal Titration Calorimetry Research Articles

Assessing the molecular interaction between a COVID-19 drug, nirmatrelvir, and human serum albumin: calorimetric, spectroscopic, and microscopic investigations.

The research examined the molecular interaction between nirmatrelvir (NIR), a drug used to treat COVID-19, and human serum albumin (HSA) using various techniques, viz., isothermal titration calorimetry (ITC), absorption, fluorescence, CD spectroscopy, and atomic force microscopy (AFM). ITC analysis showed that the NIR-HSA system possessed a moderate binding affinity, with a K a value of 6.53±0.23×104 M-1 at a temperature of 300 K. The thermodynamic values demonstrated that the NIR-HSA complex was stabilized by hydrophobic contacts, hydrogen bonds, and van der Waals forces. The research also discovered modifications in the UV-Vis absorption spectrum of the protein as well as swelling of the HSA molecule when exposed to NIR, based on AFM results. The three-dimensional fluorescence spectral data indicated changes in the microenvironment around HSA's Trp and Tyr residues. Alterations in the protein structure (both secondary and tertiary structures) of HSA after NIR binding were verified using CD spectral studies in the far-UV and near-UV regions. The identification of the NIR binding site in subdomain IB (Site III) of HSA was predicted through competitive displacement experiments.

Synthesis and Metal Ion Adsorption Properties of a Dense Triazole Polymer Carrying Cysteine Residues

ABSTRACTCopper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) is promising as a reaction to synthesize functional polymers consisting of 1,2,3‐triazole units. Recently, we reported a series of dense 1,2,3‐triazole polymers of 4‐azido‐5‐hexynoic acid derivatives. Herein, we designed a new water‐soluble dense 1,2,3‐triazole polymer carrying amino acid residues in the side chains, that is, poly(N‐(4‐azido‐5‐hexynoyl)cysteine) (poly(AC)), bearing L‐cysteine as a ligand for metal ions. CuAAC polymerization of a protected monomer followed by deprotection and neutralization yielded the sodium salt of poly(AC) (poly(AC)Na). The adsorption capacity of poly(AC)Na with Cd2+ was investigated with a colorimetric assay. Furthermore, the interactions for poly(AC) with group 12 metal ions (Zn2+, Cd2+, and Hg2+) were investigated using isothermal titration calorimetry (ITC) and transmittance measurements. Utilizing the colorimetric assay data, the Cd2+ adsorption capacity per unit weight of poly(AC)Na was evaluated to be 4–5 mmol g−1, comparable to those of Cd2+ adsorbents reported previously. The ITC data demonstrated that the interactions of poly(AC)Na with Zn2+, Cd2+, and Hg2+ were predominantly exothermic. Based on the ITC data, apparent dissociation constants of interactions were roughly estimated to be in the order of 10−5–10−4 M, which are comparable to those for metal‐binding biomolecules. The transmittance data indicated that the poly(AC)Na/CdCl2 system underwent phase separation.

A High-Affinity and Selective DNA Aptamer for the N-Linked C8-Deoxyguanosine Adduct Produced by the Arylamine Carcinogen 4-Aminobiphenyl.

4-Aminobiphenyl (4-ABP) is a known human carcinogen that is implicated in the development of bladder cancers in smokers. The amine substituent undergoes bioactivation to generate nitrenium ions capable of covalently modifying DNA nucleobases. The primary adduct of 4-ABP, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), is a bulky N-linked C8-dG adduct that serves as a biomarker for assessing the cancer risk associated with aromatic amine exposure. In this study, the capture-SELEX method was utilized to isolate DNA aptamers for dG-C8-ABP with high affinity and specificity. Using thioflavin T fluorescence spectroscopy and isothermal titration calorimetry, the parent aptamer PdG-1 has a Kd value below 100 nM and over 50-fold selectivity for dG-C8-ABP against competing analytes. A turn-on fluorescent sensor for dG-C8-ABP diagnostics, developed using a strand displacement assay, is also presented with a limit of detection of 68 nM. Our work represents the first selection of a DNA aptamer for a bulky DNA adduct produced by a known human carcinogen and sets the stage for the creation of ultrasensitive aptasensor platforms to meet the challenge of dG-C8-ABP detection in clinical settings.

Protolytic Reactions at Electrified TiO2 P25 Interface: Quantitative and Thermodynamic Characterization

Protolytic reactions on the surface of a titania photocatalyst (TiO2 P25 containing chlorine impurities) were studied using potentiometric and calorimetric acid-base titration. The impurity was removed by either washing or heat treatment. The efficiency of purification was tested by chlorine (TOX) analysis and acid-base titration. Common intersection points of −0.023 and −0.021 mmol/g were obtained for the original and 400 °C heat-treated samples, which are in good agreement with the measured TOX value of 28 mmol/kg. The point of zero charge of the purified sample was determined to be 6.50. Titration data were fitted to simulate protolytic reactions during isothermal calorimetric titrations of purified titania. The evolved heat was measured, and data points were corrected with the heat of mixing and neutralization. The quantity of charged surface species formed in each step of titration was calculated using the parameters from the constant capacitance model fit. The partial molar enthalpy values of the exothermic and endothermic processes of surface protonation (ΔHpr, −17.47 to −16.10 kJ/mol) and deprotonation (ΔHdepr, 32.53 to 27.08 kJ/mol) depend slightly on the ionic strength of suspensions. The average standard enthalpy of one proton transfer reaction is −23.54 ± 1.75 kJ/mol, which is consistent with the literature.

Targeting Mycobacterium tuberculosis Parallel G-Quadruplex Motifs with Aminoglycosides Neomycin and Streptomycin: Spectroscopic and Calorimetric Aspects.

Mycobacterium tuberculosis (Mtb) contains potential G-quadruplex (PGQ) motifs in the genes espK and cyp51, which are crucial for the bacteria's virulence within host cells. Aminoglycoside molecules are commonly used as antibiotics for ribosomal targets. This study provides insight into the interactions between these aminoglycosides and Mtb-PGQ sequences (espK and cyp51), shedding light on the structural and thermodynamic dynamics of their binding. This study demonstrates the stability, affinity, and conformation of Mtb-PGQ in the presence of neomycin and streptomycin. Ultraviolet-visible spectroscopy (UV-vis), circular dichroism spectroscopy (CD), CD thermal melting, isothermal titration calorimetry (ITC), and fluorescence intercalator displacement (FID) assays were used to comprehensively examine these interactions. Our results reveal that neomycin with Mtb-PGQexhibits hypochromism accompanied by a 4-5 nm red shift in the UV-visible absorption titration, whereas streptomycin exhibits a hypochromic shift without changes in the maximum wavelength. Notably, neomycin shows a nonlinear binding isotherm, suggesting the involvement of more than one binding process in the formation of neomycin.Mtb-PGQ complexes. Scatchard plot analysis indicates higher binding affinity values for neomycin compared with weaker affinity of streptomycin. CD studies reveal that neomycin decreases the ellipticity of Mtb-PGQ with a red shift while retaining the parallel topology, ultimately enhancing the thermal stability of both espK and cyp51. In contrast, streptomycin destabilizes the cells. ITC analysis reveals that neomycin exhibits the strongest binding affinity for cyp51, with the relative order being NEO-cyp51 > NEO-espk > STR-cyp51 > STR-espk. Moreover, thermodynamic analysis reveals that neomycin possesses a unique dual mode of binding through grooves as well as stacking. FID studies further confirm a lower DC50 value for neomycin than for streptomycin, suggesting that neomycin is a strong displacer of thiazole orange. Thus, the results show that neomycin with amino groups selectively recognizes the grooves of cyp51 over espK.

Platinum as both a drug and its modulator - Do platinum nanoparticles influence cisplatin activity?

Breast cancer was the most frequent cause of cancer death in females in 2022. Despite the development of personalized therapies, chemotherapy frequently remains the only available treatment method. However, the administration of classic antineoplastic drugs, like cisplatin (CDDP), often causes severe side effects and may lead to drug resistance making the therapy inefficient. Therefore, there is a great need for new, effective treatment regimens development. For this reason, we applied platinum nanoparticles (PtNPs) to verify if they can influence the CDDP activity with particular emphasis on the differences due to nanoparticles' sizes. We employed a broad spectrum of physicochemical methods, including Dynamic Light Scattering, Atomic Force Microscopy, Isothermal Titration Calorimetry, Fourier Transform Infrared Spectroscopy, and Near Infrared Spectroscopy and also Differential Scanning Calorimetry, to characterize the possible interactions between nanoparticles and CDDP. Moreover, the impact of PtNPs on CDDP biological activity was investigated using the Ames mutagenicity test on Salmonella enterica serovar Typhimurium TA102 and MTT assay on two breast cancer cell lines MDA-MB-231 and SKBR3. The obtained results revealed PtNPs direct interactions with CDDP dependent on the nanoparticles' size. Despite the lack of explicit confirmation of PtNPs aggregation by AFM imaging and DLS, further physicochemical methods indicated structural changes between nanoparticles alone and PtNPs-CDDP mixtures. Moreover, the biological assays confirmed that PtNPs decrease CDDP mutagenicity and also slightly increase its cytotoxicity on the chosen cell lines. The latter effects are ambiguous, nevertheless, provide a valuable basis for further research.

The evaluation of DNA-linked inhibitor antibody and AlphaScreen assays for high-throughput screening of compounds targeting the cap-binding domain in influenza a polymerase.

The PB2 subunit of the influenza virus polymerase complex is essential for viral replication, primarily through a mechanism known as cap-snatching. In this process, PB2 binds to the 5' cap structure of host pre-mRNAs, enabling the viral polymerase to hijack the host transcriptional machinery. This binding facilitates the cleavage and integration of the capped RNA fragment into viral mRNA, thereby promoting efficient viral replication. Inhibiting the PB2-cap interaction is therefore crucial, as it directly disrupts the viral replication cycle. Consequently, targeting PB2 with specific inhibitors is a promising strategy for antiviral drug development against influenza. However, there are currently no available methods for the high-throughput screening of potential inhibitors. The development of new inhibitor screening methods of potential PB2 binders is the focus of this study. In this study, we present two novel methods, DIANA and AlphaScreen, for screening influenza PB2 cap-binding inhibitors and evaluate their effectiveness compared to the established differential scanning fluorimetry (DSF) technique. Using a diverse set of substrates and compounds based on the previously described PB2 binder pimodivir, we thoroughly assessed the capabilities of these new methods. Our findings demonstrate that both DIANA and AlphaScreen are highly effective for PB2 inhibitor screening, offering distinct advantages over traditional techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). These advantages include improved scalability, reduced sample requirements, and the capacity for label-free detection. Notably, DIANA's ability to determine Ki values from a single-well measurement significantly enhances its practicality and efficiency in inhibitor screening. This research represents a significant step forward in the development of more efficient and scalable screening strategies, helping advance efforts in the discovery of antiviral drugs against influenza.

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.

Citrullination at the N-terminal region of MDM2 by the PADI4 enzyme.

PADI4 is one of the human isoforms of a family of enzymes involved in the conversion of arginine to citrulline. MDM2 is an E3 ubiquitin ligase that is critical for degradation of the tumor suppressor gene p53. We have previously shown that there is an interaction between MDM2 and PADI4 in cellulo, and that such interaction occurs through the N-terminal region of MDM2, N-MDM2, and in particular through residues Thr26, Val28, Phe91, and Lys98. Here, by using a "divide-and-conquer" approach, we have designed and synthesized peptides comprising these two polypeptide stretches (residues Ala21-Lys36, and Lys94-Val108), either in the wild-type species or in their citrullinated versions. Some of the citrullinated peptides were aggregation-prone, as suggested by DOSY-NMR experiments, but the wild-type versions of both fragments were monomeric in solution. We found out that wild-type and modified peptides were disordered in all cases, as also tested by far-UV circular dichroism (CD), and citrullination mainly affected the NMR chemical shifts of adjacent residues. Isothermal titration calorimetry (ITC) in the absence and presence of GSK484, an enzymatic PADI4 inhibitor, indicated that this compound blocked binding of the peptides to the enzyme. Binding to the active site of the N-MDM2 fragments was also confirmed by in silico experiments. The affinities of PADI4 for the wild-type peptides were more favorable than those of the corresponding citrullinated ones, but all measured values were within the micromolar range, indicating that there were no major variations in the thermodynamics of binding due to sequence effects. The kinetic dissociation rates, koff, measured by biolayer interferometry (BLI), were always one-order of magnitude faster for the citrullinated peptides than for the wild-type ones. Taken together, all these findings indicate that MDM2 is a substrate for PADI4 and is prone to citrullination in the identified (and specific) positions of its N-terminal region.

Construction of fish scale (Cyprinus carpio L.) gelatin-fatty acid conjugate for loading curcumin: Effect of alkyl chain length on the interaction and stability.

A fish scale (FS) gelatin-fatty acid conjugate (GFC) with alkyl chain lengths of 8-18 was constructed to increase the aqueous solubility of curcumin. The effect of alkyl chain length on the interaction between GFC and curcumin was characterized by dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), fluorescence spectroscopy (FS), and isothermal titration calorimetry (ITC). The surface hydrophobicity (from 4987 ± 223.79 to 9982 ± 262.78) and curcumin loading capacity (from 8.20 ± 0.54 to 31.18 ± 1.41 μg/mg) of the GFC exhibited significant enhancements through increasing alkyl chain lengths from 8 to 18. This was accompanied by a reduction in particle size (from 661.5 ± 28.9 to 329.7 ± 6.6 nm) and ζ-potential (from -2.7 ± 0.92 to -26.8 ± 0.27). FS and ITC confirmed that GOC shared an optimal binding constant (Ka, 2.40 × 108 L·mol-1 and 3.47 × 105 M-1) and binding site (n, 1.45 and 2.276) with curcumin among GFCs. Increasing GFC's alkyl chain length also boosted the stability of entrapped curcumin against the thermal environment and ultraviolet radiation. These results could be beneficial for gelatin-based nanocarrier development and application.

Dendrimer-Mediated Molecular Sieving on Avidin.

Decoration of proteins and enzymes with well-defined polymeric structures allows precise decoration of protein surfaces, enabling controlled modulation of activity. Here, the impact of dendronization on the interaction between avidin and biotin was investigated. A series of generation 3-7 bis(2,2-hydroxymethyl)propionic acid (bis-MPA) dendrons were coupled to either biotin or avidin to yield a library of dendronized avidin and biotin structures. The thermodynamics of binding each biotinylated generation to a library of avidin conjugates was probed with isothermal titration calorimetry (ITC). Dissociation constants of high-generation biotin-dendrons (G5 and G6) with higher-generation avidin-dendron conjugates (Av-G6) increased from ∼10-15 M (for the native structures) to ∼10-6 M, and binding was found to be weaker than that of the Avidin-HABA complex. Avidin-G5 and Avidin-G6 were highly size-selective for biotinylated ligands; both prevented the binding of aprotinin (6.9 kDa), bovine serum albumin (BSA), and PEG3400 while forming fractional complexes with smaller biotinylated dendrons.

Kinetic and Thermodynamic Characterization of Human 4-Oxo-l-proline Reductase Catalysis.

The enzyme 4-oxo-l-proline reductase (BDH2) has recently been identified in humans. BDH2, previously thought to be a cytosolic (R)-3-hydroxybutyrate dehydrogenase, actually catalyzes the NADH-dependent reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline, a compound with known anticancer activity. Here we provide an initial mechanistic characterization of the BDH2-catalyzed reaction. Haldane relationships show the reaction equilibrium strongly favors the formation of cis-4-hydroxy-l-proline. Stereospecific deuteration of NADH C4 coupled with mass spectrometry analysis of the reaction established that the pro-S hydrogen is transferred. NADH is co-purified with the enzyme, and a binding kinetics competition assays with NAD+ defined dissociation rate constants for NADH of 0.13 s-1 at 5 °C and 7.2 s-1 at 25 °C. Isothermal titration calorimetry at 25 °C defined equilibrium dissociation constants of 0.48 and 29 μM for the BDH2:NADH and BDH2:NAD+ complexes, respectively. Differential scanning fluorimetry showed BDH2 is highly thermostabilized by NADH and NAD+. The kcat/KM pH-rate profile indicates that a group with a pKa of 7.3 and possibly another with a pKa of 8.7 must be deprotonated and protonated, respectively, for maximum binding of 4-oxo-l-proline and/or catalysis, while the kcat profile is largely insensitive to pH in the pH range used. The single-turnover rate constant is only 2-fold higher than kcat. This agrees with a pre-steady-state burst of substrate consumption, suggesting that a step after chemistry, possibly product release, contributes to limit kcat. A modest solvent viscosity effect on kcat indicates that this step is only partially diffusional. Taken together, these data suggest chemistry does not limit the reaction rate but may contribute to it.

Design of a light and Ca2+ switchable organic–peptide hybrid

The design of organic-peptide hybrids has the potential to combine our vast knowledge of protein design with small molecule engineering to create hybrid structures with complex functions. Here, we describe the computational design of a photoswitchable Ca2+-binding organic-peptide hybrid. The designed molecule, designated Ca2+-binding switch (CaBS), combines an EF-hand motif from classical Ca2+-binding proteins such as calmodulin with a photoswitchable group that can be reversibly isomerized between a spiropyran (SP) and merocyanine (MC) state in response to different wavelengths of light. The MC/SP group acts both as a photoswitch as well as an optical sensor of Ca2+ binding. Photoconversion of the SP to the corresponding MC unmasks an acidic phenol, which CaBS uses as an integral part of both its Ca2+-binding site as well as its tertiary and quaternary structure. By design, the SP state of CaBS is monomeric, while the Ca2+-bound form of the MC state is an obligate dimer, with two Ca2+-binding sites formed at the interface of a domain-swapped dimer. Thus, light and Ca2+ were expected to serve as an "AND gate" that powers a change in backbone structure/dynamics, oligomerization state, and fluorescence properties of the designed molecule. CaBS was designed using Rosetta and molecular dynamics simulations, and experimentally characterized by nuclear magnetic resonance, isothermal titration calorimetry, and optical titrations. These data illustrate the potential of combining small molecule engineering with de novo protein design to develop sensors whose conformation, association state, and optical properties respond to multiple environmental cues.

C-di-GMP phosphodiesterase ProE interacts with quorum sensing protein PqsE to promote pyocyanin production in Pseudomonas aeruginosa.

The universal bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays critical roles in regulating a variety of bacterial functions such as biofilm formation and virulence. The metabolism of c-di-GMP is inversely controlled by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). Recently, increasing studies suggested that the protein-protein interactions between DGCs/PDEs and their partners appear to be a common way to achieve specific regulation. In this work, we showed that the PDE ProE can interact with PQS quorum sensing protein PqsE to regulate pyocyanin production in Pseudomonas aeruginosa. Our bacterial two-hybrid assay demonstrated that ProE directly interacts with PqsE, and isothermal titration calorimetry and surface plasmon resonance assay further confirmed that the binding affinity of ProE with PqsE is at micromolar level. Both ProE and PqsE negatively regulate intracellular c-di-GMP levels. Furthermore, our transcriptomic study showed that co-expression of ProE and PqsE significantly changes the gene expression profiles in P. aeruginosa, especially with increased expression of pyocyanin genes, and the qPCR and phenotypic results confirmed the transcriptome data. Taken together, our study suggested that the interaction between ProE and PqsE plays a critical role in regulation of pyocyanin production and highlights the importance of protein-protein interaction mediated c-di-GMP signaling in P. aeruginosa.IMPORTANCEc-di-GMP is pivotal in orchestrating various bacterial functions. In Pseudomonas aeruginosa, the nuanced balance of intracellular c-di-GMP is maintained by approximately 41 diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). Emerging studies indicate that the c-di-GMP metabolic DGCs and PDEs may be involved in the signal transduction process by directly binding to the target protein, thus influencing downstream function. Despite their known importance, the precise functions of these proteins, especially their interacting partners, remain unclear. In this study, we identified that PQS quorum sensing system protein PqsE is a binding partner of c-di-GMP phosphodiesterase ProE; further analysis suggested that the ProE specifically interacts with PqsE to promote pyocyanin production. Our study extended the regulatory mechanism of the c-di-GMP signal transduction and quorum sensing in governing bacterial physiology.

Predicting the Key Properties of a Modified Product to Pre-Select a Pluronic F127 Modification Scheme for Preparing High-Quality Nano-Micelles

Hydrophobic modification alters the properties of Pluronic F127 to form micelles more efficiently and enhances its drug-loading capacity. However, selecting the appropriate hydrophobic group for modification is laborious. In this paper, we propose an efficient approach for predicting key parameters to select hydrophobic groups for F127 modification prior to synthesis, in order to improve the formability and stability of the micelles. The results of nuclear magnetic resonance and isothermal titration calorimetry were utilized to establish a function for predicting the hydrophile–lipophile balance, critical micelle concentration, and Gibbs free energy of the products based on the structure of raw material. These predicted values can assist us in selecting suitable hydrophobic groups for F127 modification. Subsequently, we successfully tested our method and validated our work using pharmaceutical evaluation methods, such as appearance observation, particle size measurement, drug loading determination, equilibrium binding rate assessment, storage stability testing, and the plotting of accumulation release curves. Therefore, we suggest that our work could provide a model linking the molecular structure to properties, with the purpose of pre-selecting modification products that have advantages in micelle preparation. This can facilitate the application of F127 in preparing nano-micelles.

Conformational dynamics in specialized C2H2 zinc finger domains enable zinc-responsive gene repression in S. pombe.

Loz1 is a zinc-responsive transcription factor in fission yeast that maintains cellular zinc homeostasis by repressing the expression of genes required for zinc uptake in high zinc conditions. Previous deletion analysis of Loz1 found a region containing two tandem C2H2 zinc-fingers and an upstream "accessory domain" rich in histidine, lysine, and arginine residues to be sufficient for zinc-dependent DNA binding and gene repression. Here we report unexpected biophysical properties of this pair of seemingly classical C2H2 zinc fingers. Isothermal titration calorimetry and NMR spectroscopy reveal two distinct zinc binding events localized to the zinc fingers. NMR spectra reveal complex dynamic behavior in this zinc-responsive region spanning time scales from fast 10-12-10-10 to slow >100 s. Slow exchange due to cis-trans isomerization of the TGERP linker results in the doubling of many signals in the protein. Conformational exchange on the 10-3 s timescale throughout the first zinc finger distinguishes it from the second and is linked to a weaker affinity for zinc. These findings reveal a mechanism of zinc sensing by Loz1 and illuminate how the protein's rough free-energy landscape enables zinc sensing, DNA binding and regulated gene expression.

Affinity Maturation for Antibody Engineering: The Critical Role of Residues on CDR Loops of Antibodies in Antigen Binding

During the course of affinity maturation, antibodies exhibit enhanced antigen-binding affinities by altering the amino acids in their variable regions. Understanding the structural basis of these antibodies can be beneficial for antibody engineering. We determined the crystal structures of single-chain Fv (scFv) antibodies against (4-hydroxy-3-nitrophenyl)acetyl, C6 and E11, which had undergone affinity maturation. Compared with germline-type antibodies, the affinity-matured antibodies with somatic hypermutation from Lys58 to Arg58 of the heavy chain located in the complementarity-determining region 2 (CDR2) seemed to be critical for increasing the antigen-binding affinity. E11 possessed a disulfide bond at the base of CDR3 in the heavy chain, which contributed to a further increase in its antigen-binding affinity compared with that of C6. In this study, we generated several mutant scFvs of C6 and E11 and analyzed their antigen-binding thermodynamics using isothermal titration calorimetry. The results indicated that the CDR conformations could adjust antigen-binding not only at the mutated sites but also at the surrounding residues. The analysis of folding thermodynamics showed that the stability of the affinity-matured antibodies was lower than that of the germline-type antibodies and remarkably increased upon strong antigen binding. The results also indicated that the structural dynamics of the affinity-matured antibodies were greater than those of the germline-type antibodies and decreased upon antigen binding.

Cellular uptake and transport mechanism of lycopene-loaded nanomicelles formed by amphiphilic peptide self-assembly in the intestinal epithelium.

This study aimed to elucidate the transport mechanism of lycopene-loaded nanomicelles to improve intestinal absorption of lycopene. The interactive mechanism between lycopene and nanomicelles was investigated through isothermal titration calorimetry (ITC). The cytotoxicity, cellular uptake, endocytosis, and intracellular transport pathways of lycopene-loaded nanomicelles were investigated using the Caco-2 cell model. The ITC results demonstrated that nanomicelles/lycopene binding was an entropy-driven spontaneous exothermic reaction, and hydrophobic interactions were the main driving force. Lycopene-loaded nanomicelles were not cytotoxic, and uptake of lycopene by Caco-2 cells increased 2.20-fold after nanoencapsulation. The results of intracellular transport of lycopene-loaded nanomicelles indicated that the endoplasmic reticulum, Golgi apparatus, and lysosomes play key roles in this process. The intracellular transport results showed that the endoplasmic reticulum, Golgi apparatus, and lysosomes were important organelles for intracellular transport of lycopene-loaded nanomicelles. Nanomicelles effectively enhance the cellular uptake of lycopene and are promising carriers for its delivery. This study contributes to the understanding of the transport mechanism of nanomicelles through intestinal epithelial cells. © 2025 Society of Chemical Industry.

A Roadmap of Responses to Asymmetry Stress in Lipid Membranes.

The selective insertion of membrane-impermeant amphiphiles such as detergents, (lipo)peptides, drugs, etc. into the cis leaflet of a membrane causes an imbalance between the intrinsic areas of the cis and trans leaflet, referred to as asymmetry stress or differential stress. The literature provides individual mechanisms of how membranes respond to such stress, which are relevant to membrane remodeling processes and leakage phenomena. By studying vesicle budding, membrane leakage, and isothermal titration calorimetry of liposomes interacting with digitonin, alkyl maltosides, miltefosine, and octyl glucoside, we developed a roadmap linking the stress-response mechanisms to each other. Initially, lateral compression or stretching of the leaflets accommodates a minor asymmetry stress. Then, either molecules flip to the trans leaflet or the membrane bends to form buds. Fast flip leads to the classic three-stage model. Budding proceeds up to its limit at 20-40% of the lipid. Beyond, insertion of further detergent is opposed by the pressure in the overpopulated leaflet. This "staying out" state can persist over hours or days and up to high detergent concentrations before detergent micelles induce "micellar solubilization". Alternatively, the stress can be reduced by a transient failure of the membrane, allowing for "cracking in" of molecules, transferring them to the trans side.

Arrhythmogenic calmodulin variants D131E and Q135P disrupt interaction with the L-type voltage-gated Ca2+ channel (Cav1.2) and reduce Ca2+-dependent inactivation.

Long QT syndrome (LQTS) and catecholaminergic polymorphism ventricular tachycardia (CPVT) are inherited cardiac disorders often caused by mutations in ion channels. These arrhythmia syndromes have recently been associated with calmodulin (CaM) variants. Here, we investigate the impact of the arrhythmogenic variants D131E and Q135P on CaM's structure-function relationship. Our study focuses on the L-type calcium channel Cav1.2, a crucial component of the ventricular action potential and excitation-contraction coupling. We used circular dichroism (CD), 1H-15N HSQC NMR, and trypsin digestion to determine the structural and stability properties of CaM variants. The affinity of CaM for Ca2+ and interaction of Ca2+/CaM with Cav1.2 (IQ and NSCaTE domains) were investigated using intrinsic tyrosine fluorescence and isothermal titration calorimetry (ITC), respectively. The effect of CaM variants of Cav1.2 activity was determined using HEK293-Cav1.2 cells (B'SYS) and whole-cell patch-clamp electrophysiology. Using a combination of protein biophysics and structural biology, we show that the disease-associated mutations D131E and Q135P mutations alter apo/CaM structure and stability. In the Ca2+-bound state, D131E and Q135P exhibited reduced Ca2+ binding affinity, significant structural changes, and altered interaction with Cav1.2 domains (increased affinity for Cav1.2-IQ and decreased affinity for Cav1.2-NSCaTE). We show that the mutations dramatically impair Ca2+-dependent inactivation (CDI) of Cav1.2, which would contribute to abnormal Ca2+ influx, leading to disrupted Ca2+ handling, characteristic of cardiac arrhythmia syndromes. These findings provide insights into the molecular mechanisms behind arrhythmia caused by calmodulin mutations, contributing to our understanding of cardiac syndromes at a molecular and cellular level.