20 research outputs found
Conformational Dynamics of <i>Escherichia coli</i> Flavodoxins in Apo- and Holo-States by Solution NMR Spectroscopy
<div><p>Flavodoxins are a family of small FMN-binding proteins that commonly exist in prokaryotes. They utilize a non-covalently bound FMN molecule to act as the redox center during the electron transfer processes in various important biological pathways. Although extensive investigations were performed, detailed molecular mechanisms of cofactor binding and electron transfer remain elusive. Herein we report the solution NMR studies on <i>Escherichia coli</i> flavodoxins FldA and YqcA, belonging to the long-chain and short-chain flavodoxin subfamilies respectively. Our structural studies demonstrate that both proteins show the typical flavodoxin fold, with extensive conformational exchanges observed near the FMN binding pocket in their apo-forms. Cofactor binding significantly stabilizes both proteins as revealed by the extension of secondary structures in the holo-forms, and the overall rigidity shown by the backbone dynamics data. However, the 50 s loops of both proteins in the holo-form still show conformational exchanges on the µs-ms timescales, which appears to be a common feature in the flavodoxin family, and might play an important role in structural fine-tuning during the electron transfer reactions.</p></div
Mapping of the internal dynamics of <i>E. coli</i> YqcA onto the structures.
<p>(A, C) Ribbon diagrams of the apo- (A) and holo-YqcA (C) representing the generalized order parameter <i>S<sup>2</sup></i> values. Colors ranging from green to blue correspond to <i>S<sup>2</sup></i> values from 0.75 to 1, and red corresponds to <i>S<sup>2</sup></i><0.75. Missing residues in apo-YqcA are shown in pink. (B, D) Ribbon diagrams of the apo- (B) and holo-YqcA (D) representing the <i>R<sub>ex</sub></i> values. Colors ranging from yellow to red correspond to <i>R<sub>ex</sub></i> values from 1 s<sup>−1</sup> to 15 s<sup>−1</sup>. Residues with <i>R<sub>ex</sub></i>>15 s<sup>−1</sup> are also shown in red. Missing residues in apo-YqcA are shown in pink.</p
Backbone relaxation data and internal dynamic parameters of <i>E. coli</i> YqcA.
<p>(A) Longitudinal relaxation rates (<i>R</i><sub>1</sub>), transverse relaxation rates (<i>R</i><sub>2</sub>), and heteronuclear {<sup>1</sup>H}-<sup>15</sup>N NOE values of the apo- (black) and holo-form (red) of <i>E. coli</i> YqcA <i>versus</i> the amino acid sequence. The data were recorded on a Bruker Avance 800-MHz spectrometer at 25°C. (B) The backbone dynamic parameters <i>S<sup>2</sup></i>, <i>τ<sub>e</sub></i>, and <i>R<sub>ex</sub></i> of the apo- (black) and holo-form (red) of <i>E. coli</i> YqcA <i>versus</i> the amino acid sequence.</p
Backbone relaxation data and reduced spectral density functions of <i>E. coli</i> FldA.
<p>(A) Longitudinal relaxation rates (<i>R</i><sub>1</sub>), transverse relaxation rates (<i>R</i><sub>2</sub>), and heteronuclear {<sup>1</sup>H}-<sup>15</sup>N NOE values of the apo- (black) and holo-FldA (red) <i>versus</i> the amino acid sequence. The data were recorded on a Bruker Avance 800-MHz spectrometer at 25°C. (B) The extracted spectral density functions <i>J(0)</i>, <i>J(0.87ω<sub>H</sub>)</i> and <i>J(ω<sub>N</sub>)</i> of the apo- (black) and holo-FldA (red) <i>versus</i> the amino acid sequence. The grey-colored background columns in both panels represent the missing residues in apo-FldA.</p
Internal dynamic parameters of holo-FldA.
<p>(A) The backbone dynamic parameters <i>S<sup>2</sup></i>, <i>τ<sub>e</sub></i>, and <i>R<sub>ex</sub></i> of holo-FldA <i>versus</i> the amino acid sequence. (B) Ribbon diagrams of holo-FldA representing the generalized order parameter <i>S<sup>2</sup></i>. Colors ranging from green to blue correspond to <i>S<sup>2</sup></i> values from 0.75 to 1, and red corresponds to <i>S<sup>2</sup></i><0.75. (C) Ribbon diagrams of holo-FldA representing the <i>R<sub>ex</sub></i> values. Colors ranging from yellow to red correspond to <i>R<sub>ex</sub></i> values from 1 s<sup>−1</sup> to 15 s<sup>−1</sup>. Residues with <i>R<sub>ex</sub></i>>15 s<sup>−1</sup> are also shown in red.</p
Solution structures of the apo- and holo-forms of <i>E. coli</i> YqcA.
<p>(A, C) Superimpositions of the 20 representative solution structures of YqcA in the apo- (A) and holo-forms (C). The FMN molecule is shown in red. (B, D) Ribbon diagram representations of YqcA in the apo- (B) and holo-forms (D). The secondary structural elements and the loops around the FMN-binding site are labeled in B and D, respectively. (E) An overlay of the ribbon diagram of apo- (red) and holo-YqcA (blue). The FMN molecule is not shown.</p
Structural characterizations of the apo- and holo-forms of <i>E. coli</i> FldA.
<p>(A) Superimposition of the 20 representative solution structures of holo-FldA. The FMN molecule is shown in red. (B) Ribbon diagram representation of holo-FldA, with secondary structural elements and loops labeled. (C) An overlay of the ribbon diagram of the solution structure (red) and crystal structure (yellow, PDB code 1AHN). The FMN molecule is not shown. (D–E) Chemical shift differences of the apo- and holo-FldA (E) and mapping onto the holo-FldA structure (D). The composite chemical shift changes were calculated using the empirical equation, , where Δδ<sub>H</sub> and Δδ<sub>N</sub> are the chemical shift changes of <sup>1</sup>H and <sup>15</sup>N, respectively. The grey-colored background columns in (E) and the blue-colored segments in (D) represent the missing residues in apo-FldA. The residues showing little changes in chemical shifts (Δδ<sub>comp</sub><0.1 ppm) are colored red in (D). The residues showing large chemical shift changes (Δδ<sub>comp</sub>>0.5 ppm) are show as purple balls in (D).</p
Interface of TMH regions in dimeric TatA.
<p>A) Dimerization interface between the TMH segments of two subunits. Only TMH segments are shown for clarity. The omitted APHs are positioned below the paper in the upper panel. B) Representative strips from <sup>13</sup>C/<sup>15</sup>N-filtered, <sup>13</sup>C-edited NOESY experiment using a d-MCG-TatA sample with a mixture of uniformly <sup>13</sup>C/<sup>15</sup>N-labeled and unlabeled MCG-TatA (mixing time 300 ms). The experiment selectively detects inter-proton NOEs between a proton attached to a <sup>13</sup>C-labeled carbon (the ω<sub>3</sub> dimension) and protons attached to unlabeled carbon or nitrogen atoms (the ω<sub>1</sub> dimension). All protons attached to <sup>13</sup>C/<sup>15</sup>N-isotopes are filtered out in the ω<sub>1</sub> dimension. Therefore only the intermolecular NOEs between a uniformly labeled subunit and an unlabeled subunit are retained, whereas intramolecular NOEs or intermolecular NOEs in homo-labeled or homo-unlabeled dimers do not show up. C) Structure-based helical wheel diagrams of the dimer interface, with representative NOE interactions shown in dashed lines. Positively and negatively charged residues are shown in blue and red, respectively. The helical wheel diagram was drawn using an in-house written script based on the atomic coordinates of the dimeric TatA structure. D) Structural comparison of the TMH dimerization interface. The dimeric TatA structures determined in the present study are shown in green and pale green. The atomic coordinates of two adjacent subunits are subtracted from 9-mer TatA structure (PDB 2LZS) and shown in blue and sky blue. Side chains observed to show inter-molecular NOEs are shown in sticks and labeled.</p
Characterization of the activity of MCG-TatA.
<p>A) Blue-native PAGE of wt-TatA, MCG-TatA and d-MCG-TatA. The mutant proteins retain the characteristic ladder pattern as the wild-type TatA. The molecular masses (kDa) of marker proteins are given on the left. B) Tenfold serial dilution of <i>ΔtatA</i> mutant strain expressing TatA variants on SDS-containing medium. The plates were anaerobically incubated at 35°C for 72 hours and then photographed (see more details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103157#s2" target="_blank">Materials and Methods</a>). The upmost panel corresponds to a positive control experiment using the <i>ΔtatE</i> mutant strain transformed with empty pET-21a plasmids. The lower panel corresponds to the <i>ΔtatA</i> mutant strain transformed with either empty pET-21a plasmids or plasmids carrying wt-TatA gene or mutated variants.</p
Structural Basis for TatA Oligomerization: An NMR Study of <i>Escherichia coli</i> TatA Dimeric Structure
<div><p>Many proteins are transported across lipid membranes by protein translocation systems in living cells. The twin-arginine transport (Tat) system identified in bacteria and plant chloroplasts is a unique system that transports proteins across membranes in their fully-folded states. Up to date, the detailed molecular mechanism of this process remains largely unclear. The <i>Escherichia coli</i> Tat system consists of three essential transmembrane proteins: TatA, TatB and TatC. Among them, TatB and TatC form a tight complex and function in substrate recognition. The major component TatA contains a single transmembrane helix followed by an amphipathic helix, and is suggested to form the translocation pore via self-oligomerization. Since the TatA oligomer has to accommodate substrate proteins of various sizes and shapes, the process of its assembly stands essential for understanding the translocation mechanism. A structure model of TatA oligomer was recently proposed based on NMR and EPR observations, revealing contacts between the transmembrane helices from adjacent subunits. Herein we report the construction and stabilization of a dimeric TatA, as well as the structure determination by solution NMR spectroscopy. In addition to more extensive inter-subunit contacts between the transmembrane helices, we were also able to observe interactions between neighbouring amphipathic helices. The side-by-side packing of the amphipathic helices extends the solvent-exposed hydrophilic surface of the protein, which might be favourable for interactions with substrate proteins. The dimeric TatA structure offers more detailed information of TatA oligomeric interface and provides new insights on Tat translocation mechanism.</p></div