Solvent-Dependent Emissions Properties of a Model Aurone Enable Use in Biological Applications.

Fluorophores are powerful visualization tools and the development of novel small organic fluorophores are in great demand. Small organic fluorophores have been derived from the aurone skeleton, 2-benzylidenebenzofuran-3(2H)-one. In this study, we have utilized a model aurone derivative with a methoxy group at the 3' position and a hydroxyl group at the 4' position, termed vanillin aurone, to develop a foundational understanding of structural factors impacting aurone fluorescence properties. The fluorescent behaviors of the model aurone were characterized in solvent environments differing in relative polarity and dielectric constant. These data suggested that hydrogen bonding or electrostatic interactions between excited state aurone and solvent directly impact emissions properties such as peak emission wavelength, emission intensity, and Stokes shift. Time-dependent Density Functional Theory (TD-DFT) model calculations suggest that quenched aurone emissions observed in water are a consequence of stabilization of a twisted excited state conformation that disrupts conjugation. In contrast, the calculations indicate that low polarity solvents such as toluene or acetone stabilize a brightly fluorescent planar state. Based on this, additional experiments were performed to demonstrate use as a turn-on probe in an aqueous environment in response to conditions leading to planar excited state stabilization. Vanillin aurone was observed to bind to a model ATP binding protein, YME1L, leading to enhanced emissions intensities with a dissociation equilibrium constant equal to ~ 30 µM. Separately, the aurone was observed to be cell permeable with significant toxicity at doses exceeding 6.25 µM. Taken together, these results suggest that aurones may be broadly useful as turn-on probes in aqueous environments that promote either a change in relative solvent polarity or through direct stabilization of a planar excited state through macromolecular binding.

Risk Factors for Revision Surgery Following Revision Total Knee Arthroplasty Using a Hinged Knee Prosthesis for Septic and Aseptic Indications.

INTRODUCTION: The use of hinged knee replacements (HKRs) for limb salvage is a popular option for revision total knee arthroplasty (RTKA). Although recent literature focuses on the outcomes of HKR for septic and aseptic RTKAs, little is reported on the risk factors of returning to the operating room. The purpose of this study was to evaluate risk factors of revision surgery and revision after receiving HKR for septic versus aseptic etiology. METHODS: A multicenter, retrospective review was conducted on consecutive patients who received HKR from January 2010 to February 2020 with a minimum follow-up of 2 years. Patients were separated into two groups: septic and aseptic RTKAs. Demographic, comorbidity, perioperative, postoperative, and survivorship data were collected and compared between groups. Cox hazard regression was used to identify risk factors associated with revision surgery and revision. RESULTS: One-hundred fifty patients were included. Eighty-five patients received HKR because of prior infection, and 65 received HKR for aseptic revision. A larger proportion of septic RTKA returned to the OR versus aseptic RTKA (46% vs 25%, P = 0.01). Survival curves revealed superior revision surgery-free survival favoring the aseptic group ( P = 0.002). Regression analysis revealed that HKR with concomitant flap reconstruction was associated with a three-fold increased risk of revision surgery ( P < 0.0001). DISCUSSION: HKR implantation for aseptic revision is more reliable with a lower revision surgery rate. Concomitant flap reconstruction increased the risk of revision surgery, regardless of indication for RTKA using HKR. Although surgeons must educate patients about these risk factors, HKR remains a successful treatment option for RTKA when indicated. LEVEL OF EVIDENCE: prognostic, level III evidence.

Mini-Open Achilles Tendon Repair: Improving Outcomes While Decreasing Complications.

An acute rupture of the Achilles tendon is a traumatic injury that can cause considerable morbidity and reduced function. Nonoperative intervention may put patients at higher risk of rerupture, whereas surgical intervention carries risk of infection, wound complications, and iatrogenic nerve injury. The mini-open Achilles tendon repair technique has been popularized in helping decrease complications. The goal of this study was to examine and compare the functional outcomes and rate of complications in patients treated with a mini-open repair technique versus a traditional open repair for acute Achilles tendon ruptures. A retrospective review was performed of all patients with a complete Achilles tendon rupture that were treated by a single foot and ankle fellowship-trained surgeon. Functional outcome scores were assessed using the American Orthopaedic Foot and Ankle scoring system (AOFAS) and the Achilles Tendon Rupture Score (ATRS). Eighty-one patients with a complete Achilles tendon rupture underwent mini-open repair and 22 patients underwent traditional open repair surgery between 2013 and 2020. The mean follow-up was 38.40 months (range, 12-71). Mean preoperative AOFAS and ATRS improved in the mini-open group from 45.60 and 47.18 respectively, to 90.29 and 87.97 after surgery (p < .05). Mean preoperative AOFAS and ATRS scores for the traditional open repair (n = 22) cohort were 44.02 and 42.27, respectively. Postoperatively, the AOFAS and ATRS scores improved to 85.27 and 86.64 (P value < .05), respectively. There was no statistically significant difference in postoperative ATRS scores. However, the mini-open repair group showed a statistically higher postoperative AOFAS score (90.30) than the traditional open-repair group (85.27) (P value < .05). The overall complication rate for our study was 2.9% (2 mini-open repair and 1 traditional open repair). The complication rate in the mini-open repair group and traditional open repair cohort were 2.4% and 4.5%, respectively (P value > .05). One patient in the mini-open repair cohort (1.2%) reruptured his Achilles tendon 4 months postoperatively. A second patient in the mini-open repair group (1.2%) developed a superficial skin infection and suture irritation. One patient (4.5%) in the traditional open repair group developed a superficial skin infection. There were no sural nerve injuries in our series. The mini-open approach to repair a ruptured Achilles tendon is a viable treatment option to decrease the incidence rate of postoperative complications and rerupture rates while also producing a superior cosmetic result.Level of Evidence: 3 (retrospective cohort study N ≥ 30).

Aurone-derived 1,2,3-triazoles as potential fluorescence molecules in vitro.

Aurones are a class of well-studied natural compounds primarily responsible for the yellow pigment in flowering plants and have been shown to have fluorescent properties as well as beneficial biological effects. Traditionally, aurones can be easily synthesized through a Knoevenagel condensation of benzofuranones with arylaldehydes. Recently, Kafle et al. unexpectedly synthesized a new aurone derivative containing a 1,2,3-triazole within its backbone. Since, 1,2,3-triazole containing structures have been shown to be useful as fluorophores with large Stokes shifts, we hypothesized that these new aurone-derived triazole compounds (ATs) could be utilized as potential fluorophores. Here we describe a newly-synthesized fluorescent compound which has potential for use as a live-cell probe, having a large Stokes shift of 118.3 ± 1.01 nm in phosphate-buffered saline with the benefit of increased fluorescence in protic environments, which is uncommon in aurone-derived fluorophores.

A monomeric mycobacteriophage immunity repressor utilizes two domains to recognize an asymmetric DNA sequence.

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.

Engineered Sphingomonas sp. KT-1 PahZ1 monomers efficiently degrade poly(aspartic acid).

In recent years, there has been an effort toward creating and utilizing novel biodegradable polymeric materials. As products become available, it is necessary to concurrently search for novel biodegradation catalysts and further investigate the properties of known biodegradation enzymes. Regarding the latter, we recently reported the crystal structure of a dimeric enzyme, Sphingomonas sp. KT-1 PahZ1, capable of degrading poly(aspartic acid), a green alternative to non-biodegradable polycarboxylates. However, the role of the dimeric state in catalytic function remained unclear. Here we report PahZ1KT-1 constructs with either single or multiple mutation(s) at the dimer interface yield active monomers. Our data indicates PahZ1KT-1 monomers and dimers catalyze PAA degradation at equivalent rates. Unfolding experiments reveal differences where the activation energy for monomers is ~ 46 kJ mol-1 lower than for dimers despite similar thermodynamic properties. Characterization of this biodegradation enzyme and others is critical for future protein engineering efforts toward polymer remediation.

Sphingomonas sp. KT-1 PahZ2 Structure Reveals a Role for Conformational Dynamics in Peptide Bond Hydrolysis.

Poly(aspartic acid) (PAA) is a common water-soluble polycarboxylate used in a broad range of applications. PAA biodegradation and environmental assimilation were first identified in river water bacterial strains, Sphingomonas sp. KT-1 and Pedobacter sp. KP-2. Within Sphingomonas sp. KT-1, PahZ1KT-1 cleaves ß-amide linkages to oligo(aspartic acid) and then is degraded by PahZ2KT-1. Recently, we reported the first structure for PahZ1KT-1. Here, we report novel structures for PahZ2KT-1 bound to either Gd3+/Sm3+ or Zn2+ cations in a dimeric state consistent with M28 metallopeptidase family members. PahZ2KT-1 monomers include a dimerization domain and a catalytic domain with dual Zn2+ cations. MD methods predict the putative substrate binding site to span across the dimerization and catalytic domains, where NaCl promotes the transition from an open conformation to a closed conformation that positions the substrate adjacent to catalytic zinc ions. Structural knowledge of PahZ1KT-1 and PahZ2KT-1 will allow for protein engineering endeavors to develop novel biodegradation reagents.

Examination of the Role of Mg2+ in the Mechanism of Nucleotide Binding to the Monomeric YME1L AAA+ Domain.

The first line of defense in the mitochondrial quality control network involves the stress response from a family of ATP-dependent proteases. We have reported that a solubilized version of the mitochondrial inner membrane ATP-dependent protease YME1L displays nucleotide binding kinetics that are sensitive to the reactive oxygen species hydrogen peroxide under a limiting ATP concentration. Our observations were consistent with an altered YME1L conformational ensemble leading to increased nucleotide binding site accessibility under oxidative stress conditions. To examine this hypothesis further, we report here the results of a comprehensive study of the thermodynamic and kinetic properties underlying the binding of nucleoside di- and triphosphate to the isolated YME1L AAA+ domain (YME1L-AAA+). A combination of fluorescence titrations, molecular dynamics, and stopped-flow fluorescence experiments have demonstrated similarity between nucleotide binding behaviors for YME1L under oxidative conditions and the isolated AAA+ domain. Our data demonstrate that YME1L-AAA+ binds ATP and ADP with affinities equal to ∼30 and 5 µM, respectively, in the absence of Mg2+. We note a negative heterotropic linkage effect between Mg2+ and ATP that arises as the MgCl2 concentration is increased such that the affinity of YME1L-AAA+ for ATP decreases to ∼60 µM in the presence of 10 mM MgCl2. Molecular dynamics methods allow for structural rationalization by revealing condition-dependent conformational populations for YME1L-AAA+. Taken together, these data suggest a preliminary model in which YME1L modulates its affinity for the nucleotide to stabilize against degradation or instability inherent to such stress conditions.

Distally Unlocked Intramedullary Nailing With Cement Fixation for Impending and Actual Pathologic Humerus Fractures: A Retrospective Case Series.

The humerus is a common site of metastatic tumor involvement and pathologic fracture. Intramedullary nailing is a treatment option that offers the benefit of protecting a long segment of diseased bone, but it is not without complications. This study aims to examine the survival, functional outcomes, and complications of patients treated with cement-augmented unlocked intramedullary nailing for actual and impending pathologic fractures of the humeral shaft. Methods: From 2014 to 2019, 26 patients were treated with this technique. Functional outcomes were assessed using the Musculoskeletal Tumor Society scoring system. Outcome scores, complications, reoperations, and mortality were determined by retrospective chart reviews and direct patient examinations. Results: The mean age at the time of surgery was 66.8 years. The mean follow-up was 20.2 months. Patients reported significant improvement in the mean Musculoskeletal Tumor Society score from 10.5 preoperatively to 26.1 after surgery (P < 0.001). Five patients died of disease during the follow-up period. One patient had intraoperative fracture propagation during implant placement, and one patient experienced a postoperative rotator cuff tear. Discussion: Unlocked intramedullary nailing with cement augmentation is a reliable treatment method for actual and impending pathologic fractures of the humerus that results in favorable outcomes, including consistent pain relief and restoration of function.

Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding.

ATP-dependent proteases are ubiquitous across all kingdoms of life and are critical to the maintenance of intracellular protein quality control. The enzymatic function of these enzymes requires structural stability under conditions that may drive instability and/or loss of function in potential protein substrates. Thus, these molecular machines must demonstrate greater stability than their substrates in order to ensure continued function in essential quality control networks. We report here a role for ATP in the stabilization of the inner membrane YME1L protease. Qualitative fluorescence data derived from protein unfolding experiments with urea reveal non-standard protein unfolding behavior that is dependent on [ATP]. Using multiple fluorophore systems, stopped-flow fluorescence experiments demonstrate a depletion of the native YME1L ensemble by urea-dependent unfolding and formation of a non-native conformation. Additional stopped-flow fluorescence experiments based on nucleotide binding and unfoldase activities predict that unfolding yields significant loss of active YME1L hexamers from the starting ensemble. Taken together, these data clearly define the stress limits of an important mitochondrial protease.

Hydrogen sulfide sensing using an aurone-based fluorescent probe.

Hydrogen sulfide detection and sensing is an area of interest from both an environmental and a biological perspective. While many methods are currently available, the most sensitive and biologically applicable ones are fluorescence based. In general, these fluorescent probes are based upon large, high-molecular weight, well-characterized fluorescent scaffolds that are synthetically demanding to prepare and difficult to tune and modify. In this study, we have reported a new reduction-based, rationally designed and synthesized turn-on fluorescent probe (Z)-2-(4'-azidobenzylidene)-5-fluorobenzofuran-3(2H)-one (6g) utilizing a low molecular weight aurone fluorophore. During these studies, the modular nature of the synthesis was used to quickly overcome problems with solubility, overlap of excitation of the probe and reduced product, and rate of reaction, resulting in a final compound that is efficient and sensitive for the detection of hydrogen sulfide. The limitation of slow reaction and the reduced fluorescence in a biologically relevent medium was solved by employing cationic surfactant cetyltrimethyl ammonium bromide (CTAB). The probe features a high fluorescence enhancement, fast response (10-30 min), and good sensitivity (1 µm) and selectivity for hydrogen sulfide.

Characterization of Mitochondrial YME1L Protease Oxidative Stress-Induced Conformational State.

Oxidative stress is a common challenge to mitochondrial function where reactive oxygen species are capable of significant organelle damage. The generation of mitochondrial reactive oxygen species occurs in the inner membrane and matrix compartments as a consequence of subunit function in the electron transport chain and citric acid cycle, respectively. Maintenance of mitochondrial proteostasis and stress response is facilitated by compartmentalized proteases that couple the energy of ATP hydrolysis to unfolding and the regulated removal of damaged, misfolded, or aggregated proteins. The mitochondrial protease YME1L functions in the maintenance of proteostasis in the intermembrane space. YME1L is an inner membrane-anchored hexameric protease with distinct N-terminal, transmembrane, AAA+ (ATPases associated with various cellular activities), and C-terminal M41 zinc-dependent protease domains. The effect of oxidative stress on enzymes such as YME1L tasked with maintaining proteostasis is currently unclear. We report here that recombinant YME1L undergoes a reversible conformational change in response to oxidative stress that involves the interaction of one hydrogen peroxide molecule per YME1L monomer with affinities equal to 31 ±â€¯2 and 26 ±â€¯1 mM for conditions lacking or including nucleotide, respectively. Our data also reveal that oxidative stress does not significantly impact nucleotide binding equilibria, but does stimulate a 2-fold increase in the rate constant for high-affinity ATP binding from (8.9 ±â€¯0.2) × 105 M-1 s-1 to (1.5 ±â€¯0.1) × 106 M-1 s-1. Taken together, these data may suggest a mechanism for the regulated processing of YME1L by other inner membrane proteases such as OMA1.

Mycobacterium tuberculosis ClpC1 N-Terminal Domain Is Dispensable for Adaptor Protein-Dependent Allosteric Regulation.

ClpC1 hexamers couple the energy of ATP hydrolysis to unfold and, subsequently, translocate specific protein substrates into the associated ClpP protease. Substrate recognition by ATPases associated with various cellular activities (AAA+) proteases is driven by the ATPase component, which selectively determines protein substrates to be degraded. The specificity of these unfoldases for protein substrates is often controlled by an adaptor protein with examples that include MecA regulation of Bacillus subtilis ClpC or ClpS-mediated control of Escherichia coli ClpA. No adaptor protein-mediated control has been reported for mycobacterial ClpC1. Using pulldown and stopped-flow fluorescence methods, we report data demonstrating that Mycobacterium tuberculosis ClpC1 catalyzed unfolding of an SsrA-tagged protein is negatively impacted by association with the ClpS adaptor protein. Our data indicate that ClpS-dependent inhibition of ClpC1 catalyzed SsrA-dependent protein unfolding does not require the ClpC1 N-terminal domain but instead requires the presence of an interaction surface located in the ClpC1 Middle Domain. Taken together, our results demonstrate for the first time that mycobacterial ClpC1 is subject to adaptor protein-mediated regulation in vitro.

Phylogenetic analysis predicts structural divergence for proteobacterial ClpC proteins.

Regulated proteolysis is required in all organisms for the removal of misfolded or degradation-tagged protein substrates in cellular quality control pathways. The molecular machines that catalyze this process are known as ATP-dependent proteases with examples that include ClpAP and ClpCP. Clp/Hsp100 subunits form ring-structures that couple the energy of ATP binding and hydrolysis to protein unfolding and subsequent translocation of denatured protein into the compartmentalized ClpP protease for degradation. Copies of the clpA, clpC, clpE, clpK, and clpL genes are present in all characterized bacteria and their gene products are highly conserved in structure and function. However, the evolutionary relationship between these proteins remains unclear. Here we report a comprehensive phylogenetic analysis that suggests divergent evolution yielded ClpA from an ancestral ClpC protein and that ClpE/ClpL represent intermediates between ClpA/ClpC. This analysis also identifies a group of proteobacterial ClpC proteins that are likely not functional in regulated proteolysis. Our results strongly suggest that bacterial ClpC proteins should not be assumed to all function identically due to the structural differences identified here.

Fundamental Characteristics of AAA+ Protein Family Structure and Function.

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins.

Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2-7 Helicase to Reveal Essential Features of Structure and Function.

In eukaryotes, the replicative helicase is the large multisubunit CMG complex consisting of the Mcm2-7 hexameric ring, Cdc45, and the tetrameric GINS complex. The Mcm2-7 ring assembles from six different, related proteins and forms the core of this complex. In archaea, a homologous MCM hexameric ring functions as the replicative helicase at the replication fork. Archaeal MCM proteins form thermostable homohexamers, facilitating their use as models of the eukaryotic Mcm2-7 helicase. Here we review archaeal MCM helicase structure and function and how the archaeal findings relate to the eukaryotic Mcm2-7 ring.

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.

Analysis of the crystal structure of an active MCM hexamer.

In a previous Research article (Froelich et al., 2014), we suggested an MCM helicase activation mechanism, but were limited in discussing the ATPase domain because it was absent from the crystal structure. Here we present the crystal structure of a nearly full-length MCM hexamer that is helicase-active and thus has all features essential for unwinding DNA. The structure is a chimera of Sulfolobus solfataricus N-terminal domain and Pyrococcus furiosus ATPase domain. We discuss three major findings: 1) a novel conformation for the A-subdomain that could play a role in MCM regulation; 2) interaction of a universally conserved glutamine in the N-terminal Allosteric Communication Loop with the AAA+ domain helix-2-insert (h2i); and 3) a recessed binding pocket for the MCM ssDNA-binding motif influenced by the h2i. We suggest that during helicase activation, the h2i clamps down on the leading strand to facilitate strand retention and regulate ATP hydrolysis.

ATPγS competes with ATP for binding at Domain 1 but not Domain 2 during ClpA catalyzed polypeptide translocation.

ClpAP is an ATP-dependent protease that assembles through the association of hexameric rings of ClpA with the cylindrically-shaped protease ClpP. ClpA contains two nucleotide binding domains, termed Domain 1 (D1) or 2 (D2). We have proposed that D1 or D2 limits the rate of ClpA catalyzed polypeptide translocation when ClpP is either absent or present, respectively. Here we show that the rate of ClpA catalyzed polypeptide translocation depends on [ATPγS] in the absence of ClpP, but not in the presence of ClpP. We observe that ATPγS non-cooperatively binds to ClpA during polypeptide translocation with an apparent affinity of ~6 µM, but that introduction of ClpP shifts this affinity such that translocation is not affected. Interpreting these data with our proposed model for translocation catalyzed by ClpA vs. ClpAP suggests that ATPγS competes for binding at D1 but not at D2.

E. coli ClpA catalyzed polypeptide translocation is allosterically controlled by the protease ClpP.

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.