In-cell thermodynamics and a new role for protein surfaces

Understanding protein thermodynamics as it occurs inside cells is a fundamental goal of biophysics, and, from a practical point of view, will facilitate the design and improvement of protein-based drugs and catalysts. By measuring the temperature dependence of protein stability inside Escherichia coli cells, we show, contrary to predictions, that proteins are not necessarily stabilized inside cells compared with buffer alone. We also show that crowding-induced charge–charge interactions slow folding because of preferential interactions with the unfolded ensemble, and reducing these interactions increases protein stability

Protein stability in crowding and confinement

In ihrer natürlichen Umgebung sind Proteine von einer Vielzahl verschiedener gelöster Stoffe umgeben (Crowding durch Cosolvens-Moleküle). Außerdem kann das verfügbare Volumen des Proteins innerhalb von Poren oder durch Membranen eingeschränkt sein (Confinement). Beide Effekte werden im Allgemeinen mit Hilfe der Volumenrestriktion beschrieben. Diese Arbeit behandelt die Frage, in welchem Ausmaß die Proteinstabilität durch Crowding und Cofinement beeinflusst wird. Die Proteinstabilität wird durch Messungen der Freien Enthalpie der Proteinfaltung quantifiziert. Die Freie Enthalpie wird in die enthalpischen und entropischen Beiträge aufgeteilt, um die zugrundeliegenden Protein-(Co-)Solvens Wechselwirkungen zu beschreiben. Die Ergebnisse zeigen, dass die Volumenrestriktion nicht die Ursache für die veränderte Proteinstabilität ist. Das Verständnis der veränderten Proteinstabilität verlangt eine Analyse aller chemischen Wechselwirkungen unter expliziter Berücksichtigung des Lösungsmittels

Templating effects in aristolochene synthase catalysis: elimination versus cyclisation

Analysis of the products generated by mutants of aristolochene synthase from P. roqueforti (PR-AS) revealed the prominent structural role played by the aliphatic residue Leu 108 in maintaining the productive conformation of farnesyl diphosphate to ensure C1–C10 (σ-bond) ring-closure and hence (+)-aristolochene production

In-cell thermodynamics and a new role for protein surfaces

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge–charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells

Protein Stabilization by Macromolecular Crowding through Enthalpy Rather Than Entropy

The interior of the cell is a densely crowded environment in which protein stability is affected differently than in dilute solution. Macromolecular crowding is commonly understood in terms of an entropic volume exclusion effect based on hardcore repulsions among the macromolecules. We studied the thermal unfolding of ubiquitin in the presence of different cosolutes (glucose, dextran, poly­(ethylene glycol), KCl, urea). Our results show that for a correct dissection of the cosolute-induced changes of the free energy into its enthalpic and entropic contributions, the temperature dependence of the heat capacity change needs to be explicitly taken into account. In contrast to the prediction by the excluded volume theory, we observed an enthalpic stabilization and an entropic destabilization for glucose, dextran, and poly­(ethylene glycol). The enthalpic stabilization mechanism induced by the macromolecular crowder dextran was similar to the enthalpic stabilization mechanism of its monomeric building block glucose. In the case of poly­(ethylene glycol), entropy is dominating over enthalpy leading to an overall destabilization. We propose a new model to classify cosolute effects in terms of their enthalpic contributions to protein stability

Galileo Imaging of Jupiter’s Atmosphere: The Great Red Spot, Equatorial Region, and White Ovals

During the first six orbits of the Galileo spacecraft's prime mission, the Solid State Imaging (SSI) system acquired multispectral image mosaics of Jupiter's Great Red Spot, an equatorial belt/zone boundary, a “5-μm hot spot” similar to the Galileo Probe entry site, and two of the classic White Ovals. We present mosaics of each region, approximating their appearance at visible wavelengths and showing cloud height and opacity variations. The local wind field is derived by tracking cloud motions between multiple observations of each region with time separations of roughly 1 and 10 hr. Vertical cloud structure is derived in a companion paper by Banfieldet al. (Icarus135, 230–250). Galileo's brief, high-resolution observations complement Earth-based and Voyager studies and offer local meteorological context for the Galileo Probe results. Our results show that the dynamics of the zonal jets and large vortices have changed little since Voyager, with a few exceptions. We detect a cyclonic current within the center of the predominantly anticyclonic Great Red Spot. The zonal velocity difference between 0° S and 6° S has increased by 20 m sec^(−1). We measure a strong northeast flow approaching the hot spot. This flow indicates either massive horizontal convergence or the presence of a large anticyclonic vortex southeast of the hot spot. The current compact arrangement of two White Ovals and a cyclonic structure greatly perturbs the zonal jets in that region

Circular trimers of gelatinase B/ matrix metalloproteinase-9 constitute a distinct population of functional enzyme molecules differentially regulated by tissue inhibitor of metalloproteinases-1

Gelatinase B/ matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many substrates and is produced by most cell types as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers, and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical, and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast to a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, atomic force microscopy (AFM) and transmission electron microscopy (TEM), we generated a 3D structure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers versus monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1.status: publishe

Venus Corona and Tessera Explorer (VeCaTEx)

Venus Corona and Tessera Explorer (VeCaTEx) would use an aerobot to descend repeatedly beneath the dense clouds for imaging targeted area of the surface in the near infrared to address six of the prime investigations prioritized by VEXAG. The technologies needed could be matured during the next decade

Impact features on Europa: results of the Galileo Europa Mission (GEM)

During the Galileo Europa Mission (GEM), impact features on Europa were observed with improved resolution and coverage was compared with Voyager or the Galileo nominal mission. We surveyed all primary impact features >4 km in diameter seen on Europa (through orbit E19). The transition from simple to complex crater morphology occurs at a diameter of about 5 km. We calculated the transient crater dimensions and excavation depths of all craters surveyed. The largest impact feature (Tyre) probably had a transient crater depth between 5 and 10 km and transported material to the surface from a depth of not greater than ∼4 km. Craters <30 km in diameter, such as Manannàn and Pwyll, formed within targets whose immediate subcrater materials exhibited nonfluid behavior on time scales of the impact event, and that are capable, especially in the case of Pwyll, of supporting significant local topographic loads such as a central peak. These craters are nevertheless quite shallow, with very subdued floors, and we suspect that Manannàn and Pwyll's small depth-to-diameter ratios are due to the isostatic adjustment of large-scale topography, facilitated by warm, plastically deformable ice at depth. Morphological similarities between Callanish and Tyre strongly imply that conclusions reached regarding Callanish in J. Moore et al. (1998, Icarus135, 127–145) also apply to Tyre, which was that Callanish is the consequence of impact into target materials that are mechanically very weak at depth. New evidence that Callanish's circumferential rings formed before the proximal ejecta became immobile implies a low-viscosity substrate at the time of impact. We also report additional evidence that a component of the proximal ejecta of Callanish was emplaced as a fluid. Our observations of Pwyll secondaries support the conclusions stated in Alpert and Melosh (1999) that impacts on icy bodies eject smaller fragments and that fragment size decreases more gradually as velocity increases than observed for impacts on silicate bodies at equivalent ejection velocities. Examination of Pwyll's secondary craters reveals azimuthal variations, with ejecta fragment sizes being larger near the center of a ray than off the ray. Our initial analysis of the characteristic size distribution of Pwyll's secondary craters shows that they form a differential slope slightly shallower than −4. Similar steep slopes for small craters on Ganymede imply that small craters there are mostly formed by secondary impact, and the jovian system may thus be deficient in small impacts relative to the environment of the terrestrial planets