Key processes required for the different stages of fungal carnivory by a nematode-trapping fungus.

Nutritional deprivation triggers a switch from a saprotrophic to predatory lifestyle in soil-dwelling nematode-trapping fungi (NTF). In particular, the NTF Arthrobotrys oligospora secretes food and sex cues to lure nematodes to its mycelium and is triggered to develop specialized trapping devices. Captured nematodes are then invaded and digested by the fungus, thus serving as a food source. In this study, we examined the transcriptomic response of A. oligospora across the stages of sensing, trap development, and digestion upon exposure to the model nematode Caenorhabditis elegans. A. oligospora enacts a dynamic transcriptomic response, especially of protein secretion-related genes, in the presence of prey. Two-thirds of the predicted secretome of A. oligospora was up-regulated in the presence of C. elegans at all time points examined, and among these secreted proteins, 38.5% are predicted to be effector proteins. Furthermore, functional studies disrupting the t-SNARE protein Sso2 resulted in impaired ability to capture nematodes. Additionally, genes of the DUF3129 family, which are expanded in the genomes of several NTF, were highly up-regulated upon nematode exposure. We observed the accumulation of highly expressed DUF3129 proteins in trap cells, leading us to name members of this gene family as Trap Enriched Proteins (TEPs). Gene deletion of the most highly expressed TEP gene, TEP1, impairs the function of traps and prevents the fungus from capturing prey efficiently. In late stages of predation, we observed up-regulation of a variety of proteases, including metalloproteases. Following penetration of nematodes, these metalloproteases facilitate hyphal growth required for colonization of prey. These findings provide insights into the biology of the predatory lifestyle switch in a carnivorous fungus and provide frameworks for other fungal-nematode predator-prey systems.

Downregulation of TAZ elicits a mitochondrial redox imbalance and ferroptosis in lung epithelial cells exposed to diesel exhaust particles.

Mitochondrial dysfunction was reported to be involved in the development of lung diseases including chronic obstructive pulmonary disease (COPD). However, molecular regulation underlying metabolic disorders in the airway epithelia exposed to air pollution remains unclear. In the present study, lung bronchial epithelial BEAS-2B and alveolar epithelial A549 cells were treated with diesel exhaust particles (DEPs), the primary representative of ambient particle matter. This treatment elicited cell death accompanied by induction of lipid reactive oxygen species (ROS) production and ferroptosis. Lipidomics analyses revealed that DEPs increased glycerophospholipid contents. Accordingly, DEPs upregulated expression of the electron transport chain (ETC) complex and induced mitochondrial ROS production. Mechanistically, DEP exposure downregulated the Hippo transducer transcriptional co-activator with PDZ-binding motif (TAZ), which was further identified to be crucial for the ferroptosis-associated antioxidant system, including glutathione peroxidase 4 (GPX4), the glutamate-cysteine ligase catalytic subunit (GCLC), and glutathione-disulfide reductase (GSR). Moreover, immunohistochemistry confirmed downregulation of GPX4 and upregulation of lipid peroxidation in the bronchial epithelium of COPD patients and Sprague-Dawley rats exposed to air pollution. Finally, proteomics analyses confirmed alterations of ETC-related proteins in bronchoalveolar lavage from COPD patients compared to healthy subjects. Together, our study discovered that involvement of mitochondrial redox dysregulation plays a vital role in pulmonary epithelial cell destruction after exposure to air pollution.

Effects of colonization-associated gene yqiC on global transcriptome, cellular respiration, and oxidative stress in Salmonella Typhimurium.

BACKGROUND: yqiC is required for colonizing the Salmonella enterica serovar Typhimurium (S. Typhimurium) in human cells; however, how yqiC regulates nontyphoidal Salmonella (NTS) genes to influence bacteria-host interactions remains unclear. METHODS: The global transcriptomes of S. Typhimurium yqiC-deleted mutant (ΔyqiC) and its wild-type strain SL1344 after 2 h of in vitro infection with Caco-2 cells were obtained through RNA sequencing to conduct comparisons and identify major yqiC-regulated genes, particularly those involved in Salmonella pathogenicity islands (SPIs), ubiquinone and menaquinone biosynthesis, electron transportation chains (ETCs), and carbohydrate/energy metabolism. A Seahorse XFp Analyzer and assays of NADH/NAD+ and H2O2 were used to compare oxygen consumption and extracellular acidification, glycolysis parameters, adenosine triphosphate (ATP) generation, NADH/NAD+ ratios, and H2O2 production between ΔyqiC and SL1344. RESULTS: After S. Typhimurium interacts with Caco-2 cells, yqiC represses gene upregulation in aspartate carbamoyl transferase, type 1 fimbriae, and iron-sulfur assembly, and it is required for expressing ilvB operon, flagellin, tdcABCD, and dmsAB. Furthermore, yqiC is required for expressing mainly SPI-1 genes and specific SPI-4, SPI-5, and SPI-6 genes; however, it diversely regulates SPI-2 and SPI-3 gene expression. yqiC significantly contributes to menD expression in menaquinone biosynthesis. A Kyoto Encyclopedia of Genes and Genomes analysis revealed the extensive association of yqiC with carbohydrate and energy metabolism. yqiC contributes to ATP generation, and the analyzer results demonstrate that yqiC is required for maintaining cellular respiration and metabolic potential under energy stress and for achieving glycolysis, glycolytic capacity, and glycolytic reserve. yqiC is also required for expressing ndh, cydA, nuoE, and sdhB but suppresses cyoC upregulation in the ETC of aerobically and anaerobically grown S. Typhimurium; priming with Caco-2 cells caused a reversed regulation of yiqC toward upregulation in these ETC complex genes. Furthermore, yqiC is required for maintaining NADH/NAD+ redox status and H2O2 production. CONCLUSIONS: Specific unreported genes that were considerably regulated by the colonization-associated gene yqiC in NTS were identified, and the key role and tentative mechanisms of yqiC in the extensive modulation of virulence factors, SPIs, ubiquinone and menaquinone biosynthesis, ETCs, glycolysis, and oxidative stress were discovered.

Measuring membrane protein stability under native conditions.

The thermodynamic stability of proteins is typically measured at high denaturant concentrations and then extrapolated back to zero denaturant conditions to obtain unfolding free energies under native conditions. For membrane proteins, the extrapolations are fraught with considerable uncertainty as the denaturants may have complex effects on the membrane or micellar structure. We therefore sought to measure stability under native conditions, using a method that does not perturb the properties of the membrane or membrane mimetics. We use a technique called steric trapping to measure the thermodynamic stability of bacteriorhodopsin in bicelles and micelles. We find that bacteriorhodopsin has a high thermodynamic stability, with an unfolding free energy of ∼11 kcal/mol in dimyristoyl phosphatidylcholine bicelles. Nevertheless, the stability is much lower than predicted by extrapolation of measurements made at high denaturant concentrations. We investigated the discrepancy and found that unfolding free energy is not linear with denaturant concentration. Apparently, long extrapolations of helical membrane protein unfolding free energies must be treated with caution. Steric trapping, however, provides a method for making these measurements.

Ligand concentration regulates the pathways of coupled protein folding and binding.

Coupled ligand binding and conformational change plays a central role in biological regulation. Ligands often regulate protein function by modulating conformational dynamics, yet the order in which binding and conformational change occurs are often hotly debated. Here we show that the "conformational selection versus induced fit" distinction on which this debate is based is a false dichotomy because the mechanism depends on ligand concentration. Using the binding of pyrophosphate (PPi) to Bacillus subtilis RNase P protein as a model, we show that coupled reactions are best understood as a change in flux between competing pathways with distinct orders of binding and conformational change. The degree of partitioning through each pathway depends strongly on PPi concentration, with ligand binding redistributing the conformational ensemble toward the folded state by both increasing folding rates and decreasing unfolding rates. These results indicate that ligand binding induces marked and varied changes in protein conformational dynamics, and that the order of binding and conformational change is ligand concentration dependent.

Effects of stand-alone polar residue on membrane protein stability and structure.

Helical membrane proteins generally have a hydrophobic nature, with apolar side chains comprising the majority of the transmembrane (TM) helices. However, whenever polar side chains are present in the TM domain, they often exert a crucial role in structural interactions with other polar residues, such as TM helix associations and oligomerization. Moreover, polar residues in the TM region also often participate in protein functions, such as the Schiff base bonding between Lys residues and retinal in rhodopsin-like membrane proteins. Although many studies have focused on these functional polar residues, our understanding of stand-alone polar residues that are energetically unfavored in TM helixes is limited. Here, we adopted bacteriorhodopsin (bR) as a model system and systematically mutated 17 of its apolar Leu or Phe residues to polar Asn. Stability measurements of the resulting mutants revealed that all of these polar substitutions reduced bR stability to various extents, and the extent of destabilization of each mutant bR is also correlated to different structural factors, such as the relative accessible surface area and membrane depth of the mutation site. Structural analyses of these Asn residues revealed that they form sidechain-to-backbone hydrogen bonds that alleviate the unfavorable energetics in hydrophobic and apolar surroundings. Our results indicate that membrane proteins are able to accommodate certain stand-alone polar residues in the TM region without disrupting overall structures.

Development and functional evaluation of a hyaluronic acid coated nano-formulation with kaempferol as a novel intra-articular agent for Knee Osteoarthritis treatment.

Knee osteoarthritis (OA) involves articular cartilage degradation driven mainly by inflammation. Kaempferol (KM), known for its anti-inflammatory property, holds potential for OA treatment. This study investigated the potential of hyaluronic acid (HA)-coated gelatin nanoparticles loaded with KM (HA-KM GNP) for treating knee OA. KM was encapsulated into gelatin nanoparticles (KM GNP) and then coated with HA to form HA-KM GNPs. Physical properties were characterized, and biocompatibility and cellular uptake were assessed in rat chondrocytes. Anti-inflammatory and chondrogenic properties were evaluated using IL-1ß-stimulated rat chondrocytes, compared with HA-coated nanoparticles without KM (HA GNP) and KM alone. Preclinical efficacy was tested in an anterior cruciate ligament transection (ACLT)-induced knee OA rat model treated with intra-articular injection of HA-KM GNP. Results show spherical HA-KM GNPs (88.62 ± 3.90 nm) with positive surface charge. Encapsulation efficiency was 98.34 % with a sustained release rate of 18 % over 48 h. Non-toxic KM concentration was 2.5 µg/mL. In IL-1ß-stimulated OA rat chondrocytes, HA-KM GNP significantly down-regulated RNA expression of IL-1ß, TNF-α, COX-2, MMP-9, and MMP-13, while up-regulating SOX9 compared to HA GNP, and KM. In vivo imaging demonstrated significantly higher fluorescence intensity within rat knee joints for 3 hours post HA-KM GNP injection compared with KM GNP (185.2% ± 34.1% vs. 45.0% ± 16.7%). HA-KM GNP demonstrated significant effectiveness in reducing subchondral sclerosis, attenuating inflammation, inhibiting matrix degradation, restoring cartilage thickness, and reducing the severity of OA in the ACLT rat model. In conclusion, HA-KM GNP holds promise for knee OA therapy.

The nematode-trapping fungus Arthrobotrys oligospora detects prey pheromones via G protein-coupled receptors.

The ability to sense prey-derived cues is essential for predatory lifestyles. Under low-nutrient conditions, Arthrobotrys oligospora and other nematode-trapping fungi develop dedicated structures for nematode capture when exposed to nematode-derived cues, including a conserved family of pheromones, the ascarosides. A. oligospora senses ascarosides via conserved MAPK and cAMP-PKA pathways; however, the upstream receptors remain unknown. Here, using genomic, transcriptomic and functional analyses, we identified two families of G protein-coupled receptors (GPCRs) involved in sensing distinct nematode-derived cues. GPCRs homologous to yeast glucose receptors are required for ascaroside sensing, whereas Pth11-like GPCRs contribute to ascaroside-independent nematode sensing. Both GPCR classes activate conserved cAMP-PKA signalling to trigger trap development. This work demonstrates that predatory fungi use multiple GPCRs to sense several distinct nematode-derived cues for prey recognition and to enable a switch to a predatory lifestyle. Identification of these receptors reveals the molecular mechanisms of cross-kingdom communication via conserved pheromones also sensed by plants and animals.

Salmonella YqiC exerts its function through an oligomeric state.

Protein oligomerization occurs frequently both in vitro and in vivo, with specific functionalities associated with different oligomeric states. The YqiC protein from Salmonella Typhimurium forms a homotrimer through its C-terminal coiled-coil domain, and the protein is closely linked to the colonization and invasion of the bacteria to the host cells. To elucidate the importance of the oligomeric state of YqiC in vivo and its relation with bacterial infection, we mutated crucial residues in YqiC's coiled-coil region and confirmed the loss of trimer formation using chemical crosslinking and size exclusion chromatography coupled with multiple angle light scattering (SEC-MALS) techniques. The yqiC-knockout strain complemented with mutant YqiC showed significantly reduced colonization and invasion of Salmonella to host cells, demonstrating the critical role of YqiC oligomerization in bacterial pathogenesis. Furthermore, we conducted a protein-protein interaction study of YqiC using a pulled-down assay coupled with mass spectrometry analysis to investigate the protein's role in bacterial virulence. The results reveal that YqiC interacts with subunits of Complex II of the electron transport chain (SdhA and SdhB) and the ß-subunit of F0 F1 -ATP synthase. These interactions suggest that YqiC may modulate the energy production of Salmonella and subsequently affect the assembly of crucial virulence factors, such as flagella. Overall, our findings provide new insights into the molecular mechanisms of YqiC's role in S. Typhimurium pathogenesis and suggest potential therapeutic targets for bacterial infections.

Persistence and dynamic structures of diverse cephalosporinase genes in nontyphoidal Salmonella in cross-sectional surveillance in Taiwan.

OBJECTIVES: Nontyphoidal Salmonella (NTS) is a major foodborne pathogen causing from acute gastroenteritis to bacteraemia, particularly in paediatric and elderly patients. Antimicrobial resistance of NTS, especially resistance to extended-spectrum cephalosporins, has emerged over the past decades. METHODS: Thirteen NTS isolates resistant to ceftriaxone or cefotaxime were collected from a teaching hospital in Taipei, and another three from a tertiary hospital, in New Taipei City, Taiwan, from September 2018 to December 2019. Ten other archived isolates from 2000 to 2017 were also obtained. Complete genomes of the 26 isolates were obtained. Serovars, sequence types, resistomes, genetic relatedness, and sequence comparison of plasmids were analyzed. RESULTS: Serogroups B, C2 and E were significantly associated with ampicillin resistance. Over 90% of these 26 isolates are susceptible to carbapenems and colistin. Genomic epidemiology of these isolates shows that blaCMY-2-harbouring isolates in different serovars were prevalent over two decades, presumably resulting from highly mobile IncI1 plasmid harbouring blaCMY-2. One type of the IncI1 plasmids contained a mobile element, IS26, which might be involved in the acquisition of antimicrobial resistance genes. Two emerging serovars, S. Goldcoast ST358 harbouring blaCTX-M-55 on IncHI2 plasmids and S. Anatum ST64 harbouring blaDHA-1 on IncA/C2 plasmids persisted in Taiwan, possibly through the clonal spread. Integration of complete or partial plasmid sequences into host chromosomes or multiplications of the antimicrobial resistance genes also appears to be mediated by IS26, in the two emerging clones. CONCLUSION: The dynamic movement of cephalosporinase genes mediated by IS26 in NTS is of great concern.

A carnivorous mushroom paralyzes and kills nematodes via a volatile ketone.

The carnivorous mushroom Pleurotus ostreatus uses an unknown toxin to rapidly paralyze and kill nematode prey upon contact. We report that small lollipop-shaped structures (toxocysts) on fungal hyphae are nematicidal and that a volatile ketone, 3-octanone, is detected in these fragile toxocysts. Treatment of Caenorhabditis elegans with 3-octanone recapitulates the rapid paralysis, calcium influx, and neuronal cell death arising from fungal contact. Moreover, 3-octanone disrupts cell membrane integrity, resulting in extracellular calcium influx into cytosol and mitochondria, propagating cell death throughout the entire organism. Last, we demonstrate that structurally related compounds are also biotoxic to C. elegans, with the length of the ketone carbon chain being crucial. Our work reveals that the oyster mushroom has evolved a specialized structure containing a volatile ketone to disrupt the cell membrane integrity of its prey, leading to rapid cell and organismal death in nematodes.

Conformational selection or induced fit: a flux description of reaction mechanism.

The mechanism of ligand binding coupled to conformational changes in macromolecules has recently attracted considerable interest. The 2 limiting cases are the "induced fit" mechanism (binding first) or "conformational selection" (conformational change first). Described here are the criteria by which the sequence of events can be determined quantitatively. The relative importance of the 2 pathways is determined not by comparing rate constants (a common misconception) but instead by comparing the flux through each pathway. The simple rules for calculating flux in multistep mechanisms are described and then applied to 2 examples from the literature, neither of which has previously been analyzed using the concept of flux. The first example is the mechanism of conformational change in the binding of NADPH to dihydrofolate reductase. The second example is the mechanism of flavodoxin folding coupled to binding of its cofactor, flavin mononucleotide. In both cases, the mechanism switches from being dominated by the conformational selection pathway at low ligand concentration to induced fit at high ligand concentration. Over a wide range of conditions, a significant fraction of the flux occurs through both pathways. Such a mixed mechanism likely will be discovered for many cases of coupled conformational change and ligand binding when kinetic data are analyzed by using a flux-based approach.

PRMT1 Confers Resistance to Olaparib via Modulating MYC Signaling in Triple-Negative Breast Cancer.

Treatment of triple-negative breast cancer (TNBC) remains an unmet clinical need owing to its lack of an efficient therapeutic target. The targeting of DNA repair by poly(ADP-ribose) polymerase (PARP) inhibitors has shown benefit for patients with the BRCA variation. However, sensitivities to the PARP inhibitors were reported regardless of BRCA status. Thus, exploring the underlying mechanisms is imperative. Herein, we identified that breast cancer cells with an elevated expression of protein arginine methyl transferase 1 (PRMT1) was associated with therapeutic sensitivity to the PARP inhibitor olaparib. The results of cell viability and colony formation assays indicated that the suppression of PRMT1 by small hairpin RNA or by the chemical inhibitor increased sensitivity to olaparib in human TNBC MDA-MB-231 and BT549 cells. Bioinformatic analysis revealed that PRMT1 expression was significantly associated with the MYC signature, and TNBC cells with higher PRMT1 and the MYC signature were associated with therapeutic sensitivity to olaparib. Mechanistic studies further demonstrated that knockdown of PRMT1 reduced the c-Myc protein level and downregulated the expression of MYC downstream targets, whereas overexpression of PRMT1 enhanced c-Myc protein expression. Moreover, the overexpression of PRMT1 promoted c-Myc protein stability, and the inhibition of PRMT1 downregulated c-Myc protein stability. Accordingly, the knockdown of PRMT1 inhibited homologous recombination gene expression. These data indicate that PRMT1 is instrumental in regulating DNA repair, at least in part, by modulating c-Myc signaling. Our data highlighted the PRMT1/c-Myc network as a potential therapeutic target in patients with TNBC.

Prenatal ultrasound diagnosis of neural tube defects in the era of intrauterine repair - Eleven years' experiences.

OBJECTIVE: To modify the current neural tube defect (NTD) classification for fetal medicine specialists, and to investigate the impact of prenatal ultrasound conus medullaris position screening on the detection rate of closed spinal dysraphism and pregnancy outcomes. MATERIALS AND METHODS: The clinical data of 112 patients prenatally diagnosed with neural tube defects in Taiji clinic from 2008 to 2018 were retrospectively analyzed. All cases were classified following the modified classification. We compared the detection rate before and after introducing the conus medullaris screening and pregnancy outcomes for NTD types. RESULTS: Closed spinal dysraphism type prevailed in our sample (43.8%). The median gestational age at the time of detection for cranial dysraphism was 13.3 weeks, open spinal dysraphism was 22.0 weeks, and closed spinal dysraphism was 22.6 weeks. All cranial dysraphism (n = 43) and open spinal dysraphism cases (n = 20) had pregnancies terminated. For closed spinal dysraphism Class 1, the live-birth rate was 100.0% in the cases without other anomalies and 33.3% in the cases with other anomalies, respectively (X2 = 17.25, p < 0.001). Similarly, for Class 2, pregnancy continuation rate was 50.0% in cases without other anomalies and 20.0% in cases with other anomalies, yet it failed to reach statistical significance (X2 = 0.9, p = 0.524). CONCLUSION: Our case series may help to improve early screening and prenatal diagnosis of NTDs. Modified classification is adjusted for use in ultrasound fetal care facilities, which could be used for predicting pregnancy outcome. We suggest promoting first-trimester anatomical screening in order to make an earlier diagnosis and therefore provide better prenatal care for open spinal dysraphism cases in the era of intrauterine repair. Our findings imply that the use of fetal conus medullaris position as a marker for closed spinal dysraphism improves the detection rate and would unlikely lead to a higher termination rate.

Genotypic Diversity of Ciprofloxacin Nonsusceptibility and Its Relationship with Minimum Inhibitory Concentrations in Nontyphoidal Salmonella Clinical Isolates in Taiwan.

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6')-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25-2 µg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 µg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25-2 µg/mL), thus providing insights into mechanisms underlying CIP resistance.

Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand.

Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.

Probing the folding intermediate of Bacillus subtilis RNase P protein by nuclear magnetic resonance.

Protein folding intermediates are often imperative for overall folding processes and consequent biological functions. However, the low population and transient nature of the intermediate states often hinder their biochemical and biophysical characterization. Previous studies have demonstrated that Bacillus subtilis ribonuclease P protein (P protein) is conformationally heterogeneous and folds with multiphasic kinetics, indicating the presence of an equilibrium and kinetic intermediate in its folding mechanism. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to study the ensemble corresponding to this intermediate (I). The results indicate that the N-terminal and C-terminal helical regions are mostly unfolded in I. 1H−15N heteronuclear single-quantum coherence NMR spectra collected as a function of pH suggest that the protonation of His 22 may play a major role in the energetics of the equilibria among the unfolded, intermediate, and folded state ensembles of P protein. NMR paramagnetic relaxation enhancement experiments were also used to locate the small anion binding sites in both the intermediate and folded ensembles. The results for the folded protein are consistent with the previously modeled binding regions. These structural insights suggest a possible role for I in the RNase P holoenzyme assembly process.

Folding kinetics of the lipoic acid-bearing domain of human mitochondrial branched chain alpha-ketoacid dehydrogenase complex.

A reversible two-step (native state<-->denatured state) folding mechanism based on equilibrium and stopped flow experiments is proposed for urea denaturation of the lipoyl-bearing domain (hbLBD) of human mitochondrial branched chain alpha-ketoacid dehydrogenase (BCKD) complex. The results from this circular dichroism (CD) and fluorescence study have ruled out populated kinetic or equilibrium intermediates on folding pathway of this beta-barrel domain under experimental conditions. Both studies suggested mono-exponential kinetics without any burst phases. Moreover the thermodynamic parameters DeltaG(H(2)O) and m obtained from the kinetic analysis are consistent with the equilibrium measurements.

Measuring transmembrane helix interaction strengths in lipid bilayers using steric trapping.

We have developed a method to measure strong transmembrane (TM) helix interaction affinities in lipid bilayers that are difficult to measure by traditional dilution methods. The method, called steric trapping, couples dissociation of biotinylated TM helices to a competitive binding by monovalent streptavidin (mSA), so that dissociation is driven by the affinity of mSA for biotin and mSA concentration. By adjusting the binding affinity of mSA through mutation, the method can obtain dissociation constants of TM helix dimers (K d,dimer) over a range of six orders of magnitudes. The K d,dimer limit of measurable target interaction is extended 3-4 orders of magnitude lower than possible by dilution methods. Thus, steric trapping opens up new opportunities to study the folding and assembly of α-helical membrane proteins in lipid bilayer environments. Here we provide detailed methods for applying steric trapping to a TM helix dimer.