Energetic driving force for LHCII clustering in plant membranes.

Plants capture and convert solar energy in a complex network of membrane proteins. Under high light, the luminal pH drops and induces a reorganization of the protein network, particularly clustering of the major light-harvesting complex (LHCII). While the structures of the network have been resolved in exquisite detail, the thermodynamics that control the assembly and reorganization had not been determined, largely because the interaction energies of membrane proteins have been inaccessible. Here, we describe a method to quantify these energies and its application to LHCII. Using single-molecule measurements, LHCII proteoliposomes, and statistical thermodynamic modeling, we quantified the LHCII-LHCII interaction energy as ~-5 kBT at neutral pH and at least -7 kBT at acidic pH. These values revealed an enthalpic thermodynamic driving force behind LHCII clustering. Collectively, this work captures the interactions that drive the organization of membrane protein networks from the perspective of equilibrium statistical thermodynamics, which has a long and rich tradition in biology.

Controlling and exploiting intrinsic unpaired electrons in metalloproteins.

Electron paramagnetic resonance spectroscopy encompasses a versatile set of techniques that allow detailed insight into intrinsically occurring paramagnetic centers in metalloproteins and enzymes that undergo oxidation-reduction reactions. In this chapter, we discuss the process from isolating the protein to acquiring and analyzing pulse EPR spectra, adopting a practical perspective. We start with considerations when preparing the protein sample, explain techniques and procedures available for determining the reduction potential of the redox-active center of interest and provide details on methodologies to trap a given paramagnetic state for detailed pulse EPR studies, with an emphasis on biochemical and spectroscopic tools available when multiple EPR-active species are present. We elaborate on some of the most commonly used pulse EPR techniques and the choices the user has to make, considering advantages and disadvantages and how to avoid pitfalls. Examples are provided throughout.

Functional basis of electron transport within photosynthetic complex I.

Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme - the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions.

A conserved arginine residue is critical for stabilizing the N2 FeS cluster in mitochondrial complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase), the first enzyme of the electron-transport chain, captures the free energy released by NADH oxidation and ubiquinone reduction to translocate protons across an energy-transducing membrane and drive ATP synthesis during oxidative phosphorylation. The cofactor that transfers the electrons directly to ubiquinone is an iron-sulfur cluster (N2) located in the NDUFS2/NUCM subunit. A nearby arginine residue (R121), which forms part of the second coordination sphere of the N2 cluster, is known to be posttranslationally dimethylated but its functional and structural significance are not known. Here, we show that mutations of this arginine residue (R121M/K) abolish the quinone-reductase activity, concomitant with disappearance of the N2 signature from the electron paramagnetic resonance (EPR) spectrum. Analysis of the cryo-EM structure of NDUFS2-R121M complex I at 3.7 Å resolution identified the absence of the cubane N2 cluster as the cause of the dysfunction, within an otherwise intact enzyme. The mutation further induced localized disorder in nearby elements of the quinone-binding site, consistent with the close connections between the cluster and substrate-binding regions. Our results demonstrate that R121 is required for the formation and/or stability of the N2 cluster and highlight the importance of structural analyses for mechanistic interpretation of biochemical and spectroscopic data on complex I variants.

Structure and functional analysis of the Legionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation.

Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires' disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface, but this is not dependent on ChiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. We also show that the ChiA C-terminal chitinase domain has novel Zn2+-dependent peptidase activity against mammalian mucin-like proteins, namely MUC5AC and the C1-esterase inhibitor, and that ChiA promotes bacterial penetration of mucin gels. Our findings suggest that ChiA can facilitate passage of L. pneumophila through the alveolar mucosa, can modulate the host complement system and that ChiA may be a promising target for vaccine development.

Microbial Degradation of Plastic in Aqueous Solutions Demonstrated by CO2 Evolution and Quantification.

The environmental accumulation of plastics worldwide is a consequence of the durability of the material. Alternative polymers, marketed as biodegradable, present a potential solution to mitigate their ecological damage. However, understanding of biodegradability has been hindered by a lack of reproducible testing methods. We developed a novel method to evaluate the biodegradability of plastic samples based on the monitoring of bacterial respiration in aqueous media via the quantification of CO2 produced, where the only carbon source available is from the polymer. Rhodococcus rhodochrous and Alcanivorax borkumensis were used as model organisms for soil and marine systems, respectively. Our results demonstrate that this approach is reproducible and can be used with a variety of plastics, allowing comparison of the relative biodegradability of the different materials. In the case of low-density polyethylene, the study demonstrated a clear correlation between the molecular weight of the sample and CO2 released, taken as a measure of biodegradability.

Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition during Legionella pneumophila Intracellular Infection of Amoebas.

Legionella pneumophila genes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection of Acanthamoeba castellanii, although these genes had been considered dispensable on the basis of the behavior of mutants lacking either lapA and lapB or plaC A plaC mutant showed even higher levels of lapA and lapB transcripts, and a lapA lapB mutant showed heightening of plaC mRNA levels, suggesting that the role of the LapA/B aminopeptidase is compensatory with respect to that of the PlaC acyltransferase. Hence, we made double mutants and found that lapA plaC mutants have an ~50-fold defect during infection of A. castellanii These data revealed, for the first time, the importance of LapA in any sort of infection; thus, we purified LapA and defined its crystal structure, activation by another T2S-dependent protease (ProA), and broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of the lapA plaC mutant was reversed, implying that LapA generates amino acids for nutrition. Since the LapA and PlaC data did not fully explain the role of T2S in infection, we identified, via proteomic analysis, a novel secreted protein (NttD) that promotes infection of A. castellanii A lapA plaC nttD mutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data explain, to a significant degree, the importance of T2S. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside the Legionella genus. LapA was notable for having as its closest homologue an A. castellanii protein.IMPORTANCE Transmission of L. pneumophila to humans is facilitated by its ability to grow in Acanthamoeba species. We previously documented that type II secretion (T2S) promotes L. pneumophila infection of A. castellanii Utilizing transcriptional analysis and proteomics, double and triple mutants, and crystal structures, we defined three secreted substrates/effectors that largely clarify the role of T2S during infection of A. castellanii Particularly interesting are the unique functional overlap between an acyltransferase (PlaC) and aminopeptidase (LapA), the broad substrate specificity and eukaryotic-protein-like character of LapA, and the novelty of NttD. Linking LapA to amino acid acquisition, we defined, for the first time, the importance of secreted aminopeptidases in intracellular infection. Bioinformatic investigation, not previously applied to T2S, revealed that effectors originate from diverse sources and distribute within the Legionella genus in unique ways. The results of this study represent a major advance in understanding Legionella ecology and pathogenesis, bacterial secretion, and the evolution of intracellular parasitism.