Nickel(II), zinc(II) and cadmium(II) complexes of hexapeptides containing separate histidyl and cysteinyl binding sites

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Inhibition of protein phosphatase-1 and -2A by ellagitannins: structure-inhibitory potency relationships and influences on cellular systems

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Investigation of the Molecular Details of the Interactions of Selenoglycosides and Human Galectin-3

Human galectin-3 (hGal-3) is involved in a variety of biological processes and is implicated in wide range of diseases. As a result, targeting hGal-3 for clinical applications has become an intense area of research. As a step towards the development of novel hGal-3 inhibitors, we describe a study of the binding of two Se-containing hGal-3 inhibitors, specifically that of di(β-Dgalactopyranosyl)selenide (SeDG), in which two galactose rings are linked by one Se atom and a di(β-D-galactopyranosyl)diselenide (DSeDG) analogue with a diseleno bond between the two sugar units. The binding affinities of these derivatives to hGal-3 were determined by 15N-1H HSQC NMR spectroscopy and fluorescence anisotropy titrations in solution, indicating a slight decrease in the strength of interaction for SeDG compared to thiodigalactoside (TDG), a well-known inhibitor of hGal-3, while DSeDG displayed a much weaker interaction strength. NMR and FA measurements showed that both seleno derivatives bind to the canonical S face site of hGal-3 and stack against the conserved W181 residue also confirmed by X-ray crystallography, revealing canonical properties of the interaction. The interaction with DSeDG revealed two distinct binding modes in the crystal structure which are in fast exchange on the NMR time scale in solution, explaining a weaker interaction with hGal-3 than SeDG. Using molecular dynamics simulations, we have found that energetic contributions to the binding enthalpies mainly differ in the electrostatic interactions and in polar solvation terms and are responsible for weaker binding of DSeDG compared to SeDG. Selenium-containing carbohydrate inhibitors of hGal-3 showing canonical binding modes offer the potential of becoming novel hydrolytically stable scaffolds for a new class of hGal-3 inhibitors

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule

Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC

Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be “silent” in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated

Inhibition of protein phosphatase-1 and -2A by ellagitannins: structure-inhibitory potency relationships and influences on cellular systems

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Introducing 77Se Spectroscopy to Analyzing Galectin–Ligand Interaction

Their emerging nature as multifunctional effectors explains the large interest to monitor glycan binding to galectins and to define bound-state conformer(s) of their ligands in solution. Basically, NMR spectroscopy facilitates respective experiments. Towards developing new and even better approaches for these purposes, extending the range of exploitable isotopes beyond1H,13C, and15N offers promising perspectives. Having therefore prepared selenodigalactoside and revealed its bioactivity as galectin ligand, monitoring of its binding by77Se NMR spectroscopy at a practical level becomes possible by setting up a 2D1H,77Se CPMG-HSQBMC experiment including CPMG-INEPT long-range transfer. This first step into applying77Se as sensor for galectin binding substantiates its potential for screening relative to inhibitory potencies in compound mixtures and for achieving sophisticated epitope mapping. The documented strategic combination of synthetic carbohydrate chemistry and NMR spectroscopy prompts to envision to work with isotopically pure77Se-containing β-galactosides and to build on the gained experience with77Se by adding19F as second sensor in doubly labeled glycosides. © 2022, Springer Science+Business Media, LLC, part of Springer Nature

Nickel(II), zinc(II) and cadmium(II) complexes of hexapeptides containing separate histidyl and cysteinyl binding sites

Nickel(II), zinc(II) and cadmium(II) complexes of two N-terminally free hexapeptides AAHAAC and AHAAAC containing separate histidyl and cysteinyl residues have been studied using potentiometric and various spectroscopic (UV-Vis, CD, 1H-NMR) techniques. Both peptides have outstanding metal binding ability but the speciation of the systems and the binding sites of peptides reveal a significant specificity. In the nickel(II)–AAHAAC system the amino terminus is the primary nickel(II) binding site in the form of the (NH2,N−,N−,Nim) chelate. However, the C-terminal thiolate function can bind another nickel(II) ion with the involvement of amide nitrogens in metal binding. Zinc(II) and cadmium(II)ions are not able to promote deprotonation and coordination of the peptide amide groups of AAHAAC and only mononuclear complexes are formed, in which imidazole-N and thiolate-S− donors are the primary metal binding sites. In the case of AHAAAC both nickel(II) and zinc(II) ions can induce deprotonation and coordination of the first amide bond in the sequence. This results in the enhanced stability of the corresponding species, [MH−1L], containing a tridentate (NH2,N−,Nim) binding mode at the amino terminus supported by a macrochelate from the distant thiolate group

The effect of carboxylate groups on the complexation of metal ion with oligopeptides - Potentiometric investigation

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The effect of carboxylate groups on the complexation of metal ion with oligopeptides - Potentiometric investigation

Iron(II), iron(III), cobalt(II), zinc(II) cadmium(II) and lead(II) complexes of Asp2 and Asp3 and other Asp/Glu containing ligands were studied by potentiometry. Our goal was to study the influence of β- and γ-carboxylate groups as well as the increased negative charge of the ligands on the complex formation processes. Mainly 1:1 species are present, the formation of bis(ligand) complexes is not typical for these systems. It can be concluded that the effect of side chain carboxylate groups on the stability of complexes is especially significant in the case of lead(II) and cadmium(II). The greater the number of carboxylic groups there are in the system, the more stable complexes are formed with the studied metal ions. The influence of β-carboxylate groups of aspartic acid is unambiguous, they are directly bounded to the metal ion, which results in increased stability of the complexes. On the other hand, the repulsion between the negative charged residues becomes conspicuous resulting less stable species in the case of peptides containing glutamic acid. © 2017 Elsevier B.V