Glycocin F (GccF), a ribosomally synthesized, post-translationally modified peptide secreted by Lactobacillus plantarum KW30, rapidly inhibits the growth of susceptible bacteria at nanomolar ...concentrations. Previous studies have highlighted structural features important for its activity and have shown the absolute requirement for the Ser18 O-linked GlcNAc on the eight-residue loop linking the two short helices of the (C–X6–C)2 structure. Here, we show that an ostensibly very small chemical modification to Ser18, the substitution of the Cα proton with a methyl group, reduces the antimicrobial activity of GccF 1000-fold (IC50 1.5 μM cf. 1.5 nM). A comparison of the GccF α‑methylSer18 NMR structure (PDB 8DFZ) with that of the native protein (PDB 2KUY) showed a marked difference in the orientation and mobility of the loop, as well as a markedly different positioning of the GlcNAc, suggesting that loop conformation, dynamics, and glycan presentation play an important role in the interaction of GccF with as yet unknown but essential physiological target molecules.
Single-molecule studies continue to grow in popularity. In cases where biopolymer samples of interest exhibit variations in fine-structure between individual chains such single-molecule studies ...uniquely offer the promise of revealing deep structure-function relationships. Polysaccharides are typically studied in bulk and, as such, their study could greatly benefit from the application of single-molecule techniques. However, while for example single-molecule optical tweezers (OT) studies have become commonplace for DNA, studies of polysaccharides have lagged behind somewhat, complicated by the difficulty of studying molecules that amongst other things have more complex end-group chemistry. Recently, divalent streptavidin linkers have been shown to be capable of concatenating two pieces of biotin-terminated DNA to produce robust composite strings that run intact through conventional gels, and can be used in single-molecule OT experiments (Mohandas, Kent, Raudsepp, Jameson, & Williams, 2022). By using two such streptavidin linkers, biotin-terminated polymers could be inserted between two sections of DNA in order to facilitate single-molecule experiments on biopolymers that are currently difficult to address by other means. Here, we describe a generic approach for placing the required biotin moieties at both ends of polysaccharide chains, producing plug-and-play polysaccharide inserts that can be incorporated into composite polymer strings using streptavidin linking hubs.
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APOBEC3 proteins are double-edged swords. They deaminate cytosine to uracil in single-stranded DNA and provide protection, as part of our innate immune system, against viruses and retrotransposons, ...but they are also involved in cancer evolution and development of drug resistance. We report a solution-state model of APOBEC3A interaction with its single-stranded DNA substrate obtained with the 'method of small changes'. This method compares pairwise the 2D 15N-1H NMR spectra of APOBEC3A bearing a deactivating mutation E72A in the presence of 36 slightly different DNA substrates. From changes in chemical shifts of peptide N-H moieties, the positions of each nucleotide relative to the protein can be identified. This provided distance restraints for molecular-dynamic simulations to derive a 3-D molecular model of the APOBEC3A-ssDNA complex. The model reveals that loops 1 and 7 of APOBEC3A move to accommodate substrate binding, indicating an important role for protein-DNA dynamics. Overall, our method may prove useful to study other DNA-protein complexes where crystallographic techniques or full NMR structure calculations are hindered by weak binding or other problems. Subsequent to submission, an APOBEC3A structure with a bound DNA oligomer was published and coordinates released, which has provided an unbiased validation of the 'method of small changes'.
Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates ...protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1‐Gcn2 interaction by binding to Gcn1, thus preventing Gcn2‐mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.
The thermodynamic stability of a cytosine(C)‐rich i‐motif tract of DNA, which features pH‐sensitive C..H..C+ moieties, has been studied as function of both pressure (0.1–200 MPa) and pH (3.7–6.2). ...Careful attention was paid to correcting citrate buffer pH for known variations that stem from changes in pressure. Once pH‐corrected, (i) at pH >4.6 the i‐motif becomes less stable as pressure is increased (KD decreases), giving a small negative volume change for dissociation (ΔDV°) of the i‐motif – a conclusion opposite to that which would be drawn if the buffer pH was not corrected for the effects of pressure; (ii) the i‐motif's melting temperature increases by more than 30 K between pH 6.5 and 4.5, the consequence of an enthalpy for dissociation (ΔDH°) of 77(3) and 90(3) kJ (mol H+)−1 at 0.1 and 200 MPa, respectively; (iii) below pH 4.6 at 0.1 MPa (pH 4.3 at 200 MPa) the melting temperature decreases as a result of double protonation of cytosine pairs, and ΔDH° and ΔDV° change signs; and (iv) the combination of ΔDH° and ΔDV° lead to the melting temperature at pH 4.3 being 3 K higher at 200 MPa than at 0.1 MPa.
Pressure correction: By means of 1H NMR spectroscopy of cytosine‐rich i‐motif C…H…C+ moieties, the melting point Tm as a function of pH and pressure reveals a strong dependence on pH and, when pH is properly corrected for the effects of pressure, a moderate destabilisation of the i‐motif as pressure is increased at a given pH.
The synthesis and characterization of the fluxional, d10 cyclotriphosphazene complexes, MLCl2 (M = Zn, Cd, and Hg; L = ...spiro-(1,1′-biphenyl)-2,2′-dioxytetrakis(4-methyl-2-pyridyloxy)cyclotriphosphazene), are described. Single-crystal X-ray structures show that the zinc complex has crystallized into two crystal forms: one as a tetrahedral species, with a N2Cl2 donor set in which a geminal pair of the pendant pyridyloxy nitrogen atoms binds to the zinc, and the other as a trigonal-bipyramidal (tbp) one, with an N3Cl2 donor set. The third nitrogen atom comes from the phosphazene ring and the two pyridyl ligands are non-geminal. The asymmetric unit of the cadmium complex contains three structurally distinct molecules. One molecule has a tbp structure similar to that of the zinc complex. The second molecule has a six-coordinate, distorted octahedral geometry around the cadmium center with a N4Cl2 donor set, with three of the nitrogen donor atoms coming from the pendant pyridyloxy arms. The third site contains a tbp complex and a distorted octahedral species with a relative occupancy of 3:1. The identification of these three different forms in the one crystal suggests that the energy difference between the tbp and distorted octahedral isomers is not large. Quantitative analysis of the 1H NMR and variable-temperature 31P NMR spectra of the zinc, cadmium, and mercury complexes in a CD2Cl2 solution, coupled with the X-ray structural results, shows that an associative fluxional mechanism (ΔS ⧧ < −65 J mol–1 K–1) is operating.
Complex formation between sodium caseinate and gum arabic as a function of temperature was investigated using dynamic light scattering, fluorescence and NMR. At neutral pH, the turbidity and the ...particle size increased when sodium caseinate and gum arabic mixtures were heated
in situ at temperatures above a critical temperature. The increases in turbidity and particle size were reversible. This effect was considered to be due to hydrophobic interactions, leading to the formation of a complex between sodium caseinate and gum arabic.
1H NMR spectroscopy showed that ANS, which bound to caseinate at low temperatures in caseinate solution or a caseinate–gum arabic mixture, was released at high temperatures upon formation of a caseinate or caseinate–gum arabic complex. This supported changes observed in the fluorescence of 8-anilino-1-naphthalene sulfonate upon binding to caseinate, which decreased at high temperatures for caseinate alone or when sodium caseinate was mixed with gum arabic. Light-scattering (turbidity) and dynamic light-scattering studies show that the temperature-dependent complexation between sodium caseinate and gum arabic was sensitive to the mass ratio of protein to gum arabic (greater complexation at a 1:5 ratio than a 1:1 ratio) and the pH (maximum complexation at pH 6.5).
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The relatively low chemical stability of cytosine compared with other nucleobases is a key concern in origin‐of‐life scenarios, but the effect of pressure on the rate of hydrolysis of cytosine to ...uracil remains unknown. Through in situ NMR spectroscopy measurements, it has been determined that the half‐life of cytosine at 373.15 K decreases from (18.0±0.7) days at ambient pressure (0.1 MPa) to (8.64±0.18) days at high pressure (200 MPa). This yields an activation volume for hydrolysis of (−11.8±0.5) cm3 mol−1; a decrease that is similar to the molar volume of water (18.0 cm3 mol−1) and consistent with a tetrahedral 3,3‐hydroxyamine transition‐state/intermediate species. Similar behaviour was also observed for cytidine. At both ambient and high pressures, the half‐life of cytosine decreases significantly as the pH decreases from 7.0 to 6.0. These results provide scant support for the notion that RNA‐based life forms originated in high‐temperature, high‐pressure, acidic environments.
Back to the beginning: The relatively low chemical stability of cytosine compared with other nucleobases is a key concern in origin‐of‐life scenarios, but the effect of pressure on the rate of hydrolysis of cytosine to uracil has remained unknown. At high (200 MPa) pressures, the half‐life of cytosine at 100 °C is halved to 8.6 days compared with that at ambient pressure.
The production of soluble and correctly folded eukaryotic proteins in prokaryotic systems has always been hampered by the difference in or lack of cell machinery responsible for folding, ...post-translation modification and secretion of the proteins involved. In the case of bovine β-lactoglobulin (BLG), a major cow’s milk allergen and a protein widely used for protein folding studies, a eukaryotic yeast expression system has been the preferred choice of many researchers, particularly for the production of isotopically labeled protein required for NMR studies. Although this system yields high amounts of recombinant protein, the BLG produced is usually associated with extracellular polysaccharides, which is problematic for NMR analysis. In our study we show that when co-expressed with the signal-sequence-less disulfide bond isomerase (ΔssDsbC) in the dual expression vector, pETDUET-1, both BLG A and BLG B can be reproducibly produced in a soluble form. Expression was carried out in
Escherichia coli Origami(DE3)
, a
trxB/
gor mutant for thioredoxin- and glutathione reductase, which allows for proper formation of disulfide bonds in the cytoplasm. The protein was purified by anion exchange chromatography followed by salting-out at low pH and size exclusion chromatography. Our expression system is able to consistently produce milligram quantities of correctly folded BLG A and B with no additional amino acid residues at the N-terminus, except for a methionine.
15N-labeled BLG A and B, prepared and purified using this method, produced HSQC spectra typical of native bovine BLG.
Tyrosine 34 and glutamine 146 are highly conserved outer sphere residues in the mononuclear manganese active site of Escherichia coli manganese superoxide dismutase. Biochemical and spectroscopic ...characterization of site-directed mutants has allowed functional characterization of these residues in the wild-type (wt) enzyme. X-ray crystallographic analysis of three mutants (Y34F, Q146L, and Q146H) reveal subtle changes in the protein structures. The Y34A mutant, as well as the previously reported Y34F mutant, retained essentially the full superoxide dismutase activity of the wild-type enzyme, and the X-ray crystal structure of Y34F manganese superoxide dismutase shows that mutation of this strictly conserved residue has only minor effects on the positions of active site residues and the organized water in the substrate access funnel. Mutation of the outer sphere solvent pocket residue Q146 has more dramatic effects. The Q146E mutant is isolated as an apoprotein lacking dismutase activity. Q146L and Q146H mutants retain only 5−10% of the dismutase activity of the wild-type enzyme. The absorption and circular dichroism spectra of the Q146H mutant resemble corresponding data for the superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilum, which is active in both Mn and Fe forms. Interestingly, the iron-substituted Q146H protein also exhibits low dismutase activity, which increases at lower pH. Mutation of glutamine 146 disrupts the hydrogen-bonding network in the active site and has a greater effect on protein structure than does the Y34F mutant, with rearrangement of the tyrosine 34 and tryptophan 128 side chains.
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