Hsp90 functions to facilitate the folding of newly synthesized and denatured proteins. Hsp90 function is modulated through its interactions with cochaperones and the binding and hydrolysis of ATP. Recently, novobiocin has been shown to bind to a second nucleotide binding site located within the C-terminal domain of Hsp90. In this report, we have examined the effect of novobiocin on Hsp90 function in reticulocyte lysate. Novobiocin specifically inhibited the maturation of the heme-regulated eIF2alpha kinase (HRI) in a concentration-dependent manner. Novobiocin induced the dissociation of Hsp90 and Cdc37 from immature HRI, while the Hsp90 cochaperones p23, FKBP52, and protein phosphatase 5 remained associated with immature HRI. Proteolytic fingerprinting of Hsp90 indicated that novobiocin had a distinct effect on the conformation of Hsp90, and molybdate lowered the concentration of novobiocin required to alter Hsp90's conformation by 10-fold. The recombinant C-terminal domain of Hsp90 adopted a proteolytic resistant conformation in the presence of novobiocin, indicating that alteration of Hsp90/cochaperone interactions was not the cause of the novobiocin-induced protease resistance within Hsp90's C-terminal domain. The concentration dependence of this novobiocin-induced conformation change correlated with the dissociation of Hsp90 and Cdc37 from immature HRI and novobiocin-induced inhibition of Hsp90/Cdc37-dependent activation of HRI's autokinase activity. The data suggest that binding of novobiocin to the C-terminal nucleotide binding site of Hsp90 induces a change in Hsp90's conformation leading to the dissociation of bound kinase. The unique structure and properties of novobocin-bound Hsp90 suggest that it may represent the "client-release" conformation of the Hsp90 machine.

Nanostructured thin films fabricated from semiconductor nanoparticles (NPs) are of great interest for biomedical applications, but NP materials based on heavy metals can be cytotoxic. In this work, the preparation of semiconductor NPs followed the protocol of layer-by-layer (LBL) assembly, which alleviates this problem. Collagen/poly(acrylic acid) bilayers were added to CdTe/polycation LBL films to produce porous collagen bilayers. Such stratified multilayer systems showed successful cell attachment and survival while native NP films were strongly cytotoxic.

Titania nanoshells with an external diameter of 10–30 nm and a wall thickness of 3–5 nm were prepared by dissolving the silver cores of Ag@TiO2 nanoparticles in a concentrated solution of ammonium hydroxide. The nanoshells were assembled layer‐by‐layer (LBL), with negatively charged poly(acrylic acid) (PAA) to produce coatings with a network of voids and channels in the interior of the film. The diameter of the channels in the titania shells was comparable to the thickness of the electrical double layer in porous matter (0.3–30 nm). The prepared nanoparticulate films demonstrated strong ion‐sieving properties due to the exclusion of some ions from the diffuse region of the electrical double layer. The permeation of ions could be tuned effectively by the pH and ionic strength of a solution between “open” and “closed” states. The ion‐separation effect was utilized for the selective determination of one of the most important neurotransmitters, dopamine, on a background of ascorbic acid. Under physiological conditions, the negative charge on the surface of TiO2 facilitated the permeation of positively charged dopamine through the LBL film to the electrode, preventing the access of the negatively charged ascorbic acid. The deposition of the nanoshell/polyelectrolyte film resulted in a significant improvement to the selectivity of dopamine determination. The prepared nanoshell films were also found to be compatible with nervous tissue secreting dopamine. Although the obtained data demonstrated the potential of TiO2 LBL films for implantable biomedical devices for nerve tissue monitoring, the problem of electrode poisoning by the by‐products of dopamine reduction has yet to be resolved.

Previously, we have demonstrated that the renaturation of heat denatured firefly luciferase is dependent upon the activity of Hsp90 in rabbit reticulocyte lysate. Here, we demonstrate that this assay may identify inhibitors that obstruct the chaperone activity of Hsp90 either by direct binding to its N-terminal or C-terminal nucleotide binding sites or by interference with the ability of the chaperone to switch conformations. The assay was adapted and optimized for high-throughput screening. Greater than 20,000 compounds were screened to demonstrate the feasibility of using this assay on a large scale. The assay was reproducible (av Z-factor=0.62) and identified 120 compounds that inhibited luciferase renaturation by greater than 70% at a concentration of 12.5 microg/mL. IC50 values for twenty compounds with varying structures were determined for inhibition of luciferase refolding and in cell-based assays for Hsp90 inhibition. Several compounds had IC50 values <10 microM and represent a number of new lead structures with the potential for further development and optimization as potent Hsp90 inhibitors.

The 90 kDa heat shock protein (Hsp90) cooperates with its co-chaperone Cdc37 to provide obligatory support to numerous protein kinases involved in the regulation of cellular signal transduction pathways. In this report, crystal structures of protein kinases were used to guide the dissection of two kinases [the Src-family tyrosine kinase, Lck, and the heme-regulated eIF2alpha kinase (HRI)], and the association of Hsp90 and Cdc37 with these constructs was assessed. Hsp90 interacted with both the N-terminal (NL) and C-terminal (CL) lobes of the kinases' catalytic domains. In contrast, Cdc37 interacted only with the NL. The Hsp90 antagonist molybdate was necessary to stabilize the interactions between isolated subdomains and Hsp90 or Cdc37, but the presence of both lobes of the kinases' catalytic domain generated a stable salt-resistant chaperone-client heterocomplex. The Hsp90 co-chaperones FKBP52 and p23 interacted with the catalytic domain and the NL of Lck, whereas protein phosphatase 5 demonstrated unique modes of kinase binding. Cyp40 was a salt labile component of Hsp90 complexes formed with the full-length, catalytic domains, and N-terminal catalytic lobes of Lck and HRI. Additionally, dissections identify a specific kinase motif that triggers Hsp90's conformational switching to a high-affinity client binding state. Results indicate that the Hsp90 machine acts as a versatile chaperone that recognizes multiple regions of non-native proteins, while Cdc37 binds to a more specific kinase segment, and that concomitant recognition of multiple client segments is communicated to generate or stabilize high-affinity chaperone-client heterocomplexes.