Isothermal Titration Calorimetry Measurement Research Articles

Investigation on the Combined Effect of Hydroxypropyl Beta-Cyclodextrin (HPβCD) and Polysorbate in Monoclonal Antibody Formulation.

Monoclonal antibodies require careful formulation due to their inherent stability limitations. Polysorbates are commonly used to stabilize mAbs, but they are prone to degradation, which results in unwanted impurities. KLEPTOSE® HPβCD (hydroxypropyl beta-cyclodextrin) has functioned as a stable stabilizer for protein formulations in our previous research. The current study investigates the collaborative impact of combining polysorbates and HPβCD as excipients in protein formulations. The introduction of HPβCD in formulations showed it considerably reduced aggregation in two model proteins, bevacizumab and ipilimumab, following exposure to various stress conditions. The diffusion interaction parameter revealed a reduction in protein-protein interactions by HPβCD. In bevacizumab formulations, the subvisible particle counts per 0.4 mL of samples in commercial formulations vs. formulations containing both HPβCD and polysorbates subjected to distinct stressors were as follows: agitation, 87,308 particles vs. 15,350 particles; light, 25,492 particles vs. 6765 particles; and heat, 1775 particles vs. 460 particles. Isothermal titration calorimetry (ITC) measurement indicated a weak interaction between PS 80 and HPβCD, with a KD value of 74.7 ± 7.5 µM and binding sites of 5 × 10-3. Surface tension measurements illustrated that HPβCD enhanced the surface activity of polysorbates. The study suggests that combining these excipients can improve mAb stability in formulations, offering an alternative for the biopharmaceutical industry.

Direct Determination of High-Affinity Binding Constants by Continuous Injection Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) is a method to determine thermodynamic values (ΔG, ΔH, and ΔS) for ligand-receptor binding in biological and abiological systems. It is challenging to directly determine subnanomolar dissociation constants using a standard incremental injection approach ITC (IIA-ITC) measurement. We recently demonstrated a continuous injection approach ITC (CIA-ITC) [ J. Phys. Chem. B 2021, 125, 8075-8087]enables the estimation of thermodynamic parameters in situ. In this work, we demonstrate a label-free and surface modification-free CIA-ITC to determine the complete binding thermodynamics of a ligand with a subnanomolar dissociation constant KD. The KD for desthiobiotin (DTB)-avidin binding was determined to be 6.5 pM with respect to the ligand by CIA-ITC, a quantity unsuccessfully measured with IIA-ITC and surface plasmon resonance spectroscopy (SPR). This value compares well with literature-reported spectroscopic determination of DTB-avidin binding. Criteria with respect to the concentration of the ligand and receptor and flow rate for obtaining true equilibrium dissociation constants without displacement titration are presented.

Utilization of self-assembled soy protein nanoparticles as carriers for natural pigments: Examining non-interaction mechanisms and stability

In this study, we made self-assembled soy protein nanoparticles (SPN) by partially hydrolyzing soy protein isolate (SPI) using Flavourzyme at different hydrolysis times (0, 0.5, 1, 1.5, 2, 3, and 4 h). Compared with SPI, SPN (hydrolysis time, 1.5 h) was spherical with smaller particle size, better dispersion, and higher surface hydrophobicity. The interaction and stability of SPN loaded with different concentrations (0, 0.05, 0.1, 0.3, and 0.5 mmol/L) of lutein (Lut), β-carotene (β-Car), and curcumin (Cur) were then characterized. The encapsulation efficiency, load capacity, and X-ray diffraction measurements showed that natural pigments were successfully encapsulated by SPN in amorphous form, with the highest encapsulation efficiency observed at 0.05 mmol/L with 94.3%, 88.7%, and 96.0% for Lut, β-Car, and Cur, respectively. Fluorescence, ultraviolet, and infrared spectroscopy revealed that natural pigments were loaded into SPN mainly by hydrophobic interactions, and hydrogen bonding was also involved in the loading process of Lut and Cur. Moreover, the outcomes of the isothermal titration calorimetry measurement validated the spontaneous and entropy-driven binding process between SPN and natural pigments. Due to its low molecular weight, a high number of phenolic hydroxyl groups, and low spatial resistance, Cur was most encapsulated in SPN, while the strongest interactions were observed. Lut, β-Car, and Cur were encapsulated by SPN with significantly better thermal and light stability, especially Cur. This study may open a new avenue for the use of SPN as product formulations to extend lipophilic natural edible pigments in food products.

Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis

Oriented ligand immobilization is one of the most effective strategies used in the design and construction of a high-capacity protein A chromatography. In this work, cysteine was introduced as anchoring sites by substituting a specific residue on Helix I, II, and at C-terminus of antibody binding domain Z from protein A, respectively, to investigate structural evolution and binding behavior of protein A ligands at liquid–solid interfaces. Among the three affinity dextran-coated Fe3O4 magnetic nanoparticles (Fe3O4@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix I (Fe3O4@Dx-Z1 MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix II (Fe3O4@Dx-Z2 MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe3O4@Dx-Z1 MNPs because of less positive charged residues on Helix I (K6) than Helix II (R27/K35). Among the three affinity MNPs, moreover, the highest affinity to immunoglobulin G (IgG) binding was observed on Fe3O4@Dx-Z1 MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe3O4@Dx-Z1 MNPs. Finally, the study of IgG binding on MNPs and 96-well plates showed that anchoring sites for ligand immobilization had distinct influences on IgG binding and IgG-mediated antigen binding. This work illustrated that anchoring sites of the ligands had a striking significance for the molecular structure of the ligand at liquid–solid interfaces and raised an important implication for the design and optimization of protein A chromatography and protein A-based immunoassay analysis.

Interaction of sugar beet pectin and pea protein: Impact of neutral sugar side chains and acetyl groups

Sugar beet pectin (SBP) is becoming increasingly interesting due to its complex molecular structure and emulsifying properties. Compared to citrus pectin, SBP differs, for example, in the content of neutral sugar side chains and acetyl groups. In this study it was assumed that side chains lead to steric hindrance between SBP and pea protein and the electrostatic interactions are reduced. Therefore, the aim of the present study was to investigate the impact of the neutral sugar side chains of SBP on steric hindrances and the impact of the acetyl groups on the interaction with pea protein.The interaction of four SBP samples varying in the content of neutral sugar side chains and three SBP samples differing in degree of acetylation with soluble pea protein fractions were investigated. Electrostatic interaction between SBP and protein was qualitatively tested via turbidity measurements and the results were presented in state diagrams. Quantitative evaluation of the interactions was obtained by isothermal titration calorimetry (ITC).The highest interaction was obtained for both SBP sample sets by a protein-to-pectin ratio of 4:1. Less content of neutral sugars resulted in enhanced maximum turbidity. This was confirmed by the ITC measurement, where more possible binding sites and a higher binding affinity was observed. A higher content of acetyl groups increased the interaction with the pea protein, which was supported by the turbidity and ITC measurement. These effects need to be considered when using SBP for designing new systems with plant-based proteins in the food and pharmaceutical industry.

Spectroscopic investigations on fungal aspartic protease as target of gallic acid

Proteases are a major virulence factor in pathogenic fungi and can serve as a potential therapeutic target. The interaction of gallic acid (GA) with Aspartic fungal protease (PepA) was investigated using biophysical and in silico approaches. UV–Vis and fluorescence spectroscopy showed complex formation and static quenching of PepA by GA with Ka of 7.4 × 105 M−1 and stoichiometric binding site (n) of 1.67. CD-spectroscopy showed marked changes in helical content and synchronous fluorescence spectra measurements indicated significant changes in the microenvironment around tryptophan residues in the GA-PepA complex. Outcomes of Isothermal Titration Calorimetry (ITC) measurement and molecular modelling studies validated the spectroscopic results. The binding of GA to Human Serum albumin (HSA) was moderate (Ka = 1.9 × 103 M−1) and did not cause structural disruption of HSA. To conclude, gallic acid is strongly bound to fungal protease leading to structural disruption and inhibition whereas HSA structure was largely conserved. Gallic acid thus appears to be a potential therapeutic agent against fungal proteases.

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry.

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

Isothermal Titration Calorimetry Directly Measures the Selective Swelling of Block Copolymer Vesicles in the Presence of Organic Acid.

Block copolymer (BCP) vesicles loaded with drug molecules may have a nonidentical swelling behavior due to the strong interactions between BCP vesicles and loaded molecules. A thermodynamic study of the swelling for such a system is of great importance in clarifying their pH-gated drug delivery behavior. In this study, the selective swelling of polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP) vesicles in the presence of different acids was compared using dynamic light scattering, zeta-potential, and isothermal titration calorimetry (ITC) measurements. Transmission electron microscopy observation verified that these PS-b-P2VP vesicles were mainly multilamellar. Importantly, using the ITC measurement, we first compared the thermodynamic parameters, including ΔH, ΔG, and ΔS, association binding sites (N), and binding association constants (Ka) in the selective swelling of the PS-b-P2VP vesicles in low pH (pH ∼3.5), with or without a hydrogen bonding interaction. We observed that the existence of a hydrogen bonding interaction between tartaric acid/malic acid and PS-b-P2VP generates a limitation to the selective swelling of PS-b-P2VP vesicles, in which conditions will depend on the molecular structures of the organic acids and PS-b-P2VP. This work first provides a quantitative insight on the swelling of BCP vesicles in the presence of hydrogen bonding and highlights the power of ITC measurements for investigating the structural transformation of polymer nanostructures.

Monosaccharides driving the formation of conjugated linoleic acid vesicles in near-neutral solutions via weak noncovalent bonding interactions

Acted as the second component of fatty acid vesicles (FAV), almost all reported chemicals except for bio-incompatible cationic amphiphiles caused a one-side pH window extension of FAV whilst the other-side shrinkage accordingly. We recently found that dodecanol could partially compensate the alkaline-side pH window shrinkage of FAV when the acidic-side was extended by sodium dodecylsulfate (SDS). Therefore, we guess that if one single small molecule played two − in − one function as both SDS and dodecanol done on the different-side pH window, it might cause a two-side pH window extension of FAV entering into near-neutral pH. Taking conjugated linoleic acid (CLA) as the model fatty acid, ribose, glucose, glucosamine and acetyl glucosamine as the models of the second component, the influence on the pH window extension for the FAV of CLA by each of the monosaccharides and the involved noncovalent bonding interactions are investigated by laser Tyndall effect, pH titrimetry, transmission electron microscopy, dynamic light scattering, ultraviolet absorbency, isothermal titration calorimetry measurement and binding energy calculation. The two-side pH window extension for the FAV of CLA is realized by aid of the two − in − one function of monosaccharides that create H-bonding interaction with the FAV surface and thus enhancing hydrophobic self-assembly of CLA. It is crucial for self-assembling FAV as primitive cell membranes and applying it as modern nutrient carriers or drug delivery systems in the physiological pH range. We propose a practical microenvironmental polarity criterion based on the experimental results to predict how the second component to extend the pH window of FAV: If the second component made the FAV surface more hydrophobic on the alkaline-side pH window while more hydrophilic on the acidic-side via molecular interactions, it could drive the corresponding-side pH window extension, otherwise the corresponding-side pH window shrinkage.

Lipopolysaccharide-stabilized ionic colloids induce biomineralization

Biomineralization inducing by bacteria is common in nature. It involves interactions between bacterial surface and metal ions that are usually present in the form of ionic colloids. Lipopolysaccharide (LPS) is the major component of Gram-negative bacterial surfaces. In the present study, interactions between LPS and metal ions (Ag+, Fe3+, Cd2+, Co2+, Cu2+, Mn2+, Ni2+), as well as ionic colloids (Ag+/Cl-, Fe3+/OH-) were evaluated by means of isothermal titration calorimetry and Zeta potential measurement. It was found that LPS increases the energy barrier for the collapse of ionic colloids and prevents ionic colloids from aggregation. The roles of LPS-stabilized ionic colloids in inducing biomineralization on the bacterial surface were explored by means of scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray Diffraction. It showed that reducing colloidal stability by increasing the ionic strength significantly inhibited biomineralization of ionic colloids on bacterial surfaces. While the formation of ionic colloids promoted biomineralization on bacterial surfaces. This study provides a novel insight into biomineralization as well as biomineralization-based techniques with biological stabilizers for producing biominerals.

Influence of Monomer Structures for Polymeric Multivalent Ligands: Consideration of the Molecular Mobility of Glycopolymers.

Molecular mobility is important for interactions of biofunctional polymers with target molecules. Monomer structures for synthetic biofunctional polymers are usually selected based on their compatibility with polymerization systems, whereas the influence of monomer structures on the interaction with target molecules is hardly considered. In this report, we evaluate the correlation between the monomer structures of glycopolymers and their interactions with concanavalin A (ConA) with respect to the molecular mobility. Two types of glycopolymers bearing mannose are synthesized with acrylamide or acrylate monomers. Despite the similar structures, except for amide or ester bonds in the side chains, the acrylate-type glycopolymers exhibit stronger interaction with ConA both in the isothermal titration calorimetry measurement and in a hemagglutination inhibition assay. Characterization of the acrylate-type glycopolymers suggests that the higher binding constant arises from the higher molecular mobility of mannose units, which results from the rotational freedom of ester bonds in their side chains.

Insights into the cross-amyloid aggregation of Aβ40 and its N-terminal truncated peptide Aβ11-40 affected by epigallocatechin gallate

Inhibition of protein misfolding and aggregation is a great challenge in the field of biochemical and biopharmaceutical engineering. Alzheimer's disease (AD) is a protein-misfolding disease, and the interactions between 40-amino-acid-residue β-amyloid peptide (Aβ40) and its N-terminal truncated peptide Aβ11-40 demonstrate that Aβ11-40 may play an important role in the pathological process of AD. However, the effect of inhibitors on Aβ11-40 aggregation and on the cross-amyloid aggregation (co-assembly) between Aβ40 and Aβ11-40 has never been studied. Herein, coaggregation and seeding interactions between Aβ40 and Aβ11-40 as well as the effect of epigallocatechin gallate (EGCG), a small molecule inhibitor, on the cross-amyloid aggregation have been investigated by extensive analyses. It is found that Aβ11-40 participates in the aggregation of Aβ40 and leads to the formation of coaggregates that contain less β-sheet structures than pure Aβ40 aggregates. The aggregation kinetics along with morphologies and secondary structures of the coaggregates are also significantly affected by the Aβ40/Aβ11-40 ratio. EGCG accelerates the nucleation of Aβ40 but retards that of Aβ11-40 by affecting their elongation and secondary nucleation processes in solution and on solid surfaces. Meanwhile, EGCG makes the conformations of the seeding-induced Aβ aggregates more compact, especially for the homologous seedings. Isothermal titration calorimetry measurement indicates that hydrophobic interactions mainly contribute to the inhibition of the two Aβ isoforms by EGCG. The findings of this research have provided new insights into Aβ aggregation and the effect of an important inhibitor and the results would benefit in the development of potent inhibitors against co-assembly of different amyloid proteins.

Phase behavior, thermodynamic and rheological properties of ovalbumin/dextran sulfate: Effect of biopolymer ratio and salt concentration

The complexation between ovalbumin (OVA) and highly charged dextran sulfate (DS) was investigated employing phase behavior, zeta potential, confocal laser scanning microscope (CLSM), isothermal titration calorimetry (ITC) measurement, and dynamic rheometer analysis. The critical boundary pH values (pHc, pHϕ1, pHmax) were strongly dependent on the biopolymer ratio and salt concentration. Results of the phase diagram and zeta potential showed that the formation of complexes was mainly driven by strong electrostatic interactions. As the increase of the OVA/DS ratio, the critical pH values shifted to higher values. However, the addition of NaCl has a diverse effect, lower salt concentration (CNaCl <100 mM) promoted the formation of OVA/DS coacervates, while higher salt concentration (CNaCl≥ 100 mM) suppress the formation of coacervates due to the shielding effect. The ITC results revealed that DS does bind to native form of OVA at pH 7.0 through the electrostatic forces and it was a spontaneous exothermic process (△G < 0, △H < 0). Additionally, much higher viscoelasticity (G'&G″) for all OVA/DS coacervates indicates that the formation of a highly interconnected gel-like structure. The maximum viscoelasticity was shown at an OVA/DS ratio of 10:1 with 50 mM NaCl. On the other hand, the addition of salt decreased the gel transition temperature of OVA thus promote its aggregation in the mixing system. Our work provides important insight for understanding the interaction between sulfated polysaccharide with egg white protein.

Rationalizing the Role of Monosodium Glutamate in the Protein Aggregation Through Biophysical Approaches: Potential Impact on Neurodegeneration.

Monosodium glutamate (MSG) is the world’s most extensively used food additive and is generally recognized as safe according to the FDA. However, it is well reported that MSG is associated with a number of neurological diseases, and in turn, neurological diseases are associated with protein aggregation. This study rationalized the role of MSG in protein aggregation using different biophysical techniques such as absorption, far-UV CD, DLS, and ITC. Kinetic measurements revealed that MSG causes significant enhancement of aggregation of BSA through a nucleation-dependent polymerization mechanism. Also, CTAB-BSA aggregation is enhanced by MSG significantly. MSG-induced BSA aggregation also exhibits the formation of irreversible aggregates, temperature dependence, non-Arrhenius behavior, and enhancement of hydrodynamic diameter. From the isothermal titration calorimetry measurement, the significant endothermic heat of the interaction of BSA-MSG indicates that protein aggregation may be due to the coupling of MSG with the protein. The determined enthalpy change (ΔH) is largely positive, also suggesting an endothermic nature, whereas entropy change (ΔS) is positive and Gibbs free energy change (ΔG) is largely negative, suggesting the spontaneous nature of the interaction. Furthermore, even a low concentration of MSG is involved in the unfolding of the secondary structure of protein with the disappearance of original peaks and the formation of a unique peak in the far-UV CD, which is an attention-grabbing observation. This is the first investigation which links the dietary MSG with protein aggregation and thus will be very instrumental in understanding the mechanism of various MSG-related human physiological as well as neurological diseases.

Rational design and evaluation of GLP-1 derivative for treating hyperglycemia combined with overexercise-induced myocardial injury

AimsTo design and evaluate the anti-hyperglycemia and overexercise-induced myocardial injury efficacies of a novel long-acting glucagon-like peptide-1 (GLP-1)-based therapeutic peptide in rodent animals. Main methodsHere, we designed and prepared a new pro-drug, termed RYHSB-1, which was connected by a mutated GLP-1(A8G) and an albumin binding peptide via a protease-cleavable linker. Moreover, isothermal titration calorimetry (ITC) was applied to detect its binding affinity for HSA. GLP-1 release assay was conducted in mouse serum in vitro and quantified using LC-MS/MS method. Modified intraperitoneal glucose tolerance test (IPGTT), chronic efficacies study in rodent animals with overexercise-induced myocardial injury were subjected to evaluate the druggability of RYHSB-1. ResultsRYHSB-1 with purity over 99% was prepared and ITC measurement demonstrated high binding affinity for HSA with KD of 0.06 μM. Protease cleavage assay demonstrated slowly controlled-release of transient GLP-1 from RYHSB-1 under the hydrolysis catalyzed by thrombin in vitro. Moreover, IPGTT showed clearly dose-dependent glucose-lowering efficacies of RYHSB-1 within 0.1–0.9 mg/kg. The prolonged anti-diabetic efficacy of RYHSB-1 was further assessed via multiple IPGTTs and hypoglycemic duration test. Furthermore, long-term administration of RYHSB-1 in diabetic mice achieved promising efficacies on hyperglycemia and overexercise-induced myocardial injury. SignificanceRYHSB-1 holds outstanding pharmaceutical potential as an anti- overexercise-induced myocardial injury drug. The strategy of albumin-conjugation also could be applied to other active peptides develop long effecting therapeutic drugs.

Estimation of non-constant variance in isothermal titration calorimetry using an ITC measurement model.

Isothermal titration calorimetry (ITC) is the gold standard for accurate measurement of thermodynamic parameters in solution reactions. In the data processing of ITC, the non-constant variance of the heat requires special consideration. The variance function approach has been successfully applied in previous studies, but is found to fail under certain conditions in this work. Here, an explicit ITC measurement model consisting of main thermal effects and error components has been proposed to quantitatively evaluate and predict the non-constant variance of the heat data under various conditions. Monte Carlo simulation shows that the ITC measurement model provides higher accuracy and flexibility than variance function in high c-value reactions or with additional error components, for example, originated from the fluctuation of the concentrations or other properties of the solutions. The experimental design of basic error evaluation is optimized accordingly and verified by both Monte Carlo simulation and experiments. An easy-to-run Python source code is provided to illustrate the establishment of the ITC measurement model and the estimation of heat variances. The accurate and reliable non-constant variance of heat is helpful to the application of weighted least squares regression, the proper evaluation or selection of the reaction model.

Low molecular weight polyethyleneimine modified by 2-aminoimidazole achieving excellent gene transfection efficiency

The transfection efficiency and cytotoxicity of polyethyleneimine (PEI), as a non-viral vector, increase with molecular weight. Low molecular weight PEI, such as branched PEI1800 (Mw ~ 1800 kDa), has negligible transfection efficiency but low cytotoxicity. Given the low efficiency of PEI1800, a variety of modifications were explored. A structure-integrated group of guanidyl and imidazolyl, 2-aminoimidazole was designed (Scheme 1). To verify the molecular interaction between 2-aminoimidazole and gene, molecular docking experiment, 1H NMR, FTIR and isothermal titration calorimetry (ITC) measurement were carried out. Studies confirmed that 2-aminoimidazole form double hydrogen bonds with phosphate group, which enhanced the thermodynamic interaction between 2-aminoimidazole and gene. Hence, we synthesized 2-aminoimidazole modified PEI1800, and as controls, guanidyl and imidazolyl modified PEI1800 were synthesized respectively. As expected, the results showed that 2-aminoimidazole modified PEI1800 could condense DNA into a higher positively charged complex with 116 ~ 120 nm in diameter and improve the cellular uptake. EGFP assay and luciferase assay showed that 2-aminoimidazole modified PEI1800 exhibited much better transfection than PEI1800, guanidyl modified PEI1800, imidazolyl modified PEI1800 both in HEK 293 T cells and SGC-7901 cells. Besides, MTT assay showed 2-aminoimidazole modified PEI1800 exhibited better biocompatibility than PEI25 K. These results highlight the critical role that 2-aminoimidazole played in non-viral gene delivery.

The CH-π Interaction in Protein-Carbohydrate Binding: Bioinformatics and In Vitro Quantification.

The molecular recognition of carbohydrates by proteins plays a key role in many biological processes including immune response, pathogen entry into a cell, and cell-cell adhesion (e.g., in cancer metastasis). Carbohydrates interact with proteins mainly through hydrogen bonding, metal-ion-mediated interaction, and non-polar dispersion interactions. The role of dispersion-driven CH-π interactions (stacking) in protein-carbohydrate recognition has been underestimated for a long time considering the polar interactions to be the main forces for saccharide interactions. However, over the last few years it turns out that non-polar interactions are equally important. In this study, we analyzed the CH-π interactions employing bioinformatics (data mining, structural analysis), several experimental (isothermal titration calorimetry (ITC), X-ray crystallography), and computational techniques. The Protein Data Bank (PDB) has been used as a source of structural data. The PDB contains over 12 000 protein complexes with carbohydrates. Stacking interactions are very frequently present in such complexes (about 39 % of identified structures). The calculations and the ITC measurement results suggest that the CH-π stacking contribution to the overall binding energy ranges from 4 up to 8 kcal mol-1 . All the results show that the stacking CH-π interactions in protein-carbohydrate complexes can be considered to be a driving force of the binding in such complexes.

Impaired DNA-binding affinity of novel PAX6 mutations

Mutations in human PAX6 gene are associated with various congenital eye malformations including aniridia, foveal hypoplasia, and congenital nystagmus. These various phenotypes may depend on the mutation spectrums that can affect DNA-binding affinity, although this hypothesis is debatable. We screened PAX6 mutations in two unrelated patients with congenital nystagmus, and measured DNA-binding affinity through isothermal titration calorimetry (ITC). To elucidate phenotypic differences according to DNA-binding affinity, we also compared DNA-binding affinity among the previously reported PAX6 missense mutations within the linker region between two subdomains of the paired domain (PD). We identified two novel mutations of PAX6 gene: c.214 G > T (p.Gly72Cys) and c.249_250delinsCGC (p.Val84Alafs*8). Both were located within the linker region between the two subdomains of the PD. ITC measurement revealed that the mutation p.Val84Alafs*8 had no DNA-binding affinity, while the p.Gly72Cys mutation showed a decreased binding affinity (Kd = 0.58 μM) by approximately 1.4 times compared to the wild type-PAX6 (Kd = 0.41 μM). We also found that there was no close relationship between DNA-binding affinity and phenotypic differences. Our results suggest that the DNA-binding affinity alone might be insufficient to determine PAX6-related phenotypes, and that other modifier genes or environmental factors might affect phenotypes of the PAX6 gene.

Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.

SummaryElectron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.