Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α-and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.laser trapping | parallel pathways | elasticity | force | protein extension W hen looking at protein structures at atomic resolution, it is often tempting to use macroscopic mechanical analogies to describe their function as molecular machines. However, such analogies are often misleading because boundaries between independently stable subdomains cannot often be determined from structures, owing to the high cooperativity of protein folding and structural transitions. Single-molecule protein nanomechanics have emerged as a tool to force biomolecules through their conformational space and, hence, identify hinges, breaking points, and mechanically stable subdomains (1-3).A prominent example of a protein machine undergoing large conformational change during its functional cycle is the ATP-regulated Hsp70 chaperone DnaK-a central molecular chaperone of the protein quality control network in a cell (4-6). Once ATP is bound to the nucleotide binding domain (NBD, blue-yellow; Fig. 1A) of DnaK, the initially closed substrate binding domain (SBD) opens its binding cleft by engaging the β-subdomain to the NBD. In doing so, it undergoes a dramatic ∼10 Å displacement of its lid subdomain ( Fig. 1A; refs. 7 and 8) to allow exchange of substrates (9). Several crystal structures of the isolated SBD (in which the NBD is absent) have been solved (10-15). In these structures, the absence or presence of peptide clients or nonnatural ligands induce no significant structural changes in the closed conformation. There is no indication in the crystal structures of the huge conformational change of the lid domain of the SBD, seen in the ATP form of the full-length two-domain DnaK. Therefore, the large conformational change of the SBD is only observed in the two-domain DnaK after ATP binding. Thus, although the crystal structures provide us with valuable insights into the 3D arrangement of individual atoms, the thermodynamic and mechanical stability of individual substructures are difficult to predict based on this information alone. Here, we ask how the large ATP-induced changes of the SBD, as seen in the two-domain DnaK, are mirrored in the subdomain integrity and nano...
An automatic measuring apparatus called exhalometer for measurement of the radon exhalation rate from soil is introduced. It consists of a pneumatic driven accumulation chamber with an open bottom, a PC-based control system, six Lucas cells for radon measurement and sensors for environmental parameters. It allows moving the accumulation chamber and hereby opening or closing it. The exhalation rate is determined through the increase of radon in the accumulation chamber. For studying exhalation and the affecting factors, the exhalometer was placed at an undisturbed meadow for the entire year of 2015. The daily radon exhalation rate ranges from 2.5 to 50.7 Bq m-2 h-1 with an average of 25.3 Bq m-2 h-1. The exhalation rate shows daily and seasonal variations with its maximum in the afternoon and in spring. The dependence on several environmental parameters is discussed. The stable performance indicates the system's fitness for long-term measurements.
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