Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching.

DNA-based FluoroCubes were recently developed as a solution to photobleaching, a ubiquitous limitation of fluorescence microscopy (Niekamp; ; Stuurman; ; Vale Nature Methods, 2020). FluoroCubes, that is, compact ∼4 × 4 × 5.4 nm3 four-helix bundles coupled to ≤6 fluorescent dyes, remain fluorescent up to ∼50× longer than single dyes and emit up to ∼40× as many photons. The current work answers two important questions about the FluoroCubes. First, what is the mechanism by which photostability is enhanced? Second, are FluoroCubes compatible with Förster resonance energy transfer (FRET) and similar techniques? We use single particle photobleaching studies to show that photostability arises through interactions between the fluorophores and the four-helix DNA bundle. Supporting this, we discover that smaller ∼4 × 4 × 2.7 nm3 FluoroCubes also confer ultraphotostability. However, we find that certain dye-dye interactions negatively impact FluoroCube performance. Accordingly, 4-dye FluoroCubes lacking these interactions perform better than 6-dye FluoroCubes. We also demonstrate that FluoroCubes are compatible with FRET and dark quenching applications.

Sisyphus observed: Unraveling the high ATP usage of an RNA chaperone.

DEAD-box proteins are nonprocessive RNA helicases that can function as RNA chaperones by coupling ATP binding and hydrolysis to structural reorganization of RNA. Here, Jarmoskaite et al. quantify the ATP utilization of an RNA chaperone during refolding of a misfolded ribozyme substrate. Strikingly, 100 ATP hydrolysis events are needed per successfully refolded ribozyme, suggesting that each round of unfolding requires ten ATP molecules, since 90% of substrate unfolding cycles only lead back to the kinetically favored misfolded state. This near-Sisyphean effort reveals a potentially conserved model for RNA reorganization by RNA chaperones.

Examination of the nucleotide-linked assembly mechanism of E. coli ClpA.

Escherichia coli ClpA is a AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP-dependent unfolding and translocation of substrate proteins targeted for degradation by a protease, ClpP. ClpA hexamers associate with one or both ends of ClpP tetradecamers to form ClpAP complexes. Each ClpA protomer contains two nucleotide-binding sites, NBD1 and NBD2, and self-assembly into hexamers is thermodynamically linked to nucleotide binding. Despite a number of studies aimed at characterizing ClpA and ClpAP-catalyzed substrate unfolding and degradation, respectively, to date the field is unable to quantify the concentration of ClpA hexamers available to interact with ClpP for any given nucleotide and total ClpA concentration. In this work, sedimentation velocity studies are used to quantitatively examine the self-assembly of a ClpA Walker B variant in the presence of ATP. In addition to the hexamerization, we observe the formation of a previously unreported ClpA dodecamer in the presence of ATP. Further, we report apparent equilibrium constants for the formation of each ClpA oligomer obtained from direct boundary modeling of the sedimentation velocity data. The energetics of nucleotide binding to NBD1 and NBD2 are revealed by examining the dependence of the apparent association equilibrium constants on free nucleotide concentration.

ATP hydrolysis inactivating Walker B mutation perturbs E. coli ClpA self-assembly energetics in the absence of nucleotide.

E. coli ClpA is an AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP-dependent unfolding and translocation of substrate proteins for the purposes of proper proteome maintenance. Biologically active ClpA hexamers contain two nucleotide binding domains (NBD) per protomer, D1 and D2. Despite extensive study, complete understanding of how the twelve NBDs within a ClpA hexamer coordinate ATP binding and hydrolysis to polypeptide translocation is currently lacking. To examine nucleotide binding and coordination at D1 and D2, ClpA Walker B variants deficient in ATP hydrolysis at one or both NBDs have been employed in various studies. In the presence of ATP, it is widely assumed that ClpA Walker B variants are entirely hexameric. However, a thermodynamically rigorous examination of the self-assembly mechanism has not been obtained. Differences in the assembly due to the mutation can be misattributed to the active NBD, leading to potential misinterpretations of kinetic studies. Here we use sedimentation velocity studies to quantitatively examine the self-assembly mechanism of ClpA Walker B variants deficient in ATP hydrolysis at D1, D2, and both NBDs. We found that the Walker B mutations had clear, if modest, effects on the assembly. Most notably, the Walker B mutation stabilizes the population of a larger oligomer in the absence of nucleotide, that is not present for analogous concentrations of wild type ClpA. Our results indicate that Walker B mutants, widely used in studies of AAA+ family proteins, require additional characterization as the mutation affects not only ATP hydrolysis, but also the ligand linked assembly of these complexes. This linkage must be considered in investigations of unfolding or other ATP dependent functions.

Escherichia coli DnaK Allosterically Modulates ClpB between High- and Low-Peptide Affinity States.

ClpB and DnaKJE provide protection to Escherichia coli cells during extreme environmental stress. Together, this co-chaperone system can resolve protein aggregates, restoring misfolded proteins to their native form and function in solubilizing damaged proteins for removal by the cell's proteolytic systems. DnaK is the component of the KJE system that directly interacts with ClpB. There are many hypotheses for how DnaK affects ClpB-catalyzed disaggregation, each with some experimental support. Here, we build on our recent work characterizing the molecular mechanism of ClpB-catalyzed polypeptide translocation by developing a stopped-flow FRET assay that allows us to detect ClpB's movement on model polypeptide substrates in the absence or presence of DnaK. We find that DnaK induces ClpB to dissociate from the polypeptide substrate. We propose that DnaK acts as a peptide release factor, binding ClpB and causing the ClpB conformation to change to a low-peptide affinity state. Such a role for DnaK would allow ClpB to rebind to another portion of an aggregate and continue nonprocessive translocation to disrupt the aggregate.

Comparative Analysis of the Structure and Function of AAA+ Motors ClpA, ClpB, and Hsp104: Common Threads and Disparate Functions.

Cellular proteostasis involves not only the expression of proteins in response to environmental needs, but also the timely repair or removal of damaged or unneeded proteins. AAA+ motor proteins are critically involved in these pathways. Here, we review the structure and function of AAA+ proteins ClpA, ClpB, and Hsp104. ClpB and Hsp104 rescue damaged proteins from toxic aggregates and do not partner with any protease. ClpA functions as the regulatory component of the ATP dependent protease complex ClpAP, and also remodels inactive RepA dimers into active monomers in the absence of the protease. Because ClpA functions both with and without a proteolytic component, it is an ideal system for developing strategies that address one of the major challenges in the study of protein remodeling machines: how do we observe a reaction in which the substrate protein does not undergo covalent modification? Here, we review experimental designs developed for the examination of polypeptide translocation catalyzed by the AAA+ motors in the absence of proteolytic degradation. We propose that transient state kinetic methods are essential for the examination of elementary kinetic mechanisms of these motor proteins. Furthermore, rigorous kinetic analysis must also account for the thermodynamic properties of these complicated systems that reside in a dynamic equilibrium of oligomeric states, including the biologically active hexamer.

Avidity for Polypeptide Binding by Nucleotide-Bound Hsp104 Structures.

Recent Hsp104 structural studies have reported both planar and helical models of the hexameric structure. The conformation of Hsp104 monomers within the hexamer is affected by nucleotide ligation. After nucleotide-driven hexamer formation, Hsp104-catalyzed disruption of protein aggregates requires binding to the peptide substrate. Here, we examine the oligomeric state of Hsp104 and its peptide binding competency in the absence of nucleotide and in the presence of ADP, ATPγS, AMPPNP, or AMPPCP. Surprisingly, we found that only ATPγS facilitates avid peptide binding by Hsp104. We propose that the modulation between high- and low-peptide affinity states observed with these ATP analogues is an important component of the disaggregation mechanism of Hsp104.

Escherichia coli ClpB is a non-processive polypeptide translocase.

Escherichia coli caseinolytic protease (Clp)B is a hexameric AAA+ [expanded superfamily of AAA (ATPase associated with various cellular activities)] enzyme that has the unique ability to catalyse protein disaggregation. Such enzymes are essential for proteome maintenance. Based on structural comparisons to homologous enzymes involved in ATP-dependent proteolysis and clever protein engineering strategies, it has been reported that ClpB translocates polypeptide through its axial channel. Using single-turnover fluorescence and anisotropy experiments we show that ClpB is a non-processive polypeptide translocase that catalyses disaggregation by taking one or two translocation steps followed by rapid dissociation. Using single-turnover FRET experiments we show that ClpB containing the IGL loop from ClpA does not translocate substrate through its axial channel and into ClpP for proteolytic degradation. Rather, ClpB containing the IGL loop dysregulates ClpP leading to non-specific proteolysis reminiscent of ADEP (acyldepsipeptide) dysregulation. Our results support a molecular mechanism where ClpB catalyses protein disaggregation by tugging and releasing exposed tails or loops.