Heidi Korhonen

The Mechanism of Cleavage and Isomerisation of RNA Promoted by an Efficient Dinuclear Zn2+ Complex

The cleavage and isomerisation of uridine 3-alkylphosphates was studied in the presence of a dinuclear Zn(2+) complex, 3. The rate acceleration of the cleavage by 1 mM 3 is approximately 10(6)-fold under neutral conditions. Most remarkably, the complex also promotes the isomerisation of phosphodiester bonds, although the rate-enhancement is more modest: under neutral conditions complex 3 (1 mM) catalyses isomerisation by about 500-fold. The observation of this reaction shows that the reactions of these substrates catalysed by 3 proceed through a stepwise mechanism involving an intermediate phosphorane. A β(lg) value of -0.92 was determined for the 3-promoted cleavage reaction, and modest kinetic solvent deuterium isotope effects ranging from 1.5 to 2.8 were observed. Isomerisation was less sensitive to the nature of the esterifying group, with a β value of -0.5, and the kinetic solvent deuterium isotope effects were less than 1.5. Most of these characteristics of the 3-promoted cleavage are very similar to those for the cleavage of nucleoside 3-phosphotriesters. These data are explained by a mechanism in which the complex primarily acts as an electrophilic catalyst neutralising the charge on the phosphate and stabilising an intermediate phosphorane, with general acid catalysis promoting the cleavage reaction. In contrast to the behaviour of triesters, isomerisation is significantly slower than cleavage; this suggests that the changes in geometry that occur during isomerisation lead to a much less stable complex between 3 and the phosphorane intermediate.

Phosphate analogues in the dissection of mechanism.

Phosphoryl group transfer is central to genetic replication, cellular signalling and many metabolic processes. Understanding the mechanisms of phosphorylation and phosphate ester and anhydride cleavage is key to efforts towards biotechnological and biomedical exploitation of phosphate-handling enzymes. Analogues of phosphate esters and anhydrides are indispensable tools, alongside protein mutagenesis and computational methods, for the dissection of phosphoryl transfer mechanisms. Hydrolysable and non-hydrolysable phosphate analogues have provided insight into the nature and sites of phosphoryl transfer processes. Kinetic isotope effects and crystallography using transition state analogues have painted more detailed pictures of transition states and how enzymes work to stabilise them.

Detecting the Companions and Ellipsoidal Variations of RS CVn Primaries. I. σ Geminorum

To measure the properties of both components of the RS Canum Venaticorum binary σ Geminorum (σ Gem), we directly detect the faint companion, measure the orbit, obtain model-independent masses and evolutionary histories, detect ellipsoidal variations of the primary caused by the gravity of the companion, and measure gravity darkening. We detect the companion with interferometric observations obtained with the Michigan InfraRed Combiner at Georgia State Universitys Center for High Angular Resolution Astronomy Array with a primary-to-secondary H-band flux ratio of 270 ± 70. A radial velocity curve of the companion was obtained with spectra from the Tillinghast Reflector Echelle Spectrograph on the 1.5 m Tillinghast Reflector at Fred Lawrence Whipple Observatory. We additionally use new observations from the Tennessee State University Automated Spectroscopic and Photometric Telescopes (AST and APT, respectively). From our orbit, we determine model-independent masses of the components ( , ), and estimate a system age of Gyr. An average of the 27 year APT light curve of σ Gem folded over the orbital period ( days) reveals a quasi-sinusoidal signature, which has previously been attributed to active longitudes 180° apart on the surface of σ Gem. With the component masses, diameters, and orbit, we find that the predicted light curve for ellipsoidal variations due to the primary star partially filling its Roche lobe potential matches well with the observed average light curve, offering a compelling alternative explanation to the active longitudes hypothesis. Measuring gravity darkening from the light curve gives , a value slightly lower than that expected from recent theory.

Detecting the Companions and Ellipsoidal Variations of RS CVn Primaries. II. o Draconis, a Candidate for Recent Low-mass Companion Ingestion

To measure the stellar and orbital properties of the metal-poor RS CVn binary o Draconis (o Dra), we directly detect the companion using interferometric observations obtained with the Michigan InfraRed Combiner at Georgia State Universitys Center for High Angular Resolution Astronomy (CHARA) Array. The H-band flux ratio between the primary and secondary stars is the highest confirmed flux ratio (370 +/- 40) observed with long-baseline optical interferometry. These detections are combined with radial velocity data of both the primary and secondary stars, including new data obtained with the Tillinghast Reflector Echelle Spectrograph on the Tillinghast Reflector at the Fred Lawrence Whipple Observatory and the 2-m Tennessee State University Automated Spectroscopic Telescope at Fairborn Observatory. We determine an orbit from which we find model-independent masses and ages of the components (M_A = 1.35 +- 0.05 M_Sun, M_B = 0.99 +- 0.02 M_Sun, system age = 3.0 -+ 0.5 Gyr). An average of a 23-year light curve of o Dra from the Tennessee State University Automated Photometric Telescope folded over the orbital period newly reveals eclipses and the quasi-sinusoidal signature of ellipsoidal variations. The modeled light curve for our systems stellar and orbital parameters confirm these ellipsoidal variations due to the primary star partially filling its Roche lobe potential, suggesting most of the photometric variations are not due to stellar activity (starspots). Measuring gravity darkening from the average light curve gives a best-fit of beta = 0.07 +- 0.03, a value consistent with conventional theory for convective envelope stars. The primary star also exhibits an anomalously short rotation period, which, when taken with other system parameters, suggests the star likely engulfed a low-mass companion that had recently spun-up the star.

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Background: Protein allostery can be communicated purely through altered entropy. Results: Altered cAMP binding strength in CAP results in changes to entropy-driven allostery. Conclusion: The requirement to maintain allostery constrains evolution of the ligand-binding site in CAP. Significance: Entropy-driven processes can constrain amino acid covariation in evolution. Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.

PPN Pyrophosphate: A New Reagent for the Preparation of Nucleoside Triphosphates

GRAPHICAL ABSTRACT Abstract Tris{bis(triphenylphosphoranylidene) ammonium} (PPN) pyrophosphate was accessed via aqueous precipitation and desiccation. The reagent was investigated as a replacement for highly hygroscopic alkylammonium salts in Ludwig–Yoshikawa reactions for the preparation of nucleoside-5′-triphosphates.

A procedure for the preparation and isolation of nucleoside-5’-diphosphates.

Summary Tris[bis(triphenylphosphoranylidene)ammonium] pyrophosphate (PPN pyrophosphate) was used in the SN2 displacements of the tosylate ion from 5’-tosylnucleosides to afford nucleoside-5’-diphosphates. Selective precipitation permitted the direct isolation of nucleoside-5’-diphosphates from crude reaction mixtures.

Properties of stellar activity cycles

The current photometric datasets, that span decades, allow for studying long-term cycles on active stars. Complementary Ca H&K observations give information also on the cycles of normal solar-like stars, which have significantly smaller, and less easily detectable, spots. In the recent years, high precision space-based observations, for example from the Kepler satellite, have allowed also to study the sunspot-like spot sizes in other stars. Here I review what is known about the properties of the cyclic stellar activity in other stars than our Sun.

Surface magnetism of cool giant and supergiant stars

The existence of starspots on late-type giant stars in close binary systems, that exhibit rapid rotation due to tidal locking, has been known for more than five decades. Photometric monitoring spanning decades has allowed studying the long-term magnetic activity in these stars revealing complicated activity cycles. The development of observing and analysis techniques that has occurred during the past two decades has also enabled us to study the detailed starspot and magnetic field configurations on these active giants. In the recent years magnetic fields have also been detected on slowly rotating giants and supergiant stars. In this paper I review what is known of the surface magnetism in the cool giant and supergiant stars.

DNA-Templated N(Me)-Alkoxyamine Glycosylation

The potential of N(Me)-alkoxyamine glycosylation as a DNA-templated ligation has been studied. On a hairpin stem-template model, a notable rate enhancement and an increased equilibrium yield are observed compared to the corresponding reaction without a DNA catalyst. The N-glycosidic connection is dynamic at pH 5, whereas it becomes irreversible at pH 7. The N(Me)-alkoxyamine glycosylation may hence be an attractive pH controlled reaction for the assembly of DNA-based dynamic products.