Podcasts about GroES

On this week's episode, I sit down with my friend Dilly Groes, the founder of the incredible fashion brand Groes - a capsule collection created to bridge the gap between high street and luxury fashion. We explore what it truly takes to launch a fashion business and the challenges and successes Dilly has faced during the early stages of her entrepreneurial journey. Dilly uncovers her secrets on how she turned a simple online business plan into a thriving brand, her unique approach to organic promotion and how she's building her brand identity. We also talk go-to fashion tips, wellness routines, and the inspirations behind her work.IG: @dillygroesIG: @groes.ldnWww.groes.comPlease note that this episode may contain paid endorsements and advertisements for products and services. Individuals on the show may have a direct or indirect financial interest in products or services referred to in this episode. Produced by Dear Media. See Privacy Policy at https://art19.com/privacy and California Privacy Notice at https://art19.com/privacy#do-not-sell-my-info.

In this episode of iGaming Daily, SBC Media's Multimedia Editor, James Ross, is joined by EveryMatrix's CEO, Ebbe Groes, as the two go through the firm's third quarter performance. Released this morning, EveryMatrix's Q3 results highlighted the company's sixth consecutive profit margin of more than 50%, boosted by two major casio and sports acquisitions in the last five months. The duo discuss the aquisition of both FSB Technology and Fantasma Games, as well as EveryMatrix's entry into Peru and decline in profits from its affiliate division. Groes also shares insights on client retention, scalability challenges, and the organisational structure that has allowed EveryMatrix to thrive in a competitive landscape.To read more about EveryMatrix's Q3 performance, click on the following links:- https://casinobeats.com/2024/11/12/everymatrix-q3-2024-financial-results/- https://sbcnews.co.uk/technology/2024/11/12/everymatrix-q3-core-units/Host: James RossGuest: Ebbe GroesProducer: Anaya McDonaldEditor: James RossRemember to check out Optimove at https://hubs.la/Q02gLC5L0 or go to Optimove.com/sbc to get your first month free when buying the industry's leading customer-loyalty service.

In deze aflevering van de VCMS Podcast spreken Francijn de Nijs Bik (technisch lid) en Niek Schepers (VCMS Nijmegen) met Dr. ing. Sebastiaan van de Groes. Hij is werkzaam als orthopedisch chirurg in het Radboudumc. In deze podcast worden luisteraars meegenomen in de wereld van innovatie en digitalisering binnen de orthopedische chirurgie, waarbij ook de nieuwste ontwikkelingen op het gebied van robotica uitgebreid aan bod komen. We bespreken ook het gebruik van de "virtuele dokter" als oplossing voor de toenemende personeelstekorten in de zorg. Benieuwd naar deze podcast? Luister dan nu naar deze gloednieuwe aflevering!

In this episode, Sebastian Groes joins me to discuss his experience of stroke as narrated in his memoir titled Right in the Head. Bas is Professor of English Literature at the University of Wolverhampton, where he leads a Computational Literary Studies project on inclusivity and diversity. He explores the physical deficits, the emotional turmoil that accompanied his stroke, and the impact it had on his family, and on return to work.

iGaming Daily, sponsored by Optimove, heads to the Romania capital Bucharest in today's episode as SBC's Media Manager, Charlie Horner, jumped on a plane and headed to the newly opened office of EveryMatrix, to have a chat with the firm's CEO, Ebbe Groes. The duo talk about the history of EveryMatrix and how the company expanded across the globe, as well as discussing new opportunities for the igaming industry that are emerging in various markets and some high profile dealings within the lotteries sector.  To read more about the discussions talked about in today's podcast, click on the link below: - https://casinobeats.com/2024/04/12/everymatrix-peru-player-data-points-towards-enormous-potential/Host: Charlie HornerGuest: Ebbe GroesProducer: James RossEditor: James RossRemember to check out our partners Optimove at https://hubs.la/Q02gLC5L0 or go to optimove.com/sbc to get your first month free when buying the industry's leading customer-loyalty service.

Illi Gardner is the women's Everesting World Record holder. In August 2021 she set a new women's world Everesting record of 8 hours 33 minutes and 47 seconds, beating Emma Pooley's 2020 record by almost 20 minutes by riding up and down Bwlch y Groes 72 times. Illi tells us about the ride, gear choices and if she'll make another attempt.

They maybe small but Welsh spirits makers are leading the way when it comes to innovation. Across Wales there's around 25 independent and unique producers, distilling and experimenting with exciting tastes and flavours to produce a huge array of innovative drinks. Pauline Smith visits "In the Welsh Wind" a distillery in the village of Tan-y-Groes in Ceredigion, founded by Alex Jungmayr and Ellen Wakelam. The distillery prides itself on developing and distilling gins which tell the stories of the places, the people or the businesses that they represent. One of those is the "Welsh Sisters" gin in nearby New Quay. However, it's not just award winning gins the distillery make - their latest venture is developing a “grain-to-glass” whisky with locally grown barley, making it one of five whisky distilleries in Wales bidding to ringfence the quality and reputation of Welsh whisky.

Explicit. Juice and Kim are back after a week off. They discuss Huntsville's forever changing landscape, the documentary “Summer of Soul”, Lebron's Acting in Space Jam 2, Kim's nightlife experience and more Theme Music by @DJCBATTLE

BBC Wales' Environment correspondent, Steffan Messenger takes a look at the issue of climate change. Starting on a positive note he speaks to the Future Generations Commissioner about the opportunity it presents for new green jobs in Wales. We speak to 21 year old, Brittany Roberts from Rhyl who will be travelling to the G7 meeting in Cornwall, representing the Catholic international development charity CAFOD who say they want to see a global green recovery from the pandemic that includes all nations. The Size of Wales charity gets a new chair - Carwyn Jones, Wales' former first minister, says tackling climate change needs to become the world's top priority. Wales' first carbon-funded peatland restoration project at Bwlch y Groes on the edge of the Snowdonia National Park is completed following validation under the new Peatland Code. Finally, a warning that Landslides in the Brecon Beacons are themselves a "canary in the coal mine" on climate change

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 12 (Dameg y tristwch yn troi’n llawenydd) gyda Trey McCain

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 11 ( Dameg y ffŵl cyfoethog) gyda Rhys Llwyd + 2 emyn byw!

The Groes comes on to talk round one with the boys, how all the teams performed and couch materials and prices. Daneo welcomes interception chat while just dropping knowledge on the week that was.

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 10 ( Dameg y Samariad Trugarog) - Dathliad pob oed

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 9 ( Dameg yr Hedyn Mwstard) gyda Rhys Llwyd

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 8 ( Dameg y dydd a’r awr yn gyfrinach) gyda Mari Williams

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 7 (Dameg y ddau fab) gyda Rhys Llwyd

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 6 (Y ddwy sylfaen) gyda Rhys Llwyd

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 5 (Y brethyn a’r crwyn gwin) gyda Rhys Llwyd

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 4 (Y Bugail Da) gyda Arwel Jones

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 3 (Dameg y rhwyd) gyda Rhys Llwyd

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 2 (Dameg yr tenantiaid) gyda Rhys Llwyd a Menna Machreth - Mathew 21

Rhwng Bethlehem a'r Groes: Damhegion Iesu - Rhan 1 (Dameg yr Heuwr) gyda Rhys Llwyd a Menna Machreth

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.08.373423v1?rss=1 Authors: Koculi, E., Thirumalai, D. Abstract: The E. Coli. ATP-consuming chaperonin machinery, a complex between GroEL and GroES, has evolved to facilitate folding of substrate proteins (SPs) that cannot do so spontaneously. A series of kinetic experiments show that the SPs are encapsulated in the GroEL/ES nano cage for a short duration. If confining the SPs in the predominantly polar cage of GroEL in order to help folding, the assisted folding rate, relative to the bulk value, should always be enhanced. Here, we show that this is not the case for the folding of rhodanese in the presence of the full machinery of GroEL/ES and ATP. The assisted folding rate of rhodanese decreases. Based on our finding and those reported in other studies, we suggest that the ATP-consuming chaperonin machinery has evolved to optimize the product of the folding rate and the yield of the folded SPs on the biological time scale. Neither the rate nor the yield is separately maximized. Copy rights belong to original authors. Visit the link for more info

Comment vit-on à l'heure du Covid-19 ? Suite à l'évolution de la situation en France de l'épidémie du coronavirus Covid-19, La Nouvelle République et Centre Presse vous invitent à suivre l'actualité de ce virus au fil des jours...  Dans ce trente-neuvième épisode, Laurent Gaudens a questionné différentes acteurs du tourisme : Denis de Groes, propriétaire de chambres d'hôtes à Dangé-Saint-Romain, Ludivine Béauce, propriétaire de la Villa Richelieu à Châtellerault et  Isabelle Barreau, présidente de l'agence de créativité et d'attractivité du Poitou . Dans l'oeil du coronavirus est un podcast de La Nouvelle République et Centre-Presse réalisé par Laurent Gaudens. Musique : CloneMeTwice_TheDarkWoods Ce podcast est également disponible sur : Apple Podcasts, Google Podcasts, Deezer, Spotify et Podcasts Addict.  

'Sut dwi'n gwybod os ydy Duw yn fy ngharu?' Edrych ar y Groes (Luc Luc 23:44-56) gyda Rhys Llwyd

Nina Groes, Direktør for Kvinfo, taler under overskriften: Fanget af forventninger til Studenterforeningens Fredagstaler. Talen er nr. 45 i rækken af Fredagstalere, og fandt sted i Carlsberg Akademi d. 2. september 2016 Optagelse: Lasse Dahl Birch produktion, speak og musik: Anders Lundager Madsen

Direktøren for Kvinfo Nina Groes indtager ævle-pladsen i denne om Schøtministeriet Live.

Helen Mark follows Snowdonia Marathon and meets some of the people tackling this challenging course. Starting and finishing in Llanberis, the race encircles Wales' highest mountain of Snowdon, and rises to over a thousand feet in places. Andy John, Bishop of Bangor is taking on the Marathon for the third time, and he describes the sensation from running the course as being lost in the landscape but found in yourself. But he's dreading the twenty-two mile mark when he'll reach the 1200ft climb at Bwlch y Groes or "gap of the cross", before descending back into Llanberis for the finish. Helen stops at the ten-mile mark to meet Arwyn Owen at Hafod y Llan farm to find out how Hydro-Electric Power is the new cash crop in this rugged environment. She also meets Phil Owen at Llechwedd Caverns to discover how the area became the slate-mining capital of the world. Both Phil's Father and Grandfather worked in the mine, but Phil became a musician and serenades Helen on his ukulele, three hundred feet below the surface. Helen hands out water and energy gels with volunteers from the Snowdonia Society at the halfway point in Beddgelert and speaks to Margaret Thomas about Esme Kirby, the remarkable woman who set-up the organisation, before returning to Llanberis to greet a weary Andy John as he's crosses the finishing line.

Cyfres 1 Corinthiaid Rhan 2 – Grym y Groes a Pŵer yr Ysbryd 1 Corinthiaid 1:18-2:16

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli perform an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations to exclude aggregation, in combination with highly sensitive spectroscopic methods, such as single-molecule Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS), we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP), and a natural GroEL substrate - dihydrodipicolinate synthase (DapA). We show that both proteins form highly flexible, kinetically trapped folding intermediates, when folding in free solution and do not engage in inter-molecular interactions, such as aggregation, at sufficiently low concentration. We find that in the absence of aggregation, GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. The folding of DapA could be measured at physiological temperature and was found to be ~130-fold accelerated by GroEL/ES. As accelerated folding was independent of repetitive cycles of protein binding and release from GroEL, we demonstrate that iterative annealing does not significantly contribute to chaperonin assisted substrate folding. With a single molecule FRET based approach, we show that a given substrate molecule spends most of the time (~80%) during the GroEL reaction cycle inside the GroEL central cavity, in line with the inner GroEL cage being the active principle in folding catalysis. Moreover, photoinduced electron transfer experiments on DM-MBP provided direct experimental evidence that the confining environment of the chaperonin cage restricts polypeptide chain dynamics. This effect is mainly mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. Taken together, we were able to develop novel approaches, based on single molecule spectroscopy and making use of GroEL as a single molecule sorting machine, to measure GroEL substrate folding rates at sub-nanomolar concentrations. We also, for the first time, provide direct experimental evidence of conformational restriction of an encapsulated polypeptide in a chaperonin cage. Our findings suggest that global encapsulation inside the GroEL/ES cavity, not iterative cycles of annealing and forced unfolding, can accelerate substrate folding by reduction of an entropic energy barrier to the folded state, in strong support of an active chaperonin mechanism. Accelerated folding is biologically significant as it adjusts folding rates relative to the rate of protein synthesis.

To become biologically active, most proteins need to fold into precise three dimensional structures. It has been well established that all the folding information is contained within the primary structure of a protein. However, the mechanisms utilized by proteins to avoid sampling the extraordinarily large amount of possible conformations during their folding process are just beginning to be understood. Molecular chaperones assist the folding of newly synthesized and denatured proteins in acquiring their native state in the crowded intracellular environment. As a nascent chain leaves the ribosome, it is captured first by the upstream chaperones and then possibly transferred to the downstream chaperonins. GroEL-GroES, the Hsp-60 of E.coli, is one of the best studied chaperone systems. An appreciable amount of data is available providing information regarding its structure and function. GroEL encapsulates the substrate into the central cavity where folding occurs unimpaired by aggregation and unwanted inter-molecular interactions. Nevertheless, many important aspects of the GroEL mechanism remain to be addressed. Some of the open questions we have addressed in this study include: In what conformation does a substrate protein bind to the apical domains of GroEL; how is it that GroEL is able to accelerate the rate of folding of certain proteins, and how do the conformational properties of the substrate change as it undergoes repeated cycling. By using ensemble FRET and Sp-FRET (Single Pair-Fluorescence Resonance Energy Transfer), we have probed the conformation of the model substrate DM-MBP (Double Mutant Maltose Binding Protein) during different stages of the functional cycle of GroEL. With Sp-FRET coupled to PIE (Pulsed Interleaved Excitation), we have been able to explore the heterogeneity of the GroEL bound substrate protein and observed a bimodal conformational distribution. One of the two populations is as compact as the native state, whereas the other is as extended as the unfolded protein in denaturant. This unfolding is a local phenomena and can also be observed when the substrate is transferred from DnaK/J system (bacterial Hsp70) to GroEL, indicating the possibility of the existence of this conformational heterogeneity in vivo as the protein follows the cellular chaperone pathway. Subsequent to GroEL binding, there is a transient expansion of the protein upon binding of ATP to GroEL, followed by compaction when GroES triggers the encapsulation of the protein inside the chaperonin cage. This transient expansion is however found not to be a necessary event for the rate acceleration of DM-MBP folding, since ADP-AlFx (transition state analogue of ATP hydrolysis) results in a much slower rate of expansion, which does not cause a change in the folding rate. Anisotropy measurements, probing the freedom of motion of different regions of the GroEL bound protein, revealed that there is a segmental release of the substrate protein from the GroEL surface upon binding of ATP and GroES. As a consequence, the hydrophobic collapse of the protein upon encapsulation by GroES follows a step-wise mechanism. In this process, less hydrophobic regions are released upon binding of ATP, prior to more hydrophobic ones which are released only by GroES binding. Thus, the order of Hydrophobic collapse is reversed as compared to spontaneous folding possibly resulting in conformationally different folding intermediates. Evidence that the folding pathway inside the cage differs from that of spontaneous folding was obtained by observing the effect of external perturbations (e.g. mutations in substrate protein and use of different solvent conditions) on the rate of spontaneous and GroEL assisted folding reactions. These two folding reactions respond differently to the introduced perturbations. Kinetic data obtained from ensemble FRET measurements suggest that the conformation of refolding intermediate is altered by the GroEL cavity, which leads to a folding pathway that is different from the spontaneous refolding pathway. In summary, this study revealed significant novel aspects of the GroEL folding mechanism and provided insights into the basis of rate acceleration of the substrate protein by the chaperonin. This work may thus contribute to advance our fundamental knowledge of the chaperonin system and the basic mechanism of protein folding.

Thu, 2 Aug 2007 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/7304/ https://edoc.ub.uni-muenchen.de/7304/1/Tang_YunChi.pdf Tang, Yun-Chi ddc:540, ddc:500, Fakult

Chaperonins are a specific class of barrel-shaped chaperones, present in almost all organisms. Newly synthesized proteins encapsulated by the chaperonin can attain their native structure unimpaired by aggregation during repeated cycles of ATP-dependent binding and release. Chaperonins are generally divided into two groups. Group I chaperonins, such as the barrel-shaped GroEL oligomer, are found predominantly in bacteria and cooperate with cofactors of the Hsp10 familly (i.e. GroES). The Group II chaperonins, on the other hand, do not require a Hsp10- cofactor and are found in the eukaryotic cytosol and in archaea. The function of GroEL is understood in great detail and the substrate interaction proteome has been recently identified. In contrast, our knowledge about the natural substrates of Group II chaperonins is deficient and as a consequence, mechanistical studies on Group II chaperonins have been limited to using the eukaryotic model substrates actin and tubulin as well as heterologous model substrates. In the present study, the complete substrate spectrum of a Group II chaperonin, the thermosome (Ths) of the mesophilic archaeon Methanosarcina mazei (M. mazei), was analysed for the first time. In addition, the unique coexistence of both the goup I and the group II chaperonins in M. mazei, which was confirmed in the initial part of the study, provided the opportunity to obtain new insights into how the substrate selection differs between the two chaperonin groups. For these purposes, the chaperonin substrates were isolated by immunoprecipitation of the chaperonin-substrate complexes and identified by liquid chromatography coupled mass spectrometry (LC-MS) using three different approaches: LC-MS after separation of the proteins (i) by classical 2D-PAGE, (ii) by difference gel electrophoresis (Ettan DIGE) and (iii) by 1D-PAGE. Analysis of substrates of both the thermosome (MmThs) and GroEL/GroES (MmGroEL, MmGroES) of M. mazei revealed that each chaperonin handles a defined set of substrates, and both chaperonins contribute to the folding of ~17% of the proteins in the archaeal cytosol. Bioinformatic analysis revealed that the chaperonin specificity is governed by a combination of a various physical properties (hydrophobicity, net charge and size), structural features (i.e. the domain fold), and less concrete characteristics like the evolutionary status and, in this context, the phylogenetic origin of the substrate.

In Escherichia coli, the cylindrical chaperonin GroEL and its cofactor GroES promote the folding of a fraction of newly synthesized polypeptide chains by acting as an Anfinsen cage. GroEL recognizes substrate proteins with its apical domains of the tetradecameric structure. Exposed hydrophobic side chains in non-native proteins interact with GroEL and bound substrates are subsequently encapsulated under the GroES lid, where they can fold in a protected environment. Despite the detailed knowledge about structural and mechanistic features of GroEL and GroES, little is known about its genuine in vivo substrate proteins. Here, the nearly complete set of GroEL interacting proteins in vivo was identified and quantified by an approach using affinity chromatography for the isolation of GroEL/GroES/substrate complexes and subsequent analysis by mass spectrometric methods. GroEL substrate proteins were analyzed with respect to their fold types and functional classes, revealing a preference for proteins which fold into the versatile TIM barrel fold to interact with GroEL. Further in vivo and in vitro experiments with individual proteins identified as GroEL substrates verified the data obtained by the proteomic approach and allowed conclusions on the usage of the other main chaperone system in E. coli: DnaK/DnaJ/GrpE. Taken together, the results culminated in the classification of GroEL interacting proteins according to their dependence on chaperones for folding. Class I proteins are largely independent of chaperones but their folding yield can be increased by chaperone interaction. Class II proteins do not refold efficiently in the absence of chaperones in vitro, but can utilize either the DnaK or the GroEL/GroES systems for folding. Class III substrates are fully dependent on GroEL. DnaK can bind class III proteins and thus prevent their aggregation, but folding is achieved only upon transfer to GroEL.

Bartonella henselae ist der Erreger der Katzenkratzkrankheit und vaskuloproliferativer Erkrankungen des Menschen. Das Bakterium wurde erstmals 1989 korrekt klassifiziert und den oben genannten Krankheitsbildern zugeordnet. Es ist ein langsam wachsendes, nicht in Flüssigmedien kultivierbares Bakterium. Genetische Untersuchungen sind deswegen schwierig. Im Rahmen dieser Arbeit wurde daher die zweidimensionale Elektrophorese zur Untersuchung möglicher Pathogenitätsfaktoren von B. henselae etabliert. Mithilfe dieser Methode sollte es möglich sein, das gesamte Proteom von B. henselae unter verschiedenen Kulturbedingungen aufzutrennen und so Rückschlüsse auf Pathomechanismen dieses Erregers zu ziehen. Die vorliegende Dissertation lieferte folgende Ergebnisse: 1. Etablierung eines geeigneten Protokolls zum Aufschluss von B. henselae für die zweidimensionale Elektrophorese. 2. Erstellung einer Proteomkarte von auf Agarplatten kultivierten B. henselae. Hierdurch wurde die Grundvoraussetzung für weitere Proteom-basierte Studien geschaffen. Zudem wurden drei Proteine (Malatdehydrogenase, Pyruvatdehydrogenase und DnaK)erstmals bei B. henselae beschrieben. 3. Charakterisierung von vier monoklonalen anti-B. henselae-Antikörpern mithilfe von zweidimensionalen Proteom-Western-Blots. Dadurch wurden vier neue B. henselae–Proteine, unter anderem das Phagenprotein Pap31, als immunogen in der Maus beschrieben. Zudem wurde hier erstmalig die zweidimensionale Elektrophorese als schnelle und akkurate Methode zur Charakterisierung monoklonaler Antikörper eingesetzt. 4. Durch den Proteomvergleich hitzegestresster und nicht-hitzegestresster B. henselae wurde gezeigt, dass mit der hier etablierten Methodik regulative Vorgänge der Proteinsynthese von B. henselae erfasst werden können. Von den drei identifizierten Hitzestressproteinen (GroEL, GroES und DnaK) wurde DnaK für B. henselae zum ersten Mal beschrieben. 5. Zwei pilusassoziierte Proteine (220 kD und 43 kD) wurden durch Proteomvergleich gefunden. Bei dem in den zweidimensionalen Auftrennungen sichtbaren 43kD-Protein handelt es sich um die Phophoserinaminotransferase, ein bakterielles Stoffwechselenzym. Der Zusammenhang mit der Pilusexpression ist bis dato unklar. Die Identität dieses Proteins wurde mithilfe reverser Genetik verifiziert. Das zweite Protein erscheint aufgrund unterschiedlicher Gelsysteme nur in den eindimensionalen Auftrennungen und wurde daher im Rahmen dieser Arbeit nicht weiter untersucht. 6. Durch die in dieser Arbeit etablierte radioaktive Markierung von B. henselae-Proteinen mittels 35S-Pulse-Chase wurde die Untersuchung neu synthetisierter Proteine nach Kokultur mit humanen Zelllinien ermöglicht. 7. Untersuchung neu synthetisierter B. henselae-Proteine nach Kokultur des Erregers mit humanen Endothelzellen mittels 35S-Pulse-Chase. Durch Vergleich mit der Proteomkarte konnten folgende in der Kokultur neu synthetisierte Proteine identifiziert werden: die Hitzestressproteine GroES, GroEL und DnaK, die Stoffwechselenzyme Malatdehydrogenase und Pyruvatdehydrogenase, der Elongationsfaktor EF-Tu und das Phagenprotein Pap31. Die Expression von Pap31 bei B. henselae in Endothelzellkultur wurde hier erstmals gezeigt. Durch die vorliegende Arbeit wurde eine neue Methode für die weitergehende Erforschung von B. henselae etabliert, die in Zukunft die Untersuchung dieses genetisch schwer zugänglichen Organismus erleichtern wird. Eine Reihe von Proteinen wurde hier für B. henselae erstmals beschrieben bzw. wurde deren Expression unter bestimmten Lebensbedingungen zum ersten Mal beobachtet. Die Breite des methodischen Ansatzes dieser Arbeit legt den Grundstein für vielfältige weitere Untersuchungen. So konnte zwischenzeitlich die membranassoziierte Lage von Pap31 mithilfe eines in dieser Arbeit charakterisierten monoklonalen Antikörper aufgedeckt und Fibronektin als Bindungspartner von B. henselae–Pili identifiziert werden.

Untersuchung der Reaktionszyklen von Chaperoninen aus Escherichia coli und Thermoplasma acidophilum mit Hilfe der Neutronenkleinwinkelstreuung Am Thermosom wurden bisher noch keine Komplexe untersucht. Hier war von großem Vorteil, daß bei SANS kein Strahlenschaden auftritt und damit Proben modifiziert und anschließend noch einmal gemessen werden konnten. a) b) c) Abbildung 5.2: schematische Darstellung verschiedener Thermosom-Konformationen: a) offenen Konformation, b) geschlossene Konformation c) Bullet-Konformation Ergebnisse Ergebnisse zu GroE - Zum Verständnis der allosterischen Wechselwirkungen innerhalb von GroEL wurde die Lösungsstruktur einer „single-ring“-Mutante von GroEL untersucht. Die Strukturen von apo-single-ring-GroEL und ADP-Komplexen aus protoniertem singlering- GroEL und protoniertem GroES unterscheiden sich nicht oder nicht meßbar von den entsprechenden Strukturen im Kristall. Bei einer Messung mit ADP-Komplexen aus unsichtbarem single-ring-GroEL und sichtbarem GroES wurde dagegen in der Lösungsstruktur eine im Kristall nicht vorhandene Konformationsänderung von GroES beobachtet. Dies bestätigt frühere mit wild-type GroEL durchgeführte Versuche. - Symmetrische Komplexe aus GroEL und GroES, sogenannte Football-Komplexe, werden als wichtiger Zwischenschritt im GroE-Reaktionszyklus diskutiert. In Anwesenheit von AMP-PNP konnten Football-Komplexe aus protoniertem, gematchtem GroEL und deuteriertem GroES nachgewiesen werden. In der Neutronenstreuung zeigen diese Komplexe eine charakteristische Hantelstruktur. Die beiden GroES-Moleküle haben einen Abstand von ca. 200Å (siehe Pfeil in Abb. 5.1c). Mit einer Titratonsreihe konnten wir die Dissoziationskonstante des zweiten GroES-Heptamers in diesen Komplexen mit einem Wert von 2×10-7M bestimmen. - Der Phage T4 hat ein Gen für ein eigenes Co-Chaperonin, GP31, das eine starke Analogie zu GroES aufweist. Wir verglichen die beiden Co-Chaperonine, um zu verstehen, warum GP31 lebenswichtig für den Phagen ist. Aus GroEL und GP31 wurden dazu in Anwesenheit von AMP-PNP Football-Komplexe gebildet. Bei diesen Komplexen ist das Co-Chaperonin etwa 5Å weiter vom Zentrum des Gesamtkomplexes entfernt, als im Fall der GroEL-GroES-Komplexe. Dieses Ergebnis unterstützt die These, GP31 sei nötig, um für die Faltung des Phagenproteins GP23 einen größeren Anfinsen-Cage zu schaffen. GP31 scheint unter vergleichbaren Bedingungen stärker zur Bildung von Football-Komplexen zu neigen als GroES. Bei der Bindung an GroEL ändern GP31 und GroES ihre Konformation. - In Verdrängungsversuchen wurde das Bindungsverhalten von GroES und GP31 an GroEL untersucht. Hierzu wurden mit Hilfe von ADP-Komplexen aus sichtbarem GroEL mit sichtbarem Co-Chaperonin vorgeformt. Das sichtbare Co-Chaperonin wurde dann mit unsichtbarem GroES verdrängt, so daß in den neugebildeten Komplexen nur noch GroEL ein Streusignal gab. Die beobachteten Zerfallsreaktionen mit Halbwertszeiten in der Größenordnung von Stunden lassen sich mit zwei Exponentialfunktionen beschreiben. Dies ist ein Indiz für das Auftreten von verschiedenen Populationen an GroEL-Co-Chaperonin-Komplexen. In allen Fällen waren die Dissoziationsraten bei den GroEL-GP31-Komplexe geringer als bei GroEL-GroES-Komplexen. Komplexe von GroEL mit GP31 sind also stabiler als solche mit GroES. - Bei einer Reihe von Versuchen mit GroEL und Substratprotein (MPB-Y283D) konnten unter anderem 1:2 Komplexe aus GroEL und Substrat und Trans-Komplexe aus GroEL, GroES und Substrat nachgewiesen werden. Diese Experimente zeigten übereinstimmend einen Abstand von 60Å des MPB zum Zentrum von GroEL. Wegen der notwendigen Denaturierung von MBP und anderen Substratproteinen wurden jedoch die Streukurven sehr häufig von Aggregaten gestört. SANS-Experimente mit Substratprotein sind daher wesentlich schwieriger als Versuche mit GroES oder GP31. Ergebnisse zum Thermosom - Die geschlossene Konformation von Thermosom in der Kristallstruktur rührt nicht von der Bindung von Mg-ADP-AIF3 her, sondern vom für die Kristallisation verwendeten Ammoniumsulfat . In Anwesenheit von ADP mit 2M Phosphat, schließt sich das Thermosom auch in physiologischem Puffer. Durch die hohe Phosphat- Konzentration ist davon auszugehen, daß sich in den Nukleotidbindungstaschen des Proteins neben ADP freie Phosphat-Ionen befinden. Die Konformation entspricht also dem Zustand unmittelbar nach der ATP-Spaltung. Wird die Probe auf 50°, die physiologische Temperatur von T. acidophilum, erwärmt, schließt sich das Thermosom in Anwesenheit von ATP. In Kombination mit den Messungen an anderen Thermosom-Nukleotid-Komplexen konnten damit Zwischenzustände im strukturellen Reaktionszyklus des Chaperonins charakterisiert werden. - Bei einer Reihe von Pufferbedingungen wurde die Bildung von Komplexen höherer Ordnung beobachtet. Von anderen Autoren war aufgrund von EM-Arbeiten vorgeschlagen worden, daß Thermosom kein Chaperonin, sondern ein Baustein des Cytoskeletts ist (Trent et al., 1997, 1998). Für die von uns gefundenen Strukturen sind Thermosom-Filamente jedoch keine befriedigenden Modelle. Da sich diese Komplexe höherer Ordnung in der Lösung außerdem bei einer Annäherung an physiologische Bedingungen auflösen, kann davon ausgegangen werden, daß sie in vivo nicht relevant sind. methodische Ergebnisse - Es wurde gezeigt, daß GroEL und GroES einen sehr homogenen Deuterierungsgrad haben, wenn die Bakterien vor der Reinigung unter gut kontrollierten Wachstumsbedingungen im Fermenter gezogenen werden. Das Protein erscheint dann in Kontrastausgleichsexperimenten im geeigneten Puffer, in unserem Fall nahe 99% D2O, als unsichtbar. - In einer Reihe von „stopped-flow“-Messungen wurde gezeigt, daß mit SANS (bei einer Aufsummierung von Daten mehrerer Messungen) grundsätzlich eine Zeitauflösung bis in den Sekundenbereich bei Experimenten an GroEL oder vergleichbar großen Molekülen möglich ist. Um biologisch relevante Ergebnisse erreichen zu können, ist jedoch weitere Arbeit an der Instrumentierung, speziell der Mischapparatur nötig.

Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpn10; also known as groES). In pea (Pisum sativum), chloroplast cpn10 was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (ii) form a stable complex with bacterial cpn60 in the presence of Mg.ATP. The subunit size of the pea protein is approximately 24 kDa--about twice the size of bacterial cpn10. A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpn10 was isolated, sequenced, and expressed in vitro. The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide. Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpn10 molecules in tandem. Moreover, both halves of this "double" cpn10 are highly conserved at a number of residues that are present in all cpn10s that have been examined. Upon import into chloroplasts the spinach cpn10 precursor is processed to its mature form of approximately 24 kDa. N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpn10 are identical at 13 of 21 residues.