SUMMARY HSP90 is a molecular chaperone that associates with numerous substrate proteins called clients. It plays many important roles in human biology and medicine, but determinants of client recognition by HSP90 have remained frustratingly elusive. We systematically and quantitatively surveyed most human kinases, transcription factors, and E3 ligases for interaction with HSP90 and its cochaperone CDC37. Unexpectedly, many more kinases than transcription factors bound HSP90. CDC37 interacted with kinases, but not with transcription factors or E3 ligases. HSP90::kinase interactions varied continuously over a 100-fold range and provided a platform to study client protein recognition. In wild-type clients, HSP90 did not bind particular sequence motifs, but rather associated with intrinsically unstable kinases. Stabilization of the kinase in either its active or inactive conformation with diverse small molecules decreased HSP90 association. Our results establish HSP90 client recognition as a combinatorial process: CDC37 provides recognition of the kinase family, whereas thermodynamic parameters determine client binding within the family.
Prion proteins provide a unique mode of biochemical memory through self-perpetuating changes in protein conformation and function. They have been studied in fungi and mammals, but not yet identified in plants. Using a computational model, we identified candidate prion domains (PrDs) in nearly 500 plant proteins. Plant flowering is of particular interest with respect to biological memory, because its regulation involves remembering and integrating previously experienced environmental conditions. We investigated the prion-forming capacity of three prion candidates involved in flowering using a yeast model, where prion attributes are well defined and readily tested. In yeast, prions heritably change protein functions by templating monomers into higherorder assemblies. For most yeast prions, the capacity to convert into a prion resides in a distinct prion domain. Thus, new prionforming domains can be identified by functional complementation of a known prion domain. The prion-like domains (PrDs) of all three of the tested proteins formed higher-order oligomers. Uniquely, the Luminidependens PrD (LDPrD) fully replaced the prion-domain functions of a well-characterized yeast prion, Sup35. Our results suggest that prion-like conformational switches are evolutionarily conserved and might function in a wide variety of normal biological processes.prions | Luminidependens | plant prion domains
Summary Yeast prions are self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. Using magic angle spinning (MAS) nuclear magnetic resonance spectroscopy, we provide the first detailed look at the dynamic properties of these forms over a broad range of timescales. We establish that different prions strains have distinct amyloid structures, with many side chains in different chemical environments. Surprisingly, the prion strain with a larger fraction of rigid residues also has a larger fraction of highly mobile residues. Differences in mobility correlate with differences in interaction with the prion-partitioning factor Hsp104 in vivo, perhaps explaining strain-specific differences inheritance.
Background:TasA is an extracellular matrix protein that makes amyloid-like fibers in Bacillus subtilis biofilms. Results: An isolated TasA matrix precursor self-assembled in vitro into fibers on hydrophobic surfaces and in acidic solutions. Conclusion: TasA is purified as stable, structured oligomers that aggregate in response to simple physical external cues. Significance: TasA aggregation principles can be used to design new anti-biofilm drugs and surfaces.
Over 100 amino acid replacements in human Cu, Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially-folded states are primarily responsible for toxicity, the role of the structurally-important zinc ion in defining the folding free energy surface of dimeric SOD was determined by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with sub-nanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations towards more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (∼nanomolar). The significant decrease in the population of partially-folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The ∼100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a pre-organized zinc-binding loop in the transition state ensemble for the rate-limiting monomer folding reaction in this β-barrel protein.
Many proteins fold through intermediates that are populated in the submillisecond time regime. To monitor directly the formation of these kinetic intermediates, we have developed a simple, robust, easy to assemble continuous flow mixer for studying folding reactions in the 35–1000μs time regime. The mixer is constructed by laser-machining 75-μm channels in a 127-μm-thick polyimide or polyetheretherketone polymer wafer. Mixing times of ∼25to∼50μs can be achieved for a 1∕10 dilution reaction of 8M urea with flow rates of 10–20mL∕min. CCD-based steady-state and time-correlated single-photon-counting-based fluorescence detection strategies are described. Preliminary results on the early events in the refolding of cytochrome c are presented.
The earliest kinetic folding events for (␣)8 barrels reflect the appearance of off-pathway intermediates. Continuous-flow microchannel mixing methods interfaced to small-angle x-ray scattering (SAXS), circular dichroism (CD), time-resolved Förster resonant energy transfer (trFRET), and time-resolved fluorescence anisotropy (trFLAN) have been used to directly monitor global and specific dimensional properties of the partially folded state in the microsecond time range for a representative (␣) 8 barrel protein.Within 150 s, the ␣-subunit of Trp synthase (␣TS) experiences a global collapse and the partial formation of secondary structure. The time resolution of the folding reaction was enhanced with trFRET and trFLAN to show that, within 30 s, a distinct and autonomous partially collapsed structure has already formed in the N-terminal and central regions but not in the C-terminal region. A distance distribution analysis of the trFRET data confirmed the presence of a heterogeneous ensemble that persists for several hundreds of microseconds. Ready access to locally folded, stable substructures may be a hallmark of repeat-module proteins and the source of early kinetic traps in these very common motifs. Their folding free-energy landscapes should be elaborated to capture this source of frustration.FRET ͉ microsecond mixing ͉ misfolding ͉ small-angle x-ray scattering T he funnel shape of typical protein folding-energy landscapes suggests that sequences have evolved to minimize energetic and topological frustration (1, 2). This view is supported by the twostate folding of many small proteins (3) and the significant correlation of their folding rates with native topology (4, 5). Morecomplex mechanisms, typical of larger proteins, often place intermediates on a progressive path to the native conformation. Surprisingly, a few kinetic studies (6-9) and simulations (10) have revealed that off-pathway intermediates can be populated during the refolding of single-domain globular proteins (11). Delineating the relative contributions of sequence and topology to biases in the energy landscape that lead to these kinetic traps offers potential insights into factors responsible for protein misfolding and, potentially, for numerous devastating pathologies (12).(/␣) 8 TIM barrels (named after triosephosphate isomerase) are one of the most common motifs in biology (13) and are of particular interest in examining the factors leading to off-pathway folding. They constitute the most common structural class of substrates of the chaperonin GroEL, which captures and sequesters partially folded proteins to facilitate their folding to the native state (14). Although the specific partially folded forms of the Escherichia coli TIM barrels responsible for binding to GroEL are not known, in vitro studies suggest two potential candidates. The folding reactions of three TIM barrels of very low sequence identity, IOLI (a protein of unknown function corresponding to the ioll gene in Bacillus subtilis), IGPS (indole-3-glycerol phosphate synthase), a...
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