[Fe(µ2-OH)6]3- Linked Fe3O Triads: Mössbauer Evidence for Trigonal µ3-O2- or µ3-OH- Groups in Bridged versus Unbridged Complexes.

The syntheses, coordination chemistry, and Mössbauer spectroscopy of hepta-iron(III) complexes using derivatised salicylaldoxime ligands from two categories; namely, 'single-headed' (H2L) and 'double-headed' (H4L) salicylaldoximes are described. All compounds presented here share a [Fe3-µ3-O] core in which the iron(III) ions are µ3-hydroxo-bridged in the complex C1 and µ3-oxo-bridged in C2 and C3. Each compound consists of 2 × [Fe3-µ3-O] triads that are linked via a central [Fe(µ2-OH)6]3- ion. In addition to the charge balance and microanalytical evidence, Mössbauer measurements support the fact that the triads in C1 are µ3-OH bridged and are µ3-O bridged in C2 and C3.

Synthesis of 1,4-azaphosphinine nucleosides and evaluation as inhibitors of human cytidine deaminase and APOBEC3A.

Nucleoside and polynucleotide cytidine deaminases (CDAs), such as CDA and APOBEC3, share a similar mechanism of cytosine to uracil conversion. In 1984, phosphapyrimidine riboside was characterised as the most potent inhibitor of human CDA, but the quick degradation in water limited the applicability as a potential therapeutic. To improve stability in water, we synthesised derivatives of phosphapyrimidine nucleoside having a CH2 group instead of the N3 atom in the nucleobase. A charge-neutral phosphinamide and a negatively charged phosphinic acid derivative had excellent stability in water at pH 7.4, but only the charge-neutral compound inhibited human CDA, similar to previously described 2'-deoxyzebularine (Ki = 8.0 ± 1.9 and 10.7 ± 0.5 µM, respectively). However, under basic conditions, the charge-neutral phosphinamide was unstable, which prevented the incorporation into DNA using conventional DNA chemistry. In contrast, the negatively charged phosphinic acid derivative was incorporated into DNA instead of the target 2'-deoxycytidine using an automated DNA synthesiser, but no inhibition of APOBEC3A was observed for modified DNAs. Although this shows that the negative charge is poorly accommodated in the active site of CDA and APOBEC3, the synthetic route reported here provides opportunities for the synthesis of other derivatives of phosphapyrimidine riboside for potential development of more potent CDA and APOBEC3 inhibitors.

NMR Shows Why a Small Chemical Change Almost Abolishes the Antimicrobial Activity of Glycocin F.

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 GccFα-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.

Seven-membered ring nucleobases as inhibitors of human cytidine deaminase and APOBEC3A.

The APOBEC3 (APOBEC3A-H) enzyme family as a part of the human innate immune system deaminates cytosine to uracil in single-stranded DNA (ssDNA) and thereby prevents the spread of pathogenic genetic information. However, APOBEC3-induced mutagenesis promotes viral and cancer evolution, thus enabling the progression of diseases and development of drug resistance. Therefore, APOBEC3 inhibition offers a possibility to complement existing antiviral and anticancer therapies and prevent the emergence of drug resistance, thus making such therapies effective for longer periods of time. Here, we synthesised nucleosides containing seven-membered nucleobases based on azepinone and compared their inhibitory potential against human cytidine deaminase (hCDA) and APOBEC3A with previously described 2'-deoxyzebularine (dZ) and 5-fluoro-2'-deoxyzebularine (FdZ). The nanomolar inhibitor of wild-type APOBEC3A was obtained by the incorporation of 1,3,4,7-tetrahydro-2H-1,3-diazepin-2-one in the TTC loop of a DNA hairpin instead of the target 2'-deoxycytidine providing a Ki of 290 ± 40 nM, which is only slightly weaker than the Ki of the FdZ-containing inhibitor (117 ± 15 nM). A less potent but notably different inhibition of human cytidine deaminase (CDA) and engineered C-terminal domain of APOBEC3B was observed for 2'-deoxyribosides of the S and R isomers of hexahydro-5-hydroxy-azepin-2-one: the S-isomer was more active than the R-isomer. The S-isomer shows resemblance in the position of the OH-group observed recently for the hydrated dZ and FdZ in the crystal structures with APOBEC3G and APOBEC3A, respectively. This shows that 7-membered ring analogues of pyrimidine nucleosides can serve as a platform for further development of modified ssDNAs as powerful A3 inhibitors.

Assessment of Various Food Proteins as Structural Materials for Delivery of Hydrophobic Polyphenols Using a Novel Co-Precipitation Method.

In this study, sodium caseinate (NaCas), soy protein isolate (SPI), and whey protein isolate (WPI) were used as structural materials for the delivery of rutin, naringenin, curcumin, hesperidin, and catechin. For each polyphenol, the protein solution was brought to alkaline pH, and then the polyphenol and trehalose (as a cryo-protectant) were added. The mixtures were later acidified, and the co-precipitated products were lyophilized. Regardless of the type of protein used, the co-precipitation method exhibited relatively high entrapment efficiency and loading capacity for all five polyphenols. Several structural changes were seen in the scanning electron micrographs of all polyphenol-protein co-precipitates. This included a significant decrease in the crystallinity of the polyphenols, which was confirmed by X-ray diffraction analysis, where amorphous structures of rutin, naringenin, curcumin, hesperidin, and catechin were revealed after the treatment. Both the dispersibility and solubility of the lyophilized powders in water were improved dramatically (in some cases, >10-fold) after the treatment, with further improvements observed in these properties for the powders containing trehalose. Depending on the chemical structure and hydrophobicity of the tested polyphenols, there were differences observed in the degree and extent of the effect of the protein on different properties of the polyphenols. Overall, the findings of this study demonstrated that NaCas, WPI, and SPI can be used for the development of an efficient delivery system for hydrophobic polyphenols, which in turn can be incorporated into various functional foods or used as supplements in the nutraceutical industry.

Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A.

Drug resistance is a major problem associated with anticancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms have revealed that enzymes of the APOBEC3 (A3) family contribute to the development of drug resistance in multiple cancers. A3 enzymes are polynucleotide cytidine deaminases that convert cytosine to uracil (C→U) in single-stranded DNA (ssDNA) and in this way protect humans against viruses and mobile retroelements. On the other hand, cancer cells use A3s, especially A3A and A3B, to mutate human DNA, and thus by increasing rates of evolution, cancer cells escape adaptive immune responses and resist drugs. However, as A3A and A3B are non-essential for primary metabolism, their inhibition opens up a strategy to augment existing anticancer therapies and suppress cancer evolution. To test our hypothesis that pre-shaped ssDNA mimicking the U-shape observed in ssDNA-A3 complexes can provide a better binder to A3 enzymes, a Cu(I)-catalyzed azide-alkyne cycloaddition was used to cross-link two distant modified nucleobases in ssDNA. The resultant cytosine-containing substrate, where the cytosine sits at the apex of the loop, was deaminated faster by the engineered C-terminal domain of A3B than a standard, linear substrate. The cross-linked ssDNA was converted into an A3 inhibitor by replacing the 2'-deoxycytidine in the preferred TCA substrate motif by 2'-deoxyzebularine, a known inhibitor of single nucleoside cytidine deaminases. This strategy yielded the first nanomolar inhibitor of engineered A3BCTD and wild-type A3A (Ki = 690 ± 140 and 360 ± 120 nM, respectively), providing a platform for further development of powerful A3 inhibitors.

The LONELY GUY gene family: from mosses to wheat, the key to the formation of active cytokinins in plants.

LONELY GUY (LOG) was first identified in a screen of rice mutants with defects in meristem maintenance. In plants, LOG codes for cytokinin riboside 5'-monophosphate phosphoribohydrolase, which converts inactive cytokinin nucleotides directly to the active free bases. Many enzymes with the PGGxGTxxE motif have been misannotated as lysine decarboxylases; conversely not all enzymes containing this motif are cytokinin-specific LOGs. As LOG mutants clearly impact yield in rice, we investigated the LOG gene family in bread wheat. By interrogating the wheat (Triticum aestivum) genome database, we show that wheat has multiple LOGs. The close alignment of TaLOG1, TaLOG2 and TaLOG6 with the X-ray structures of two functional Arabidopsis thaliana LOGs allows us to infer that the wheat LOGs 1-11 are functional LOGs. Using RNA-seq data sets, we assessed TaLOG expression across 70 tissue types, their responses to various stressors, the pattern of cis-regulatory elements (CREs) and intron/exon patterns. TaLOG gene family members are expressed variously across tissue types. When the TaLOG CREs are compared with those of the cytokinin dehydrogenases (CKX) and glucosyltransferases (CGT), there is close alignment of CREs between TaLOGs and TaCKXs reflecting the key role of CKX in maintaining cytokinin homeostasis. However, we suggest that the main homeostatic mechanism controlling cytokinin levels in response to biotic and abiotic challenge resides in the CGTs, rather than LOG or CKX. However, LOG transgenics and identified mutants in rice variously impact yield, providing interesting avenues for investigation in wheat.

Estimating orientation of optically trapped, near vertical, microsphere dimers using central moments and off-focus imaging.

Near vertical optically trapped dimers, composed of pairs of microspheres, and constructed in situ, were imaged in bright-field in flow and at rest, and with displacement Δz from the transverse xy imaging plane of an inverted microscope. Image first central moments µ01 were measured, and their dependence on the imposed flow velocity of the surrounding fluid was calculated. This dependence was related to the at-rest restricted diffusion statistics. It was assumed that, for small perturbations, the torque T on the dimer was proportional to the velocity of flow v and resulting angular deflection Δθ so that T∝v∝Δθ. Displacements Δz at which vâˆΔµ01∝Δθ, which are typically off focus, were examined in more detail; in this range, Δθ=hΔµ01. The hydrodynamics of the dimer were modeled as that of a prolate ellipsoid, and the constant of proportionality h was determined by comparing the short-time mean-squared variation measured during diffusion to that predicted by the model calculation: h2⟨Δµ012(t)⟩=⟨Δθ2(t)⟩. With h determined, the optical trap stiffness kθ was determined from the long-time restricted diffusion of the dimer. The measured kθ and Δθ can then be used compute torque: T=kθΔθ, potentially enabling the near vertical optically trapped dimer to be used as a torque probe.

Heterochromatin protein 1α interacts with parallel RNA and DNA G-quadruplexes.

The eukaryotic genome is functionally organized into domains of transcriptionally active euchromatin and domains of highly compact transcriptionally silent heterochromatin. Heterochromatin is constitutively assembled at repetitive elements that include the telomeres and centromeres. The histone code model proposes that HP1α forms and maintains these domains of heterochromatin through the interaction of its chromodomain with trimethylated lysine 9 of histone 3, although this interaction is not the sole determinant. We show here that the unstructured hinge domain, necessary for the targeting of HP1α to constitutive heterochromatin, recognizes parallel G-quadruplex (G4) assemblies formed by the TElomeric Repeat-containing RNA (TERRA) transcribed from the telomere. This provides a mechanism by which TERRA can lead to the enrichment of HP1α at telomeres to maintain heterochromatin. Furthermore, we show that HP1α binds with a faster association rate to DNA G4s of parallel topology compared to antiparallel G4s that bind slowly or not at all. Such G4-DNAs are found in the regulatory regions of several oncogenes. This implicates specific non-canonical nucleic acid structures as determinants of HP1α function and thus RNA and DNA G4s need to be considered as contributors to chromatin domain organization and the epigenome.

The Effect of pH and Sodium Caseinate on the Aqueous Solubility, Stability, and Crystallinity of Rutin towards Concentrated Colloidally Stable Particles for the Incorporation into Functional Foods.

Poor water solubility and low bioavailability of hydrophobic flavonoids such as rutin remain as substantial challenges to their oral delivery via functional foods. In this study, the effect of pH and the addition of a protein (sodium caseinate; NaCas) on the aqueous solubility and stability of rutin was studied, from which an efficient delivery system for the incorporation of rutin into functional food products was developed. The aqueous solubility, chemical stability, crystallinity, and morphology of rutin (0.1-5% w/v) under various pH (1-11) and protein concentrations (0.2-8% w/v) were studied. To manufacture the concentrated colloidally stable rutin-NaCas particles, rutin was dissolved and deprotonated in a NaCas solution at alkaline pH before its subsequent neutralisation at pH 7. The excess water was removed using ultrafiltration to improve the loading capacity. Rutin showed the highest solubility at pH 11, while the addition of NaCas resulted in the improvement of both solubility and chemical stability. Critically, to achieve particles with colloidal stability, the NaCas:rutin ratio (w/w) had to be greater than 2.5 and 40 respectively for the lowest (0.2% w/v) and highest (4 to 8% w/v) concentrations of NaCas. The rutin-NaCas particles in the concentrated formulations were physically stable, with a size in the range of 185 to 230 nm and zeta potential of -36.8 to -38.1 mV, depending on the NaCas:rutin ratio. Encapsulation efficiency and loading capacity of rutin in different systems were 76% to 83% and 2% to 22%, respectively. The concentrated formulation containing 5% w/v NaCas and 2% w/v rutin was chosen as the most efficient delivery system due to the ideal protein:flavonoid ratio (2.5:1), which resulted in the highest loading capacity (22%). Taken together, the findings show that the delivery system developed in this study can be a promising method for the incorporation of a high concentration of hydrophobic flavonoids such as rutin into functional foods.

Exploring modular allostery via interchangeable regulatory domains.

Most proteins comprise two or more domains from a limited suite of protein families. These domains are often rearranged in various combinations through gene fusion events to evolve new protein functions, including the acquisition of protein allostery through the incorporation of regulatory domains. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of aromatic amino acid biosynthesis and displays a diverse range of allosteric mechanisms. DAH7PSs adopt a common architecture with a shared (ß/α)8 catalytic domain which can be attached to an ACT-like or a chorismate mutase regulatory domain that operates via distinct mechanisms. These respective domains confer allosteric regulation by controlling DAH7PS function in response to ligand Tyr or prephenate. Starting with contemporary DAH7PS proteins, two protein chimeras were created, with interchanged regulatory domains. Both engineered proteins were catalytically active and delivered new functional allostery with switched ligand specificity and allosteric mechanisms delivered by their nonhomologous regulatory domains. This interchangeability of protein domains represents an efficient method not only to engineer allostery in multidomain proteins but to create a new bifunctional enzyme.

Differential Inhibition of APOBEC3 DNA-Mutator Isozymes by Fluoro- and Non-Fluoro-Substituted 2'-Deoxyzebularine Embedded in Single-Stranded DNA.

The APOBEC3 (APOBEC3A-H) enzyme family is part of the human innate immune system that restricts pathogens by scrambling pathogenic single-stranded (ss) DNA by deamination of cytosines to produce uracil residues. However, APOBEC3-mediated mutagenesis of viral and cancer DNA promotes its evolution, thus enabling disease progression and the development of drug resistance. Therefore, APOBEC3 inhibition offers a new strategy to complement existing antiviral and anticancer therapies by making such therapies effective for longer periods of time, thereby preventing the emergence of drug resistance. Here, we have synthesised 2'-deoxynucleoside forms of several known inhibitors of cytidine deaminase (CDA), incorporated them into oligodeoxynucleotides (oligos) in place of 2'-deoxycytidine in the preferred substrates of APOBEC3A, APOBEC3B, and APOBEC3G, and evaluated their inhibitory potential against these enzymes. An oligo containing a 5-fluoro-2'-deoxyzebularine (5FdZ) motif exhibited an inhibition constant against APOBEC3B 3.5 times better than that of the comparable 2'-deoxyzebularine-containing (dZ-containing) oligo. A similar inhibition trend was observed for wild-type APOBEC3A. In contrast, use of the 5FdZ motif in an oligo designed for APOBEC3G inhibition resulted in an inhibitor that was less potent than the dZ-containing oligo both in the case of APOBEC3GCTD and in that of full-length wild-type APOBEC3G.

On the utility of fluorescence-detection analytical ultracentrifugation in probing biomolecular interactions in complex solutions: a case study in milk.

ß-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products. However, the physiological function of ß-lactoglobulin remains unclear. Using the fluorescence-detection system within the analytical ultracentrifuge, we observed an interaction involving fluorescently labelled ß-lactoglobulin in its native environment, i.e. cow and goat milk, for the first time. Co-elution experiments support that these ß-lactoglobulin interactions occur naturally in milk and provide evidence that the interacting partners are immunoglobulins, while further sedimentation velocity experiments confirm that an interaction occurs between these molecules. The identification of these interactions, made possible through the use of fluorescence-detected analytical ultracentrifugation, provides possible clues to the long debated physiological function of this abundant milk protein.

Inhibiting APOBEC3 Activity with Single-Stranded DNA Containing 2'-Deoxyzebularine Analogues.

APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy for augmenting existing antiviral and anticancer therapies. Here we show that, upon incorporation into short ssDNAs, the cytidine nucleoside analogue 2'-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and nuclear magnetic resonance spectroscopy assays, demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ-containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity.

Effects of Pressure and pH on the Physical Stability of an I-Motif DNA Structure.

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 (ΔD V°) 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 (ΔD H°) 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 ΔD H° and ΔD V° change signs; and (iv) the combination of ΔD H° and ΔD V° lead to the melting temperature at pH 4.3 being 3 K higher at 200 MPa than at 0.1 MPa.

Selective inhibition of APOBEC3 enzymes by single-stranded DNAs containing 2'-deoxyzebularine.

To restrict pathogens, in a normal human cell, APOBEC3 enzymes mutate cytosine to uracil in foreign single-stranded DNAs. However, in cancer cells, APOBEC3B (one of seven APOBEC3 enzymes) has been identified as the primary source of genetic mutations. As such, APOBEC3B promotes evolution and progression of cancers and leads to development of drug resistance in multiple cancers. As APOBEC3B is a non-essential protein, its inhibition can be used to suppress emergence of drug resistance in existing anti-cancer therapies. Because of the vital role of APOBEC3 enzymes in innate immunity, selective inhibitors targeting only APOBEC3B are required. Here, we use the discriminative properties of wild-type APOBEC3A, APOBEC3B and APOBEC3G to deaminate different cytosines in the CCC-recognition motif in order to best place the cytidine analogue 2'-deoxyzebularine (dZ) in the CCC-motif. Using several APOBEC3 variants that mimic deamination patterns of wild-type enzymes, we demonstrate that selective inhibition of APOBEC3B in preference to other APOBEC3 constructs is feasible for the dZCC motif. This work is an important step towards development of in vivo tools to inhibit APOBEC3 enzymes in living cells by using short, chemically modified oligonucleotides.

NMR-based method of small changes reveals how DNA mutator APOBEC3A interacts with its single-stranded DNA substrate.

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'.

A Pseudoisostructural Type II DAH7PS Enzyme from Pseudomonas aeruginosa: Alternative Evolutionary Strategies to Control Shikimate Pathway Flux.

The shikimate pathway is responsible for the biosynthesis of key aromatic metabolites in microorganisms and plants. The enzyme 3-deoxy-d- arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the pathway and DAH7PSs are classified as either type I or type II. The DAH7PSs from Pseudomonas aeruginosa are of particular interest as open reading frames encoding four putative DAH7PS isoenzymes, two classified as type Iα and two classified as type II, have been identified. Here, the structure of a type II DAH7PS enzyme from P. aeruginosa (PAO1) has been determined at 1.54 Å resolution, in complex with its allosteric inhibitor tryptophan. Structural differences in the extra-barrel elements, when compared to other type II DAH7PS enzymes, directly relate to the formation of a distinct quaternary conformation with consequences for allosteric function and the control of flux to branching pathways. In contrast to the well-characterized Mycobacterium tuberculosis type II DAH7PS, which binds multiple allosteric inhibitors, this PaeDAH7PSPA2843 is observed to be modestly allosterically inhibited by a single aromatic amino acid, tryptophan. In addition, structures in complex with tyrosine or with no allosteric ligand bound were determined. These structures provide new insights into the linkages between the active and allosteric sites. With four putative DAH7PS enzymes, P. aeruginosa appears to have evolved control of shikimate pathway flux at the genetic level, rather than control by multiple allosteric effectors to a single type II DAH7PS, as in M. tuberculosis. Type II DAH7PSs, thus, appear to have a more varied evolutionary trajectory than previously indicated.

Effects of Pressure and pH on the Hydrolysis of Cytosine: Implications for Nucleotide Stability around Deep-Sea Black Smokers.

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.

The self-association and thermal denaturation of caprine and bovine ß-lactoglobulin.

Milk components, such as proteins and lipids, have different physicochemical properties depending upon the mammalian species from which they come. Understanding the different responses of these milks to digestion, processing, and differences in their immunogenicity requires detailed knowledge of these physicochemical properties. Here we report on the oligomeric state of ß-lactoglobulin from caprine milk, the most abundant protein present in the whey fraction. At pH 2.5 caprine ß-lactoglobulin is predominantly monomeric, whereas bovine ß-lactoglobulin exists in a monomer-dimer equilibrium at the same protein concentrations. This behaviour was also observed in molecular dynamics simulations and can be rationalised in terms of the amino acid substitutions present between caprine and bovine ß-lactoglobulin that result in a greater positive charge on each subunit of caprine ß-lactoglobulin at low pH. The denaturation of ß-lactoglobulin when milk is heat-treated contributes to the fouling of heat-exchange surfaces, reducing yields and increasing cleaning costs. The bovine and caprine orthologues of ß-lactoglobulin display different responses to thermal treatment, with caprine ß-lactoglobulin precipitating at higher pH values than bovine ß-lactoglobulin (pH 7.1 compared to pH 5.6) that are closer to the natural pH of these milks (pH 6.7). This property of caprine ß-lactoglobulin likely contributes to the reduced heat stability of caprine milk compared to bovine milk at its natural pH.