A molecular mechanism of chaperone–client recognition

Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature

Two-state folding of the outer membrane protein X into a lipid bilayer membrane

Folding and insertion of β-barrel membrane proteins into native membranes is efficiently catalyzed by β-barrel assembly machineries. Understanding this catalysis requires a detailed description of the corresponding uncatalyzed folding mechanisms, which however have so far remained largely unclear. Here, we resolve folding and membrane insertion of the E. coli outer membrane protein X (OmpX) into 1,2-didecanoyl-sn-glycero-3-phosphocholine (PC10:0) membranes at the atomic level. By combining four different experimental techniques, we correlate global folding kinetics with global and local hydrogen bond formation kinetics. Under a well-defined reaction condition, these processes follow single-exponential velocity laws, with rate constants identical within experimental error. The data thus establish at atomic resolution that OmpX folds and inserts into the lipid bilayer of PC10:0 liposomes by a two-state mechanism

A Basin Analysis of the Wabigoon Area of Lake Agassiz, a Quaternary Clay Basin in Northwerstern Ontario

Information from a wide range of sources is integrated in a basin analysis of the Wabigoon Basin, a Quaternary clay basin located on the Canadian Shield in northwestern Ontario. The basin sediments were deposited between 10.9 ka and 9.5 ka, along the margin of the Rainy Lobe of the Laurentide Ice Sheet, which formed the northern boundary of proglacial Lake Agassiz. The basin architecture is dominated by four major elements: end moraines, eskers, kames and a clay plain, all of which overlie irregular bedrock topography. End moraines, eskers and kames are composed mainly of a fining upward sequence of gravels and sands. The geometry of these sedimentary units, and their sedimentary structures indicates they were deposited mainly by high and low-density turbidity currents, on ice-marginal subaqueous outwash fans. Eskers contain a core of coarse gravel and sand deposited within subglacial meltwater conduits, overlain by subaqueous fan sediments deposited at the conduit mouth. Esker ridges were formed during conduit filling events and flanking deposits were formed when a conduit remained in use during ice-marginal retreat. Where conduits were shortlived, isolated subaqueous fans (kames) were formed. A depositional model is proposed which relates moraine formation to catastrophic releases of subglacial meltwater and sediment simultaneously along the entire margin of the Rainy Lobe. The clay plain forms a broad blanket of fine-grained, rhythmically-bedded sediment which obscures bedrock topography, and often buries esker and kame deposits. Seismic profiles and overburden drilling reveal deep (50-70 m) bedrock lows beneath the clay plain. These lows, oriented sub-parallel to the ice margin, acted as sediment traps, and were infilled by the deposits of underflows generated at the ice margin.Les renseignements tirés d'un grand nombre de sources sont intégrés en vue de l'analyse du bassin de Wabigoon, situé sur le Bouclier canadien. Les sédiments qui le composent ont été déposés entre 10,9 et 9,5 ka, le long de la marge du lobe Rainy de l'Inlandsis laurentidien, qui formait la limite nord du Lac Agassiz. Les quatre formes principales qu'on y trouve sont les moraines frontales, les eskers, les kames et une plaine argileuse; ces formes recouvrent le relief irrégulier du substratum. Les moraines frontales, les eskers et les kames sont surtout composés d'une séquence de graviers et de sables s'affinant vers le haut. La géométrie de ces unités sédimentaires et leur structure montrent qu'elles ont en grande partie été déposées sur des épandages fluvioglaciaires sous-aquatiques de marge glaciaire par des courants de turbidité de haute et de basse densité. Les eskers renferment un noyau de gravier et de sable grossier déposés à l'intérieur des chenaux de fonte sous-glaciaires, recouverts par les sédiments de cône sous-aquatique déposés à l'embouchure des chenaux. Les crêtes d'eskers ont été édifiées en même temps que s'effectuait le remplissage des chenaux et les dépôts latéraux ont été formés alors qu'un chenal demeurait actif pendant le retrait glaciaire. Là où l'activité a été de courte durée, des cônes sous-aquatiques isolés (kames) se sont formés. On propose ici un modèle de mise en place des sédiments qui lie la formation des moraines à des écoulements sous-glaciaires catastrophiques et simultanés d'eau de fonte et de sédiments tout le long de la marge du lobe Rainy. La plaine argileuse forme une couverture étendue de sédiments à grains fins, à stratification rythmique, qui cache le relief du substratum et enfouit souvent les eskers et les kames. Les profils sismiques et les forages dans les dépôts meubles révèlent des dépressions profondes (50-70 m) dans le substratum sous Ia plaine argileuse.Informationen aus einer Vielfalt von Quellen sind fur die Analyse des Wabigoon-Beckens vereinigt, ein Quaternàr-Lehm-Becken, das sich auf dem kanadischen Schild befindet. Die Beckensedimente wurden zwischen 10.9 ka und 9.5 ka abgela-gert, entlang dem Rand der Rainy-Lobe der laurentidischen Eisdecke, welche die nôrdli-che Grenzlinie des proglazialen Agassiz-Sees bildete. Die Becken-Architektur wird von vier Hauptelementen beherrscht: Endmoràne, Eskers, Kames und eine Lehmebene. Die drei ersteren bestehen vor allem aus einer nach oben hin feiner werdenden Sequenz von Kies und Sand. Die Géométrie dieser Sediment-Einheiten und ihre Sediment-Strukturen zeigen, daB sie haupt-àchlich durch Dichtigkeitsstrômungen mit hoher und niedriger Dichte abgelagert wurden auf Unterwasser-Eisrand-Schwemmfàchern. Die Eskers enthalten einen Kern von grobem Kies und Sand, der innerhalb subgla-zialer Schmelzwasserkanàle abgelagert wurde, ùberlagert von Unterwasser-Fàcher-sedimenten, die an der Mùndung der Kanàle abgelagert wurden. Die Esker-Rùcken wurden wàhrend der Fùllung der Kanàle gebildet und seitliche Ablagerungen wurden gebildet, wenn ein Kanal wàhrend des Rùckzugs des Eisrands aktiv blieb. Wo Kanàle kurzlebig waren, bildeten sich isolierte Unterwasser-Fàcher (Kames). Ein Ablagerungsmodell wird vorgeschlagen, das die Bildung der Morâne mit katastrophenartigem gleichzeiti-gem Freiwerden von subglazialem Schmelz-wasser und Sedimenten entlang des ganzen RandsderRainy-Lobeverbindet. DieLehmebene bildet eine breite Decke von feinkôrnigem, rhythmisch gelagertem Sediment, welches die Topographie des anstehenden Gesteins verdeckt und hâufig Esker- und Kame-Ablagerungen ùberlagert. Die seismischen Profile und Bohrungen in den lockeren Ablagerungen lassen tiefe Niederungen (50-70 m) unter der Lehmebene im anstehenden Gestein erkennen

Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification

Filamentation induced by cyclic AMP (FIC)-domain enzymes catalyze adenylylation or other posttranslational modifications of target proteins to control their function. Recently, we have shown that Fic enzymes are autoinhibited by an α-helix (αinh) that partly obstructs the active site. For the single-domain class III Fic proteins, the αinh is located at the C terminus and its deletion relieves autoinhibition. However, it has remained unclear how activation occurs naturally. Here, we show by structural, biophysical, and enzymatic analyses combined with in vivo data that the class III Fic protein NmFic from Neisseria meningitidis gets autoadenylylated in cis, thereby autonomously relieving autoinhibition and thus allowing subsequent adenylylation of its target, the DNA gyrase subunit GyrB. Furthermore, we show that NmFic activation is antagonized by tetramerization. The combination of autoadenylylation and tetramerization results in nonmonotonic concentration dependence of NmFic activity and a pronounced lag phase in the progress of target adenylylation. Bioinformatic analyses indicate that this elaborate dual-control mechanism is conserved throughout class III Fic proteins

The dynamic dimer structure of the chaperone Trigger Factor

The chaperone Trigger Factor (TF) from Escherichia coli forms a dimer at cellular concentrations. While the monomer structure of TF is well known, the spatial arrangement of this dimeric chaperone storage form has remained unclear. Here, we determine its structure by a combination of high-resolution NMR spectroscopy and biophysical methods. TF forms a symmetric head-to-tail dimer, where the ribosome binding domain is in contact with the substrate binding domain, while the peptidyl-prolyl isomerase domain contributes only slightly to the dimer affinity. The dimer structure is highly dynamic, with the two ribosome binding domains populating a conformational ensemble in the center. These dynamics result from intermolecular in trans interactions of the TF client-binding site with the ribosome binding domain, which is conformationally frustrated in the absence of the ribosome. The avidity in the dimer structure explains how the dimeric state of TF can be monomerized also by weakly interacting clients

Regulation of chaperone function by coupled folding and oligomerization

The homotrimeric molecular chaperone Skp of Gram-negative bacteria facilitates the transport of outer membrane proteins across the periplasm. It has been unclear how its activity is modulated during its functional cycle. Here, we report an atomic-resolution characterization of the; Escherichia coli; Skp monomer-trimer transition. We find that the monomeric state of Skp is intrinsically disordered and that formation of the oligomerization interface initiates folding of the α-helical coiled-coil arms via a unique "stapling" mechanism, resulting in the formation of active trimeric Skp. Native client proteins contact all three Skp subunits simultaneously, and accordingly, their binding shifts the Skp population toward the active trimer. This activation mechanism is shown to be essential for; Salmonella; fitness in a mouse infection model. The coupled mechanism is a unique example of how an ATP-independent chaperone can modulate its activity as a function of the presence of client proteins

Concert recording 2017-04-12b

[Track 1]. Angel Falls / Lewis Songer -- [Track 2]. Grand Canyon Octet. Andante appassionato [Track 3]. Lento-allegro molto / Eric Ewazen -- [Track 4]. Quipperies / Lowell Shaw -- [Track 5]. Birdland / Josef Zawinul arranged by Kummerlander

Concert recording 2017-11-15

[Track 1]. Fanfare for barcs / Kerry Turner -- [Track 2]. Three for five / James Naigus -- [Track 3]. Big sky country / Daniel Baldwin