Biology subjects

Bricker, T. M.

Publications and source records attributed to Bricker, T. M..

6 recordsLinked to original sources

bioRxiv2025

Molecular Dynamic Studies on the Interaction of a TatA Oligomer with Tat Translocon Substrates

The Tat Translocon directly utilizes the Proton Motive Force to transport folded proteins from the n-side to the p-side of energized membranes, targeting the thylakoid lumen of chloroplasts and the periplasmic space of Bacteria and Archaea. In most organisms the Translocon consists of three subunits, TatA, TatB and TatC exhibiting a stoichiometry of [~]20-50/1/1. While TatB/TatC recognize the canonical twin-arginine motif-containing signal sequence of substrate proteins, TatA has been hypothesized to interact with TatB/TatC and translocon substrates facilitating their transport across the membrane. TatA from E.coli contains a short transmembrane helix near the N-terminus, a longer amphipathic helix and a relatively large unstructured C-terminal domain. While the transmembrane and amphipathic helixes are required for Translocon activity, the C-terminal domain is, in large measure, dispensable. TatA has been hypothesized to form higher-order oligomers in the biological membranes. In this communication we have used 1000 ns-long course-grained molecular dynamic simulations to examine the interactions between a membrane-associated E. coli TatA nonamer, alone, and in association with two Tat Translocon substrate proteins, either OEE17 or TorA. In all simulations, either in the presence or absence of substrate, the TatA nonamer markedly thinned the lipid bilayer which may facilitate substrate translocation. The pore of the nonamer was occupied by a phospholipid layer consisting of [~]6 phospholipids in the absence of substrates and [~]11 phospholipids in their presence. Structurally, the amphipathic helix of TatA were observed to exhibit significant conformational flexibility which appears to facilitate TatA-substrate interactions. In the absence of substrate the TatA nonamer was unstable with its radial architecture collapsing in 200-300 ns. In the presence of substrate, however, the radial geometry of the nonamer persists for at least 1000 ns. Interestingly, in the presence of the smaller substrate OEE17, fewer TatA monomers are retained in a radial geometry then observed in the presence of the larger substrate TorA indicating that the molecularity of the TatA oligomer can adjust to the size of the substrate. Specific hydrophilic residues of the TatA amphipathic helixes were found to interact with both substrate molecules, and these form quite stable charge-pair or hydrogen-bonding interactions. While the substrate proteins were initially placed adjacent to the amphipathic helixes of the nonamer, during the simulation trajectories the substrates moved to a more central position adjacent to, and partially entering, the oligomer pore. Concomitantly, the oligomer was observed to lose phospholipids. These latter observations may constitute a glimpse of the initial stages of protein translocation.

biochemistry↗

bioRxiv2023

A Real-Time Analysis of Protein Transport via the Twin Arginine Translocation Pathway in Response to Different Components of the Protonmotive Force in vivo

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the {Delta}{psi} component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology (NanoBiT) in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions, {Delta}pH, in addition to {Delta}{psi}, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

biochemistry↗

bioRxiv2022

High-Resolution Tandem Mass Spectrometry Indicates Rubisco Activase is Associated with PS I-LHC I-LHC II Membranes

During proteomic investigations examining the mobile LHC II chlorophyll antenna complex associated with Photosystem I (PS I), we identified, with very high confidence, the association of Rubisco Activase with PS I-LHC I-LHC II membranes. Using very rigorous criteria (p-values [≤] 10-5), a total of fifty-three high-quality Rubisco Activase peptides were identified by high resolution tandem mass spectrometry in two biological replicates, each digested with either trypsin or chymotrypsin, independently. Using these criteria, and searching the entire spinach proteome, only proteins previously known to be associated with PS I (the PS I subunits PsaD and PsaL, Lhca1-4, Lhcb1-3), the monomeric Lhcb proteins, Lhcb4-6 (which will be discussed elsewhere), and Rubisco Activase were identified. The presence of Rubisco Activase closely associated with PS I has important implications with respect to the activation of this enzyme and, consequently, the Calvin cycle by PS I, which will be discussed.

biochemistry↗

bioRxiv2021

Oxidative Modification of LHC II Associated with Photosystem II and PS I-LHC I-LHC II Membranes

Under aerobic conditions the production of Reactive Oxygen Species (ROS) by electron transport chains is unavoidable, and occurs in both autotrophic and heterotrophic organisms. In photosynthetic organisms both Photosystem II (PS II) and Photosystem I (PS I), in addition to the cytochrome b6/f complex, are demonstrated sources of ROS. All of these membrane protein complexes exhibit oxidative damage when isolated from field-grown plant material. An additional possible source of ROS in PS I and PS II is the distal, chlorophyll-containing light-harvesting array LHC II, which is present in both photosystems. These serve as possible sources of 1O2 produced by the interaction of 3O2 with 3chl* produced by intersystem crossing. We have hypothesized that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in a subset of the spinach LHC II proteins (Lhcb1 and Lhcb2) that were associated with either PS II membranes (i.e. BBYs) or PS I-LHC I-LHC II membranes, both of which were isolated from field-grown spinach. We identified oxidatively modified residues by high-resolution tandem mass spectrometry. Interestingly, two different patterns of oxidative modification were evident for the Lhcb1 and Lhcb2 proteins from these different sources. In the LHC II associated with PS II membranes, oxidized residues were identified to be located on the stromal surface of Lhcb1 and, to a much lesser extent, Lhcb2. Relatively few oxidized residues were identified as buried in the hydrophobic core of these proteins. The LHC II associated with PS I-LHC I-LHC II membranes, however, exhibited fewer surface-oxidized residues but, rather a large number of oxidative modifications buried in the hydrophobic core regions of both Lhcb1 and Lhcb2, adjacent to the chlorophyll prosthetic groups. These results appear to indicate that ROS, specifically 1O2, can modify the Lhcb proteins associated with both photosystems and that the LHC II associated with PS II membranes represent a different population from the LHC II associated with PS I-LHC I-LHC II membranes.

plant biology↗

bioRxiv2019

Natively Oxidized Amino Acid Residues in the Spinach PS I-LHC I Supercomplex

Reactive oxygen species (ROS) production is an unavoidable byproduct of electron transport under aerobic conditions. Photosystem II (PS II), the cytochrome b6f complex and Photosystem I (PS I) are all demonstrated sources of ROS. It has been proposed that PS I produces substantial levels of a variety of ROS including [Formula] 1O2, H2O2 and, possibly, [Formula], however, the site(s) of ROS production within PS I has been the subject of significant debate. We hypothesize that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in spinach PS I which was isolated from field-grown spinach. The modified residues were identified by high-resolution tandem mass spectrometry. As expected, many of the modified residues lie on the surface of the complex. However, a well-defined group of oxidized residues, both buried and surface-exposed, lead from the chl a of P700 to the surface of PS I. These residues (PsaB: 609F, 611E, 617M, 619W, 620L, and PsaF: 139L, 142A,143D) may identify a preferred route for ROS, probably 1O2, to egress the complex from the vicinity of P700. Additionally, two buried residues located in close proximity to A1B (PsaB:712H and 714S) were modified, which may be consistent with A1B being a source of [Formula]. Surprisingly, no oxidatively modified residues were identified in close proximity to the 4Fe-FS clusters FX, FA or FB. These cofactors had been identified as a principal targets for ROS damage in the photosystem. Finally, a large number of residues located in the hydrophobic cores of Lhca1-Lhca4 are oxidatively modified. These appear to be the result of 1O2 production by the distal antennae for the photosystem.

biochemistry↗

bioRxiv2017

Natively Oxidized Amino Acid Residues in the Spinach Cytochrome b6f Complex

The cytochrome b6f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b6f is 10-20 fold higher than that observed for the analogous respiratory cytochrome bc1 complex. The types of ROS produced (O2*-,1O2, and, possibly, H2O2) and the site(s) of ROS production within the b6f complex has been the subject of some debate. Proposed sources of ROS have include the heme bp, PQp*- (possible sources for O2*-), the Rieske iron-sulfur cluster (possible source of O2*- and/or H2O2), Chl a (possible source of 1O2) and heme Cn (possible source of O2*- and/or H2O2). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b6f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b6f complex.

biochemistry↗