The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a “lid” structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of 41 kBT (24 kcal/mol). The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional 12 kBT of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s−1. From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.

The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent folding intermediate comprising lobe II residues (183-383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily.

Stable anchoring of titin within the muscle Z-disk is essential for preserving muscle integrity during passive stretching. One of the main candidates for anchoring titin in the Z-disk is the actin crosslinker α-actinin. The calmodulin-like domain of α-actinin binds to the Z-repeats of titin. However, the mechanical and kinetic properties of this important interaction are still unknown. Here, we use a dual-beam optical tweezers assay to study the mechanics of this interaction at the single-molecule level. A single interaction of α-actinin and titin turns out to be surprisingly weak if force is applied. Depending on the direction of force application, the unbinding forces can more than triple. Our results suggest a model where multiple α-actinin/Z-repeat interactions cooperate to ensure long-term stable titin anchoring while allowing the individual components to exchange dynamically.M uscle is the tissue that is constantly subjected to high mechanical loads. Whereas thick and thin filaments are responsible for active force production, the passive elasticity of muscle is dominated by titin/connectin filaments (1). Hence, under passive stretching conditions the integrity of muscle relies on titin's being firmly anchored within the sarcomere, preventing the interdigitated muscle filaments from falling apart (Fig. 1A). Whereas titin is firmly attached to thick filaments in the A-band and the M-line (2-6), it is much less clear how stable anchoring is achieved in the Z-disk, where adjacent sarcomeres overlap. The superstable titin/telethonin interaction within the Z-disk was considered important for titin anchoring (7-9), but knockout mutants later showed that it is not essential for muscle integrity (10-12). Apart from a direct interaction between actin filaments and titin at the Z-disk edge (13), the most prominent candidate for the anchoring of titin within the Z-disk is its interaction with α-actinin (Fig. 1B) (6,12,14).Four isoforms of human α-actinin have been identified: the calcium-insensitive muscle isoforms 2 and 3, which cross-link actin filaments in sarcomere-delimiting Z-disk complexes, and calcium-sensitive nonmuscle isoforms 1 and 4. α-Actinin is an antiparallel homodimer whose most prominent task is crosslinking actin filaments of neighboring sarcomeres in the Z-disk ( Fig. 1B; reviewed in ref. 14). In each subunit, a flexible region called the neck separates the actin binding domain (ABD) from four spectrin-like repeats (SR) forming the rod region (Fig. 1B and Fig. S1). The rod regions of the two subunits interact and provide a rigid spacer between the actin filaments. At the other end of each subunit a calmodulin-like domain (CaMD) formed by two pairs of EF-hands (EF1-2 and EF3-4) is able to bind a Z-disk region of titin formed by the so-called Z-repeats (15-17). The current model for α-actinin 2 dynamic regulation suggests that EF3-4 hands of one subunit bind to the neck region of the juxtaposed subunit, thus not being available for the interaction with titin Z-repeats (Fig. S1) (18, 19). Upon activ...

PECVD and PEALD of ruthenium films using RuEtcp 2 as a precursor and N 2 /H 2 /Ar plasma as a reducing agent were characterized. A self-adjusting process to overcome the previously reported inhibition of Ru PEALD on TaN substrates was investigated. Ellipsometric modelling of Ru films was demonstrated providing information on both film thickness and estimated Ru content. The physical properties of PECVD/PEALD Ru films were compared to characteristics of sputtered Ru films within the categories resistivity, impurites, crystal structure, conformity and Cu plating. As a result, ToFSIMS, ERDA and 3D atomprobe revealed the presence of carbon impurities in PECVD and PEALD Ru films, dependent on deposition temperature and plasma power. Nevertheless, highly conductive Ru-C films were produced via PECVD and PEALD achieving resistivities equal to PVD Ru. For all types of Ru films, the size effect played a significant role at thicknesses below 10 nm; Cu plating and crystallization behaviour appeared similar. Direct Cu fill potential of different Ru films was discussed for damascene structures and through silicon vias.

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