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The link between cofactor binding and protein activity is well-established. However, how cofactor interactions modulate folding of large proteins remains unknown. We use optical tweezers, clustering and global fitting to dissect the folding mechanism of Drosophila cryptochrome (dCRY), a 542-residue protein that binds FAD, one of the most chemically and structurally complex cofactors in nature. We show that the first dCRY parts to fold are independent of FAD, but later steps are FAD-driven as the remaining polypeptide folds around the cofactor. FAD binds to largely unfolded intermediates, yet with association kinetics above the diffusion-limit. Interestingly, not all FAD moieties are required for folding: whereas the isoalloxazine ring linked to ribitol and one phosphate is sufficient to drive complete folding, the adenosine ring with phosphates only leads to partial folding. Lastly, we propose a dCRY folding model where regions that undergo conformational transitions during signal transduction are the last to fold.

Numerous ATPases associated with diverse cellular activities (AAA+) proteins form hexameric, ring-shaped complexes that function via ATPase-coupled translocation of substrates across the central channel. Cryo-electron microscopy of AAA+ proteins processing substrate has revealed non-symmetric, staircase-like hexameric structures that indicate a sequential clockwise/2-residue step translocation model for these motors. However, for many of the AAA+ proteins that share similar structural features, their translocation properties have not yet been experimentally determined. In the cases where translocation mechanisms have been determined, a two-residue translocation step-size has not been resolved. In this review, we explore Hsp104, ClpB, ClpA and ClpX as examples to review the experimental methods that have been used to examine, in solution, the translocation mechanisms employed by AAA+ motor proteins. We then ask whether AAA+ motors sharing similar structural features can have different translocation mechanisms. Finally, we discuss whether a single AAA+ motor can adopt multiple translocation mechanisms that are responsive to different challenges imposed by the substrate or the environment. We suggest that AAA+ motors adopt more than one translocation mechanism and are tuned to switch to the most energetically efficient mechanism when constraints are applied.

Eukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale. We analyzed

Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols’ elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I.

Eukaryotes express three DNA-dependent RNA polymerases (Pols) that are responsible for the entirety of cellular genomic expression. The three Pols have evolved to express specific cohorts of RNAs and thus have diverged both structurally and functionally to efficiently execute their specific transcriptional roles. One example of this divergence is Pol I’s inclusion of a proofreading factor as a bona fide subunit, as opposed to Pol II, which recruits a transcription factor, TFIIS, for proofreading. The A12.2 (A12) subunit of Pol I shares homology with both the Rpb9 subunit of Pol II as well as the transcription factor TFIIS, which promotes RNA cleavage and proofreading by Pol II. In this study, the functional contribution of the TFIIS-like C-terminal domain and the Rpb9-like N-terminal domain of the A12 subunit are probed through mutational analysis. We found that a Pol I mutant lacking the C-terminal domain of the A12 subunit (ΔA12CTD Pol I) is slightly faster than wild-type Pol I in single-nucleotide addition, but ΔA12CTD Pol I lacks RNA cleavage activity. ΔA12CTD Pol I is likewise similar to wild-type Pol I in elongation complex stability, whereas removal of the entire A12 subunit (ΔA12 Pol I) was previously demonstrated to stabilize transcription elongation complexes. Furthermore, the ΔA12CTD Pol I is sensitive to downstream sequence context, as ΔA12CTD Pol I exposed to AT-rich downstream DNA is more arrest prone than ΔA12 Pol I. These data demonstrate that the N-terminal domain of A12 does not stimulate Pol I intrinsic RNA cleavage activity, but rather contributes to core transcription elongation properties of Pol I.

The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) fromSaccharomyces cerevisiaehas not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausingin vitroin response to downstream DNA.In vitrostudies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity. Analysis of Pol I native elongating transcript sequencing data inSaccharomyces cerevisiaesuggests that these downstream sequence elements influence Pol Iin vivo. Native elongating transcript sequencing studies reveal that Pol I occupancy increases as downstream AT content increases and decreases as downstream GC content increases. Collectively, these data demonstrate that the downstream DNA sequence directly impacts the kinetics of transcription elongation prior to the sequence entering the active site of Pol I bothin vivoandin vitro.

Eukaryotes express three nuclear RNA polymerases (Pols I, II, and III) that are essential for cell survival. Despite extensive investigation of the three Pols, significant knowledge gaps regarding their biochemical properties remain because each Pol has been evaluated independently under disparate experimental conditions and methodologies. To advance our understanding of the Pols, we employed identical

Parameter optimization or “data fitting” is a computational process that identifies a set of parameter values that best describe an experimental data set. Parameter optimization is commonly carried out using a computer program utilizing a non-linear least squares (NLLS) algorithm. These algorithms work by continuously refining a user supplied initial guess resulting in a systematic increase in the goodness of fit. A well-understood problem with this class of algorithms is that in the case of models with correlated parameters the optimized output parameters are initial guess dependent. This dependency can potentially introduce user bias into the resultant analysis. While many optimization programs exist, few address this dilemma. Here we present a data analysis tool, MENOTR, that is capable of overcoming the initial guess dependence in parameter optimization. Several case studies with published experimental data are presented to demonstrate the capabilities of this tool. The results presented here demonstrate how to effectively overcome the initial guess dependence of NLLS leading to greater confidence that the resultant optimized parameters are the best possible set of parameters to describe an experimental data set. While the optimization strategies implemented within MENOTR are not entirely novel, the application of these strategies to optimize parameters in kinetic and thermodynamic biochemical models is uncommon. MENOTR was designed to require minimal modification to accommodate a new model making it immediately accessible to researchers with a limited programming background. We anticipate that this toolbox can be used in a wide variety of data analysis applications. Prototype versions of this toolbox have been used in a number of published investigations already, as well as ongoing work with chemical-quenched flow, stopped-flow, and molecular tweezers data sets. Statement of significance Non-linear least squares (NLLS) is a common form of parameter optimization in biochemistry kinetic and thermodynamic investigations These algorithms are used to fit experimental data sets and report corresponding parameter values. The algorithms are fast and able to provide good quality solutions for models involving few parameters. However, initial guess dependence is a well-known drawback of this optimization strategy that can introduce user bias. An alternative method of parameter optimization are genetic algorithms (GA). Genetic algorithms do not have an initial guess dependence but are slow at arriving at the best set of fit parameters. Here, we present MENOTR, a parameter optimization toolbox utilizing a hybrid GA/NLLS algorithm. The toolbox maximizes the strength of each strategy while minimizing the inherent drawbacks.

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.

RNA polymerases execute the first step in gene expression: transcription of DNA into RNA. Eukaryotes, unlike prokaryotes, express at least three specialized nuclear multisubunit RNA polymerases (Pol I, Pol II, and Pol III). RNA polymerase I (Pol I) synthesizes the most abundant RNA, ribosomal RNA. Nearly 60% of total transcription is devoted to ribosomal RNA synthesis, making it one of the cell’s most energy consuming tasks. While a kinetic mechanism for nucleotide addition catalyzed by Pol I has been reported, it remains unclear to what degree different nucleotide sequences impact the incorporation rate constants. Furthermore, it is currently unknown if the previous investigation of a single-nucleotide incorporation was sensitive to the translocation step. Here, we show that Pol I exhibits considerable variability in both k(max) and K(1/2)values using an in vitro multi-NTP incorporation assay measuring AMP and GMP incorporations. We found the first two observed nucleotide incorporations exhibited faster k(max)-values (∼200 s(−1)) compared with the remaining seven positions (∼60 s(−1)). Additionally, the average K(1/2) for ATP incorporation was found to be approximately threefold higher compared with GTP, suggesting Pol I has a tighter affinity for GTP compared with ATP. Our results demonstrate that Pol I exhibits significant variability in the observed rate constant describing each nucleotide incorporation. Understanding of the differences between the Pol enzymes will provide insight on the evolutionary pressures that led to their specialized roles. Therefore, the findings resulting from this work are critically important for comparisons with other polymerases across all domains of life.

The multitude of varied, energy-dependent processes that exist in the cell necessitate a diverse array of macromolecular machines to maintain homeostasis, allow for growth, and facilitate reproduction. ATPases associated with various cellular activity are a set of protein assemblies that function as molecular motors to couple the energy of nucleoside triphosphate binding and hydrolysis to mechanical movement along a polymer lattice. A recent boom in structural insights into these motors has led to structural hypotheses on how these motors fulfill their function. However, in many cases, we lack direct kinetic measurements of the dynamic processes these motors undergo as they transition between observed structural states. Consequently, there is a need for improved techniques for testing the structural hypotheses in solution. Here, we apply transient-state fluorescence anisotropy and total fluorescence stopped-flow methods to the analysis of polypeptide translocation catalyzed by these ATPase motors. We specifically focus on the Hsp100-Clp protein system of ClpA, which is a well-studied, model ATPases associated with various cellular activity system that has both eukaryotic and archaea homologs. Using this system, we show that we can reproduce previously established kinetic parameters from the simultaneous analysis of fluorescence anisotropy and total fluorescence and overcome previous limitations of our previous approach. Specifically, for the first time, to our knowledge, we obtain quantitative interpretations of the translocation of polypeptide substrates longer than 100 aa.

The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ClpP proteolytic chamber. Here, we determined high-resolution structures of wild-type Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis. A spiral of pore loop–substrate contacts spans both ClpA AAA+ domains. Protomers at the spiral seam undergo nucleotide-specific rearrangements, supporting substrate translocation. IGL loops extend flexibly to bind the planar, heptameric ClpP surface with the empty, symmetry-mismatched IGL pocket maintained at the seam. Three different structures identify a binding-pocket switch by the IGL loop of the lowest positioned protomer, involving release and re-engagement with the clockwise pocket. This switch is coupled to a ClpA rotation and a network of conformational changes across the seam, suggesting that ClpA can rotate around the ClpP apical surface during processive steps of translocation and proteolysis. Cryo-EM structures of Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis reveal contacts that drive substrate translocation and a dynamic switch mechanism at the ClpA–ClpP interface.

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.

Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel. Here, we apply transient state, single turnover kinetics to investigate the ATP-dependent translocation of soluble polypeptides by Hsp104 and Hsp104A503S, a potentiated variant developed to resolve misfolded conformers implicated in neurodegenerative disease. We establish that Hsp104 and Hsp104A503S can operate as nonprocessive translocases for soluble substrates, indicating a “partial threading” model of translocation. Remarkably, Hsp104A503S exhibits altered coupling of ATP binding to translocation and decelerated dissociation from polypeptide substrate compared to Hsp104. This altered coupling and prolonged substrate interaction likely increases entropic pulling forces, thereby enabling more effective aggregate dissolution by Hsp104A503S.

Eukaryotic cells express at least three nuclear RNA polymerases (Pols), each with a unique set of gene targets. Though these enzymes are homologous, there are many differences among the Pols. In this study, a novel assay for Pol I transcription elongation was developed to probe enzymatic differences among the Pols. In Saccharomyces cerevisiae, a mutation in the universally conserved hinge region of the trigger loop, E1103G, induces a gain of function in the Pol II elongation rate, whereas the corresponding mutation in Pol I, E1224G, results in a loss of function. The E1103G Pol II mutation stabilizes the closed conformation of the trigger loop, promoting the catalytic step, the putative rate-limiting step for Pol II. In single-nucleotide and multinucleotide addition assays, we observe a decrease in the rate of nucleotide addition and dinucleotide cleavage activity by E1224G Pol I and an increase in the rate of misincorporation. Collectively, these data suggest that Pol I is at least in part rate-limited by the same step as Pol II, the catalytic step.

Alkali metal salts can affect both the activities and regioselectivities of alkene hydroformylation catalysts containing polyether-functionalized phosphorus-donor ligands; however, it is unclear whether these effects arise from direct alkali metal cation binding to the active catalysts. To gain more insight into these effects, a series of phosphite-lariat ether ligands derived from the alkali metal cation binding agents 2-hydroxymethyl-12-crown-4 and 2-hydroxymethyl-15-crown-5 have been prepared. Rhodium(I) complexes of these ligands have been evaluated as styrene hydroformylation catalysts in the absence and presence of a variety of alkali metal salts. The activities of catalysts containing phosphites derived from 2,2′-biphenol or 1,1′-binaphthol increased significantly (up to 92%) in the presence of alkali metal cations that are “moderately oversized” for archetypal binding to the crown cavity. When this criterion are not met, a decrease in the catalytic activity is observed upon addition of an alkali m...

Eukaryotes express three or more multisubunit nuclear RNA polymerases (Pols) referred to as Pols I, II, and III, each of which synthesizes a specific subset of RNAs. Consistent with the diversity of their target genes, eukaryotic cells have evolved divergent cohorts of transcription factors and enzymatic properties for each RNA polymerase system. Over the years, many trans-acting factors that orchestrate transcription by the individual Pols have been described; however, little effort has been devoted to characterizing the molecular mechanisms of Pol I activity. To begin to address this gap in our understanding of eukaryotic gene expression, here we establish transient-state kinetic approaches to characterize the nucleotide incorporation mechanism of Pol I. We collected time courses for single turnover nucleotide incorporation reactions over a range of substrate ATP concentrations that provide information on both Pol I’s nucleotide addition and nuclease activities. The data were analyzed by model-independent and model-dependent approaches, resulting in, to our knowledge, the first minimal model for the nucleotide addition pathway for Pol I. Using a grid searching approach we provide rigorous bounds on estimated values of the individual elementary rate constants within the proposed model. This work reports the most detailed analysis of Pol I mechanism to date. Furthermore, in addition to their use in transient state kinetic analyses, the computational approaches applied here are broadly applicable to global optimization problems.

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.

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.

Despite sharing a highly conserved core architecture with their prokaryotic counterparts, eukaryotic multisubunit RNA polymerases (Pols) have undergone structural divergence and biological specialization. Interesting examples of structural divergence are the A12.2 and C11 subunits of Pols I and III, respectively. Whereas all known cellular Pols possess cognate protein factors that stimulate cleavage of the nascent RNA, Pols I and III have incorporated their cleavage factors as bona fide subunits. Although it is not yet clear why these polymerases have incorporated their cleavage factors as subunits, a picture is emerging that identifies roles for these subunits beyond providing nucleolytic activity. Specifically, it appears that both A12.2 and C11 are required for efficient termination of transcription by Pols I and III. Given that termination involves destabilization of the elongation complex (EC), we tested whether A12.2 influences stability of the Pol I EC. Using, to our knowledge, a novel assay to measure EC dissociation kinetics, we have determined that A12.2 is an intrinsic destabilizer of the Pol I EC. In addition, the salt concentration dependence of Pol I EC dissociation kinetics suggests that A12.2 alters electrostatic interactions within the EC. Importantly, these data present a mechanistic basis for the requirement of A12.2 in Pol I termination. Combined with recent work demonstrating the direct involvement of A12.2 in Pol I nucleotide incorporation, this study further supports the concept that A12.2 cannot be viewed solely as a cleavage factor.

The metallathiacrown ethers cis-Mo(CO)4{2,2′-(C12H8O2)PO(CH2CH2S)nCH2CH2OP(2,2′-(O2H8C12))} (n = 2, 3) and cis-Mo(CO)4{2,2′-(C12H8O2)POCH2CH2S-1-(C6H4)-2-SCH2CH2OP(2,2′-(O2H8C12))} have been prepared as soft metal selective molecular receptors. Multinuclear NMR spectroscopy and X-ray crystallography have been used to show that byproducts formed during the syntheses of the metallathiacrown ethers cis-Mo(CO)4{2,2′-(C12H8O2)PO(CH2CH2S)nCH2CH2OP(2,2′-(O2H8C12))} (n = 2, 3) are homobinuclear complexes with cis-Mo(CO)4(P-donor)(S-donor) centers. The abilities of the metallathiacrown ethers to bind PdCl2 and PtCl2 have been assessed using 31P{1H} NMR spectroscopy and X-ray crystallography. The complexes showed null results with PdCl2; however, the PtCl2 experiments showed that the complexes cis-Mo(CO)4{2,2′-(C12H8O2)PO(CH2CH2S)nCH2CH2OP(2,2′-(O2H8C12))} (n = 2, 3) formed heterobinuclear cis,cis-{[Mo(CO)4{2,2′-(C12H8O2)PO(CH2CH2S)nCH2CH2OP(2,2′-(O2H8C12))}]PtCl2} (n = 2, 3) complexes. The 31P{1H} NMR spectra of t...

Escherichia coli ClpB is a heat shock protein that belongs to the AAA+ protein superfamily. Studies have shown that ClpB and its homologue in yeast, Hsp104, can disrupt protein aggregates in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity. In addition, it is widely assumed that ClpB is uniformly hexameric in the presence of nucleotides. Here we report, in the absence of nucleotide, that increasing ClpB concentration leads to ClpB hexamer formation, decreasing NaCl concentration stabilizes ClpB hexamers, and the ClpB assembly reaction is best described by a monomer, dimer, tetramer, hexamer equilibrium under the three salt concentrations examined. Further, we found that ClpB oligomers exhibit relatively fast dissociation on the time scale of sedimentation. We anticipate our studies on ClpB assembly to be a starting point to understand how ClpB assembly is linked to the binding and disaggregation of denatured proteins.

Here we show that ClpB is a non-processive translocase that takes, at most, two steps on the polypeptide backbone before dissociation. These findings indicate that ClpB is not likely to translocate polypeptide through its axial channel as previously concluded., 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.

Enzyme-catalyzed protein unfolding is essential for a large array of biological functions, including microtubule severing, membrane fusion, morphogenesis and trafficking of endosomes, protein disaggregation, and ATP-dependent proteolysis. These enzymes are all members of the ATPases associated with various cellular activity (AAA+) superfamily of proteins. Escherichia coli ClpA is a hexameric ring ATPase responsible for enzyme-catalyzed protein unfolding and translocation of a polypeptide chain into the central cavity of the tetradecameric E. coli ClpP serine protease for proteolytic degradation. Further, ClpA also uses its protein unfolding activity to catalyze protein remodeling reactions in the absence of ClpP. ClpA recognizes and binds a variety of protein tags displayed on proteins targeted for degradation. In addition, ClpA binds unstructured or poorly structured proteins containing no specific tag sequence. Despite this, a quantitative description of the relative binding affinities for these differe...

All cellular RNA polymerases are influenced by protein factors that stimulate RNA polymerase-catalyzed cleavage of the nascent RNA. Despite divergence in amino acid sequence, these so-called “cleavage factors” appear to share a common mechanism of action. Cleavage factors associate with the polymerase through a conserved structural element of the polymerase known as the secondary channel or pore. This mode of association enables the cleavage factor to reach through the ‘ secondary channel into the polymerase active site to reorient the active site divalent metal ions. This reorientation converts the polymerase active site into a nuclease active site. Interestingly, eukaryotic RNA polymerases I and III (Pols I and III, respectively) have incorporated their cleavage factors as bona fide subunits known as A12.2 and C11, respectively. Although it is clear that A12.2 and C11 dramatically stimulate the polymerase’s cleavage activity, it is not known if or how these subunits affect the polymerization mechanism. In this work we have used transient-state kinetic techniques to characterize a Pol I isoform lacking A12.2. Our data clearly demonstrate that the A12.2 subunit profoundly affects the kinetics and energetics of the elementary steps of Pol I-catalyzed nucleotide incorporation. Given the high degree of conservation between polymerase—cleavage factor interactions, these data indicate that cleavage factor-modulated nucleotide incorporation mechanisms may be common to all cellular RNA polymerases.

For ensemble and single-molecule analyses of transcription, the use of synthetic transcription elongation complexes has been a versatile and powerful tool. However, structural analyses demonstrate that short RNA substrates, often employed in these assays, would occupy space within the RNA polymerase. Most commercial RNA oligonucleotides do not carry a 5′-triphosphate as would be present on a natural, de novo synthesized RNA. To examine the effects of 5′-moities on transcription kinetics, we measured nucleotide addition and 3′-dinucleotide cleavage by eukaryotic RNA polymerase I using 5′-hydroxyl and 5′-triphosphate RNA substrates. We found that 5′ modifications had no discernable effect on the kinetics of nucleotide addition; however, we observed clear, but modest, effects on the rate of backtracking and/or dinucleotide cleavage. These data suggest that the 5′-end may influence RNA polymerase translocation, consistent with previous prokaryotic studies, and these findings may have implications on kinetic barriers that confront RNA polymerases during the transition from initiation to elongation.

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.

The Fas death receptor-activated death-inducing signaling complex (DISC) regulates apoptosis in many normal and cancer cells. Qualitative biochemical experiments demonstrate that calmodulin (CaM) binds to the death domain of Fas. The interaction between CaM and Fas regulates Fas-mediated DISC formation. A quantitative understanding of the interaction between CaM and Fas is important for the optimal design of antagonists for CaM or Fas to regulate the CaM-Fas interaction, thus modulating Fas-mediated DISC formation and apoptosis. The V254N mutation of the Fas death domain (Fas DD) is analogous to an identified mutant allele of Fas in lpr-cg mice that have a deficiency in Fas-mediated apoptosis. In this study, the interactions of CaM with the Fas DD wild type (Fas DD WT) and with the Fas DD V254N mutant were characterized using isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), and molecular dynamics (MD) simulations. ITC results reveal an endothermic binding characteristic and an entropy-driven interaction of CaM with Fas DD WT or with Fas DD V254N. The Fas DD V254N mutation decreased the association constant (Ka) for CaM-Fas DD binding from (1.79 ± 0.20) × 10(6) to (0.88 ± 0.14) × 10(6) M(-1) and slightly increased a standard state Gibbs free energy (ΔG°) for CaM-Fas DD binding from -8.87 ± 0.07 to -8.43 ± 0.10 kcal/mol. CD secondary structure analysis and MD simulation results did not show significant secondary structural changes of the Fas DD caused by the V254N mutation. The conformational and dynamical motion analyses, the analyses of hydrogen bond formation within the CaM binding region, the contact numbers of each residue, and the electrostatic potential for the CaM binding region based on MD simulations demonstrated changes caused by the Fas DD V254N mutation. These changes caused by the Fas DD V254N mutation could affect the van der Waals interactions and electrostatic interactions between CaM and Fas DD, thereby affecting CaM-Fas DD interactions. Results from this study characterize CaM-Fas DD interactions in a quantitative way, providing structural and thermodynamic evidence of the role of the Fas DD V254N mutation in the CaM-Fas DD interaction. Furthermore, the results could help to identify novel strategies for regulating CaM-Fas DD interactions and Fas DD conformation and thus to modulate Fas-mediated DISC formation and thus Fas-mediated apoptosis.

There are five known ATP-dependent proteases in Escherichia coli (Lon, ClpAP, ClpXP, HslUV, and the membrane-associated FtsH) that catalyze the removal of both misfolded and properly folded proteins in cellular protein quality control pathways. Hexameric ClpA rings associate with one or both faces of the cylindrically shaped tetradecameric ClpP protease. ClpA catalyzes unfolding and translocation of polypeptide substrates into the proteolytic core of ClpP for degradation through repeated cycles of ATP binding and hydrolysis at two nucleotide binding domains on each ClpA monomer. We previously reported a molecular mechanism for ClpA catalyzed polypeptide translocation in the absence of ClpP, including elementary rate constants, overall rate, and the kinetic step size. However, the potential allosteric effect of ClpP on the mechanism of ClpA catalyzed translocation remains unclear. Using single-turnover fluorescence stopped-flow methods, here we report that ClpA, when associated with ClpP, translocates polypeptide with an overall rate of ~35 aa s(-1) and, on average, traverses ~5 aa between two rate-limiting steps with reduced cooperativity between ATP binding sites in the hexameric ring. This is in direct contrast to our previously reported observation that, in the absence of ClpP, ClpA translocates polypeptide substrates with a maximum translocation rate of ~20 aa s(-1) with cooperativity between ATPase sites. Our results demonstrate that ClpP allosterically impacts the polypeptide translocation activity of ClpA by reducing the cooperativity between ATP binding sites.

E. coli ClpB is a heat shock protein that belongs to the AAA+ protein family. Studies have shown that ClpB and its eukaryotic homologue, Hsp104, can disaggregate denatured proteins by themselves or cooperate with the DnaK chaperone system in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity and ClpB majorly exist as hexamer in the presence of nucleoside triphosphate. In contrast to this conclusion, our sedimentation velocity data show that ClpB resides in a monomer-dimer-tetramer-hexamer equilibrium in the presence of ATPϒS (a slowly hydrolysable ATP analog). ClpB hexamers exhibit fast subunit exchange in the absence of nucleoside triphosphate, while the exchange rates decrease when the binding of nucleotide approaching to saturation. For the first time, we determined the binding constants and stoichiometries for ATPϒS to each ClpB oligomer. The monomer is only able to bind one nucleotide whereas all twelve sites in the hexameric ring are bound by nucleotide. Interestingly, dimers and tetramers exhibit stoichiometries of ∼3 and 7, respectively, which is one fewer than the maximum number of binding sites, which suggest an open conformation for the intermediates. We also determined the assembly constants for dimers, tetramers, and hexamers and their dependencies on nucleotide. These interaction constants make it possible to predict the concentration of hexamers present and able to bind to co-chaperones and polypeptide substrates. We anticipate our studies on ClpB assembly to be a starting point for understanding how ClpB hexamers disaggregate protein aggregates.

A new metallacrown ether, cis-PdCl2{(2,2′-C12H8O2)P(OCH2CH2)4OP(2,2′-O2H8C12)}, has been synthesized and fully characterized by NMR spectroscopy and X-ray crystallography. A new synthetic method and complete NMR characterization for the previously reported and closely related cis-Mo(CO)4(2,2′-C12H8O2)P(OCH2CH2)4OP(2,2′-O2H8C12) metallacrown ether are also reported. Both complexes are monomeric, exhibiting cis coordination geometries both in solution and in the solid state, and the cis-Mo(CO)4(2,2′-C12H8O2)P(OCH2CH2)4OP(2,2′-O2H8C12) metallacrown ether does not appear to undergo cis–trans isomerization in the presence of HgCl2. These results are surprising because the closely related PdCl2(Ph2P(CH2CH2O)nCH2CH2PPh2) (n = 3–5) metallacrown ethers exhibit both cis–trans and monomer–cyclic oligomer equilibria in solution, and cis-Mo(CO)4 (Ph2P(CH2CH2O)nCH2CH2PPh2) (n = 3–5) metallacrown ethers rapidly undergo cis–trans isomerization in the presence of HgCl2. The coordination of Li+ cations to the cis-Mo(CO)4(2...

An upper-level undergraduate course that explores ethics in chemistry and the impact of chemical innovations on society has been developed. Goals of this course were to promote student recognition of ethical considerations in chemical innovations and chemical research and demonstrate the link between the application of chemical innovations and the formation of government policy. The course involved presentation and discussion on multiple topics along with student writing about a use of chemistry in society and expression of students’ own opinions based on their literature research. The SENCER-SALG assessment was used to elicit students’ opinions on their experience in this course. Student responses indicate that the course had a positive influence on their recognition of chemistry in society and their likelihood of civic engagement.

Synthetic methods have been developed for lead Sortase A inhibitors identified from previous studies. Several derivatives of the lead inhibitor were synthesized to derive preliminary structure activity relationships (SAR). Different regions of the lead inhibitor that are critical for the enzyme activity have been determined by systematic SAR studies. The E stereochemistry of the lead compound was found to be critical for its activity. Replacement of the E double bond with Z double bond or a rigid triple bond reduced the enzyme inhibitory activity in most cases. Reduction of the double bond to a C-C single bond resulted in complete loss of activity. Amide carbonyl and NH groups were also found to be crucial for the activity of this class of inhibitors, as well. The morpholine ring oxygen atom was also found to be an important factor for the activity of the lead inhibitor. Preliminary SAR studies led to the identification of compounds with improved enzyme inhibition. The most active compound was found to have an IC(50) value of 58 microM against the enzyme.

The removal of damaged or unneeded proteins by ATP-dependent proteases is crucial for cell survival in all organisms. Integral components of ATP-dependent proteases are motor proteins that unfold stably folded proteins that have been targeted for removal. These protein unfoldases/polypeptide translocases use ATP to unfold the target proteins and translocate them into a proteolytic component. Despite the central role of these motor proteins in cell homeostasis, a number of important questions regarding the molecular mechanisms of enzyme catalyzed protein unfolding and translocation remain unanswered. Here, we demonstrate that Escherichia coli ClpA, in the absence of the proteolytic component ClpP, processively and directionally steps along the polypeptide backbone with a kinetic step size of approximately 14 amino acids, independent of the concentration of ATP with a rate of approximately 19 amino acids s(-1) at saturating concentrations of ATP. In contrast to earlier studies by others, we have developed single-turnover fluorescence stopped-flow methods that allow us to quantitatively examine the molecular mechanism of the motor component ClpA decoupled from the proteolytic component ClpP. For the first time, we reveal that in the absence of ClpP ClpA translocates polypeptides directionally, processively and in discrete steps similar to other motor proteins that translocate vectorially on a linear lattice, such as nucleic acid helicases and kinesin. We believe that the methods employed here will be generally applicable to the examination of other AAA+ protein translocases involved in a variety of important biological functions where the substrate is not covalently modified; for example, membrane fusion, membrane transport, protein disaggregation, and protein refolding.

In-silico virtual screening of bacterial surface enzyme Staphylococcus aureus Sortase A against commercial compound libraries using FlexX software package has led to the identification of novel inhibitors. Inhibition of enzyme catalytic activity was determined by monitoring the steady state cleavage of a model peptide substrate. Preliminary structure activity relationship studies on the lead compound resulted in the identification of compounds with improved activity. The most active compound has an IC50 value of 58 μM against the enzyme.

The ATPases associated with diverse cellular activities (AAA+) is a large superfamily of proteins involved in a broad array of biological processes. Many members of this family require nucleotide binding to assemble into their final active hexameric form. We have been studying two example members, Escherichia coli ClpA and ClpB. These two enzymes are active as hexameric rings that both require nucleotide binding for assembly. Our studies have shown that they both reside in a monomer, dimer, tetramer, and hexamer equilibrium, and this equilibrium is thermodynamically linked to nucleotide binding. Moreover, we are finding that the kinetics of the assembly reaction are very different for the two enzymes. Here, we present our strategy for determining the self-association constants in the absence of nucleotide to set the stage for the analysis of nucleotide binding from other experimental approaches including analytical ultracentrifugation.

Escherichia coli ClpB is a heat shock protein that belongs to the AAA+ protein superfamily. Studies have shown that ClpB and its homologue in yeast, Hsp104, can disrupt protein aggregates in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity. In addition, it is widely assumed that ClpB is uniformly hexameric in the presence of nucleotides. Here we report, in the absence of nucleotide, that increasing ClpB concentration leads to ClpB hexamer formation, decreasing NaCl concentration stabilizes ClpB hexamers, and the ClpB assembly reaction is best described by a monomer, dimer, tetramer, hexamer equilibrium under the three salt concentrations examined. Further, we found that ClpB oligomers exhibit relatively fast dissociation on the time scale of sedimentation. We anticipate our studies on ClpB assembly to be a starting point to understand how ClpB assembly is linked to the binding and disaggregation of denatured proteins.

The ATPases associated with diverse cellular activities (AAA+) is a large superfamily of proteins involved in a broad array of biological processes. Many members of this family require nucleotide binding to assemble into their final active hexameric form. We have been studying two example members, Escherichia coli ClpA and ClpB. These two enzymes are active as hexameric rings that both require nucleotide binding for assembly. Our studies have shown that they both reside in a monomer, dimer, tetramer, and hexamer equilibrium, and this equilibrium is thermodynamically linked to nucleotide binding. Moreover, we are finding that the kinetics of the assembly reaction are very different for the two enzymes. Here, we present our strategy for determining the self-association constants in the absence of nucleotide to set the stage for the analysis of nucleotide binding from other experimental approaches including analytical ultracentrifugation.