Species-specific determinants in the IgG CH3 domain enable Fab-arm exchange by affecting the noncovalent CH3-CH3 interaction strength

A distinctive feature of human IgG4 is its ability to recombine half molecules (H chain and attached L chain) through a dynamic process termed Fab-arm exchange, which results in bispecific Abs. It is becoming evident that the process of Fab-arm exchange is conserved in several mammalian species, and thereby represents a mechanism that impacts humoral immunity more generally than previously thought. In humans, Fab-arm exchange has been attributed to the IgG4 core-hinge sequence (226-CPSCP-230) in combination with unknown determinants in the third constant H chain domain (CH3). In this study, we investigated the role of the CH3 domain in the mechanism of Fab-arm exchange, and thus identified amino acid position 409 as the critical CH3 determinant in human IgG, with R409 resulting in exchange and K409 resulting in stable IgG. Interestingly, studies with IgG from various species showed that Fab-arm exchange could not be assigned to a common CH3 domain amino acid motif. Accordingly, in rhesus monkeys (Macaca mulatta), aa 405 was identified as the CH3 determinant responsible (in combination with 226-CPACP-230). Using native mass spectrometry, we demonstrated that the ability to exchange Fab-arms correlated with the CH3-CH3 dissociation constant. Species-specific adaptations in the CH3 domain thus enable Fab-arm exchange by affecting the inter-CH3 domain interaction strength. The redistribution of Ag-binding domains between molecules may constitute a general immunological and evolutionary advantage. The current insights impact our view of humoral immunity and should furthermore be considered in the design and evaluation of Ab-based studies and therapeutics.     

Acclimation of aquatic microbial communities to Hg(II) and CH3Hg+ in polluted freshwater ponds

The relationship of mercury resistance to the concentration and chemical speciation of mercurial compounds was evaluated for microbial communities of mercury-polluted and control waters. Methodologies based on the direct viable counting (DVC) method were adapted to enumerate mercury-resistant communities. Elevated tolerance to Hg(II) was observed for the microbial community of one mercury-polluted pond as compared to the community of control waters. These results suggest an in situ acclimation to Hg(II). The results of the methylmercury resistance-DVC assay suggested that minimal acclimation to CH₃Hg⁺ occurred since similar concentrations of CH₃HgCl inhibited growth of 50% of organisms in both the control and polluted communities. Analyses of different mercury species in pond waters suggested that total mercury, but not CH₃Hg⁺ concentrations, approached toxic levels in the polluted ponds. Thus, microbial acclimation was specific to the chemical species of mercury present in the water at concentrations high enough to cause toxic effects to nonacclimated bacterial communities.     

Mapping the dynamics of ligand reorganization via 13CH3 and 13CH2 relaxation dispersion at natural abundance

Flexible ligands pose challenges to standard structure-activity studies since they frequently reorganize their conformations upon protein binding and catalysis. Here, we demonstrate the utility of side chain ¹³C relaxation dispersion measurements to identify and quantify the conformational dynamics that drive this reorganization. The dispersion measurements probe methylene ¹³CH₂ and methyl ¹³CH₃ groups; the latter are highly prevalent side chain moieties in known drugs. Combining these side chain studies with existing backbone dispersion studies enables a comprehensive investigation of μs–ms conformational dynamics related to binding and catalysis. We perform these measurements at natural ¹³C abundance, in congruence with common pharmaceutical research settings. We illustrate these methods through a study of the interaction of a phosphopeptide ligand with the peptidyl-prolyl isomerase, Pin1. The results illuminate the side-chain moieties that undergo conformational readjustments upon complex formation. In particular, we find evidence that multiple exchange processes influence the side chain dispersion profiles. Collectively, our studies illustrate how side-chain relaxation dispersion can shed light on ligand conformational transitions required for activity, and thereby suggest strategies for its optimization.     

Synthesis,structural characterization,and properties of the organothorium alkylthiolate complex [(CH3)5C5]2Th(SCH2CH2CH3)2

This contribution reports the synthesis and characterization of the organothorium alkylthiolate complex [(CH₃)₅C₅]₂Th(SCH₂CH₂CH₃)₂. This compound crystallizes in the monoclinic space group C2/c (#15) with four molecules in a cell of dimensions a=19.066(2), b=11.603(1), c=16.379(2) Å, and β=130.08(1)°. Least-squares refinement led to a value for the conventional R index (on F_o) of 0.040 for 132 variables and 2030 observations having F_o²⩾3σ(F_o²). The molecular structure consists of an unexceptional ‘bent sandwich’ [(CH₃)₅C₅]₂Th fragment coordinated to two n-propylthiolate ligands. The ThS bond distance is 2.718(3) Å; the SC(α) distance, 1.78(2) Å; the ThSC(α) angle, 108.3(5)°; and the SThS′ angle, 102.5(2)°. Contrasts are drawn with the structures of analogous actinide alkoxides     

In vivo comparison of N-11CH3 vs O-11CH3 radiolabeled microtubule targeted PET ligands

Altered dynamics of microtubules (MT) are implicated in the pathophysiology of a number of brain diseases. Therefore, radiolabeled MT targeted ligands that can penetrate the blood brain barrier (BBB) may offer a direct and sensitive approach for diagnosis, and assessing the clinical potential of MT targeted therapeutics using PET imaging. We recently reported two BBB penetrating radioligands, [¹¹C]MPC-6827 and [¹¹C]HD-800 as specific PET ligands for imaging MTs in brain. The major metabolic pathway of the above molecules is anticipated to be via the initial labeling site, O-methyl, compared to the N-methyl group. Herein, we report the radiosynthesis of N-¹¹CH₃-MPC-6827 and N-¹¹CH₃-HD-800 and a comparison of their in vivo binding with the corresponding O-¹¹CH₃ analogues using microPET imaging and biodistribution methods. Both O-¹¹CH₃ and N-¹¹CH₃ labeled MT tracers exhibit high specific binding and brain. The N-¹¹CH₃ labeled PET ligands demonstrated similar in vivo binding characteristics compared with the corresponding O-¹¹CH₃ labeled tracers, [¹¹C]MPC-6827 and [¹¹C]HD-800 respectively.     

The CH3PH2 and CH3PH isomers: isomerization,hydrogen release,thermodynamic, and spectroscopy properties

Curcumin has been reported to be therapeutically active but has poor bioavailability, half life, and high rate of metabolic detoxifcation. Most of the hydrophobic and acidic drugs get transported through human serum albumin (HSA). Binding of drugs to serum protein increases their half-life. The present study is focused to analyze interaction of curcumin with HSA by NMR and docking studies. In order to investigate the binding affinity of curcumin with HSA, NMR based diffusion techniques and docking study have been carried out. We report that curcumin has shown comparable binding affinity value vis-a-vis standard, the accessible surface area (ASA) of human serum albumin (uncomplexed) and its docked complex with curcumin at both binding sites was calculated and found to be close to that of warfarin and diazepam respectively. Conclusion drawn from our study demonstrates that curcumin interacts with HSA strongly thereby its poor half life is due to high rate of its metabolic detoxification as reported in literature.

Studies on the interaction between ribosomes and 14 CH 3 -F 3 initation factor

Summary Initiation factor F₃ has been purified fromEscherichia coli and labelledin vitro by reductive alkylation. The¹⁴CH₃–F₃ so obtained had a specific activity of about 1 000 cpm/g and was shown to have retained its biological activity. Labelled F₃ binds to 30S ribosomal subunits ofEscherichia coli andBacillus stearothermophilus, but does not bind to either 70S ribosomes or 50S ribosomal subunits. The stoichiometry of the binding indicates that one molecule of¹⁴CH₃–F₃ is bound to each 30S ribosomal subunit. Several antibiotics, known to interact with 30S subunits, inhibit the binding. Functional studies indicate that F₃ is released from 30S ribosomes as a result of the formation of the 70S initiation complex.     

Citrate binding of Al3+ and Fe3+

Citrate occurs at about 0.1 mM in blood plasma and is the most likely small molecule plasma binder of both Al3+ and Fe3+. This paper assesses published stability constants for citrate binding to each metal ion. From pH 2 to 5 Al3+ forms a neutral complex with citrate that may be absorbed into the body in the upper regions of the gastrointestinal tract. It is especially dangerous to ingest aluminum-containing antacids with citrus fruit or juices. Ignoring the likely occurrence of a competing 2:1 citrate-Fe3+ complex necessitates adjustments in reported stability constants for Fe3+ binding to transferrin. In the blood, plasma transferrin steals both Fe3+ and Al3+ from citrate.     

Interaction between CH2 and CH3-domains of human immunoglobulin G1 in glycosylated and aglycosylated Fc-fragments

Heat denaturation of glycosylated and aglycosylated human immunoglobulin G1 Fc fragments was investigated by differential scanning microcalorimetry. The enthalpy of the interaction between aglycosylated CH2 and CH3 domains is significantly reduced at 37 degrees C (but not at 0 degree C) as compared to the glycosylated form. The temperature dependence is consistent with the data on restricted proteolysis by trypsin.