crystal and solution structures of the multidomain cochaperone DnaJ

Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin- labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.1\. Auflag

Large-Range Movements of Neotropical Orchid Bees Observed via Radio Telemetry

Neotropical orchid bees (Euglossini) are often cited as classic examples of trapline-foragers with potentially extensive foraging ranges. If long-distance movements are habitual, rare plants in widely scattered locations may benefit from euglossine pollination services. Here we report the first successful use of micro radio telemetry to track the movement of an insect pollinator in a complex and forested environment. Our results indicate that individual male orchid bees (Exaerete frontalis) habitually use large rainforest areas (at least 42–115 ha) on a daily basis. Aerial telemetry located individuals up to 5 km away from their core areas, and bees were often stationary, for variable periods, between flights to successive localities. These data suggest a higher degree of site fidelity than what may be expected in a free living male bee, and has implications for our understanding of biological activity patterns and the evolution of forest pollinators

Climate Change Alters Seedling Emergence and Establishment in an Old-Field Ecosystem

Background: Ecological succession drives large-scale changes in ecosystem composition over time, but the mechanisms whereby climatic change might alter succession remain unresolved. Here, we asked if the effects of atmospheric and climatic change would alter tree seedling emergence and establishment in an old-field ecosystem, recognizing that small shifts in rates of seedling emergence and establishment of different species may have long-term repercussions on the transition of fields to forests in the future. Methodology/Principal Findings: We introduced seeds from three early successional tree species into constructed old-field plant communities that had been subjected for 4 years to altered temperature, precipitation, and atmospheric CO 2 regimes in an experimental facility. Our experiment revealed that different combinations of atmospheric CO2 concentration, air temperature, and soil moisture altered seedling emergence and establishment. Treatments directly and indirectly affected soil moisture, which was the best predictor of seedling establishment, though treatment effects differed among species. Conclusions: The observed impacts, coupled with variations in the timing of seed arrival, are demonstrated as predictors o

ELDOR-Abstandsmessungen an Cochaperon DnaJ und BLUF-Domäne BlrB

1 Introduction 2 Biological Systems 2.1 DnaJ 2.2 BlrB 3 EPR 3.1 Spin Hamiltonian 3.2 Bloch Equation 3.3 Pulsed EPR 3.4 Signal Relaxation 3.5 Pulsed ELDOR 3.6 cw EPR 3.7 EPR spectrometer 4 Methods and Materials 4.1 Experiments 4.2 Calculations 4.3 Samples 4.4 ELDOR distance distributions 5 DnaJ 5.1 Full length DnaJ 5.2 DnaJ storage complex 5.3 Synopsis 6 BlrB 6.1 BlrB dark state ELDOR on BlrBdark Position of the beta5-strand Position of S115 in BlrBdark Model structures from the BlrBdark ELDOR data 6.2 BlrB light state ELDOR on BlrBlight Orientation of alpha4 and beta5 in BlrBlight The role of Tryptophan92 Correlation to literature data 6.3 Synopsis 7 ConclusionProteins are building blocks of life. By their interaction with one another, they make most complex and diverse biological organisms possible. Knowledge of a protein's structure is a valuable basis for investigation and interpretation of its function. Most often, such information is attained by X-ray crystallography. This, however, does not always represent the protein's state in solution, as it is found within a cell. To be able to understand the protein mechanism it is necessary to examine, which parts of this structural model are conserved in solution and whether there are crucial differences in its conformation. With the aid of paramagnetic spin probes attached to specific protein sites, electron paramagnetic resonance (EPR) provides a snapshot of the protein's solution ensemble. EPR has been utilized in this work to examine two different proteins, Thermus thermophilus DnaJ and Rhodobacter sphaeroides BlrB. DnaJ is part of a heat chaperone system that identifies and repairs other damaged proteins, its cognates are found in prokaryotes as well as in eukaryotes including humans. In this work, a recently obtained crystal structure of Thermus thermophilus DnaJ is scrutinized and it is examined whether the formation of a stable standby complex specific to this organism leads to conformational changes in DnaJ that could point at its substrate interaction mechanism. The structure of the examined domains is found relevant, but the orientation of the domains is different and indicates unspecific, hydrophobic substrate interaction. BlrB is a photosensing BLUF protein using a flavin cofactor that is very common in nature (vitamin B2 is ribo-flavin). It undergoes a reversible photocycle that slightly rearranges the hydrogen bond network of its binding pocket in the excited state. The purpose of this work is to review the protein's available crystal structure and to study, in how far the light activation has an effect on the protein's tertiary structure. On the basis of this experimental information and in correlation with literature data, a mechanism for the signal transduction in BLUF proteins is proposed.Proteine sind eine Grundlage für die Entwicklung von Leben. Durch ihre vielfältigen strukturellen und chemischen Eigenschaften ermöglichen sie die Bildung verschiedenster biologischer Organismen. Kenntnis der Struktur eines Proteins ist eine wertvolle Basis für die Erforschung seiner Funktion. Proteinkristallstrukturen können mit sehr hoher Auflösung ermittelt werden, aber sie bilden nicht immer den Proteinzustand in Lösung ab. Um von der Kristallstruktur Rückschlüsse auf Mechanismen ziehen zu können, ist es hilfreich, seine Anordnung in Lösung zu kennen und somit eventuelle Konformationsänderungen bestimmen zu können. Durch Anbringen von paramagnetischen Spinmarkierungen an bestimmten Positionen des Proteins kann man mit Elektronen Paramagnetischer Resonanz (EPR) durch deren selektive Wechselwirkung auf die Lösungsstruktur zurückschließen, was im Rahmen dieser Arbeit angewandt wurde. DnaJ aus Thermus thermophilus ist Teil eines Hitzeschutz-Systems, das es diesem Bakterium ermöglicht, bei bis zu 75°C zu überleben. DnaJ identifiziert andere, beschädigte Proteine und stimuliert daraufhin die Reparaturaktivität seines Chaperon-Partners. Zusammen formen sie außerdem einen Speicherkomplex, der sich bei Hitzeeinfluss sofort zersetzt und direkt eine hohe Reparaturaktivität der Komponenten ermöglicht. Im Rahmen dieser Arbeit wurde DnaJ im Vergleich zu einer kürzlich gewonnenen Kristallstruktur untersucht und geprüft, ob sich ein Unterschied zwischen dem Protein in Lösung und im zusammengesetzten Komplex ergibt. Es äußert sich Variabilität der Quartärstruktur sowohl für isoliertes DnaJ als auch für im Komplex gebundenes. BlrB ist ein lichtempfindliches Flavoprotein aus dem Purpurbakterium Rhodobacter sphaeroides. Mit seinem Flavinkofaktor, einem häufig in der Natur anzutreffenden Molekül (Vitamin B2 ist Ribo-Flavin), durchschreitet es einen reversiblen Zyklus der Lichtanregung, der im aktiven Zustand die Farbe des Chromophors leicht verändert. Dies ist ein Hinweis auf eine Reorientierung der polaren Wechselwirkungen in seiner Bindungstasche. Die Motivation dieser Arbeit liegt zum Einen in der sterischen Überprüfung der vorhandenen Kristallstruktur in Lösung und zum Anderen in der Untersuchung des Einflusses der Lichtreaktion auf die äußere Konformation des Proteins. Aus den gewonnenen Erkenntnissen wurde ein Modell für den Prozess der Signalweitergabe in BLUF-Domänen erarbeitet