Far-ultra violet (UV) circular dichroism (CD) spectra of the wild type and mutant ADs.

<p>Far-ultra violet (UV) circular dichroism (CD) spectra of the wild type and mutant ADs.</p

Crystal structure of <i>Prochlorococcus marinus</i> MIT9313 aldehyde deformylating oxygenase (<i>Pm</i>AD).

<p>Cys83, Cys119, and Ala129 in <i>Pm</i>AD, which corresponds to Cys71, Cys107, and Cys117 in AD from <i>Nostoc punctiforme</i> PCC 73102 (<i>Np</i>AD), respectively, are shown as red space-fill models (PDB ID: 2OC5). The two iron atoms and the substrate are shown as purple and yellow balls, respectively. The α-helices neighboring the helix involving Cys71 (cyan) are shown in blue and yellow-green. The figure was drawn using the PyMOL Molecular Graphics System, Schrödinger, LLC.</p

Thermal denaturation curves of the wild type and mutant ADs.

<p>The denaturation curves were monitored by the CD ellipticity at 222 nm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122217#pone.0122217.s008" target="_blank">S8 Fig</a>). The values were then normalized to the baseline values of the native and unfolded states. A two-step denaturation was observed for the double mutants.</p

Role of Cysteine Residues in the Structure, Stability, and Alkane Producing Activity of Cyanobacterial Aldehyde Deformylating Oxygenase

<div><p>Aldehyde deformylating oxygenase (AD) is a key enzyme for alkane biosynthesis in cyanobacteria, and it can be used as a catalyst for alkane production <i>in vitro</i> and <i>in vivo</i>. However, three free Cys residues in AD may impair its catalytic activity by undesired disulfide bond formation and oxidation. To develop Cys-deficient mutants of AD, we examined the roles of the Cys residues in the structure, stability, and alkane producing activity of AD from <i>Nostoc punctiforme</i> PCC 73102 by systematic Cys-to-Ala/Ser mutagenesis. The C71A/S mutations reduced the hydrocarbon producing activity of AD and facilitated the formation of a dimer, indicating that the conserved Cys71, which is located in close proximity to the substrate-binding site, plays crucial roles in maintaining the activity, structure, and stability of AD. On the other hand, mutations at Cys107 and Cys117 did not affect the hydrocarbon producing activity of AD. Therefore, we propose that the C107A/C117A double mutant is preferable to wild type AD for alkane production and that the double mutant may be used as a pseudo-wild type protein for further improvement of the alkane producing activity of AD.</p></div

Activity, stability, and structural properties of the wild type and mutant aldehyde deformylating oxygenases (ADs).

<p>¹Hydrocarbon producing activity relative to that of the wild type.</p><p>²Expression level of AD protein, including the soluble and insoluble forms, in <i>Escherichia coli</i> is shown relative to that of the wild type.</p><p>³Solubility of AD when only AD was overexpressed in <i>E</i>. <i>coli</i> for <i>in vitro</i> characterization of the structure and stability. +: > 60% soluble; +/–: 20–60% soluble;–: < 20% soluble.</p><p>⁴Fraction of dimers (%), as estimated by size exclusion chromatography.</p><p>⁵Melting temperature, as measured by thermal denaturation. Errors are ±1°C. The <i>T</i>_m for the second transition is also shown for the double mutants in parenthesis.</p><p>⁶The means and standard deviations of duplicate or quadruplicate measurements are shown.</p><p>⁷not determined.</p><p>Activity, stability, and structural properties of the wild type and mutant aldehyde deformylating oxygenases (ADs).</p

Hydrocarbon producing activities of the wild type and mutant ADs.

<p>The activity value presented here is relative to that of the wild type. The data are means ± standard deviations of duplicate or quadruplicate experiments.</p

Quantitative Analysis of Multisite Protein–Ligand Interactions by NMR: Binding of Intrinsically Disordered p53 Transactivation Subdomains with the TAZ2 Domain of CBP

Determination of affinities and binding sites involved in protein–ligand interactions is essential for understanding molecular mechanisms in biological systems. Here we combine singular value decomposition and global analysis of NMR chemical shift perturbations caused by protein–protein interactions to determine the number and location of binding sites on the protein surface and to measure the binding affinities. Using this method we show that the isolated AD1 and AD2 binding motifs, derived from the intrinsically disordered N-terminal transactivation domain of the tumor suppressor p53, both interact with the TAZ2 domain of the transcriptional coactivator CBP at two binding sites. Simulations of titration curves and line shapes show that a primary dissociation constant as small as 1–10 nM can be accurately estimated by NMR titration methods, provided that the primary and secondary binding processes are coupled. Unexpectedly, the site of binding of AD2 on the hydrophobic surface of TAZ2 overlaps with the binding site for AD1, but AD2 binds TAZ2 more tightly. The results highlight the complexity of interactions between intrinsically disordered proteins and their targets. Furthermore, the association rate of AD2 to TAZ2 is estimated to be 1.7 × 10¹⁰ M^–1 s^–1, approaching the diffusion-controlled limit and indicating that intrinsic disorder plus complementary electrostatics can significantly accelerate protein binding interactions

Complexity of the Folding Transition of the B Domain of Protein A Revealed by the High-Speed Tracking of Single-Molecule Fluorescence Time Series

The equilibrium unfolding transition of the B domain of protein A (BdpA) was investigated by using single-molecule fluorescence spectroscopy based on line-confocal detection of fast-flowing samples. The method achieved the time resolution of 120 μs and the observation time of a few milliseconds in the single-molecule time-series measurements of fluorescence resonance energy transfer (FRET). Two samples of BdpA doubly labeled with donor and acceptor fluorophores, the first possessing fluorophores at residues 22 and 55 (sample 1) and the second at residues 5 and 55 (sample 2), were prepared. The equilibrium unfolding transition induced by guanidium chloride (GdmCl) was monitored by bulk measurements and demonstrated that the both samples obey the apparent two-state unfolding. In the absence of GdmCl, the single-molecule FRET measurements for the both samples showed a single peak assignable to the native state (N). The FRET efficiency for N shifts to lower values as the increase of GdmCl concentration, suggesting the swelling of the native state structure. At the higher concentration of GdmCl, the both samples convert to the unfolded state (U). Near the unfolding midpoint for sample 1, the kinetic exchange between N and U causes the averaging of the two states and the higher values of the relative fluctuation. The time series for different molecules in U showed slightly different FRET efficiencies, suggesting the apparent heterogeneity. Thus, the high-speed tracking of fluorescence signals from single molecules revealed a complexity and heterogeneity hidden in the apparent two-state behavior of protein folding

Highly Heterogeneous Nature of the Native and Unfolded States of the B Domain of Protein A Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

Elucidating the protein folding mechanism is crucial to understand how proteins acquire their unique structures to realize various biological functions. With this aim, the folding/unfolding of small globular proteins has been extensively studied. Interestingly, recent studies have revealed that even such small proteins represent considerably complex processes. In this study, we examined the folding/unfolding process of a small α-helical protein, the B domain of protein A (BdpA), at equilibrium using two-dimensional fluorescence lifetime correlation spectroscopy with 10 μs time resolution. The results showed that although the BdpA is a two-state folder, both the native and unfolded states are highly heterogeneous and the conformational conversion within each ensemble occurs within 10 μs. Furthermore, it was shown that the average structures of both ensembles gradually change and become more elongated as the denaturant concentration increases. The analysis on two mutants suggested that fraying of the N-terminal helix is the origin of the inhomogeneity of the native state. Because the direct observation of the ensemble nature of the native state at the single-molecule level has not been reported, the data obtained in this study give new insights into complex conformational properties of small proteins

An NMR spectrum and the native three-dimensional structure of GroES.

<p>(A) An HSQC spectrum of ¹⁵N-labeled GroES in 90% H₂O/10% D₂O at pH 6.5 and 25°C; and the backbone structures of heptameric GroES (B) and the GroES monomer (C); the flexible mobile loop (residues 17–34), Ala97 and Asn51 are shown in red. In (C), three residues (Ile25, Val26 and Leu27) are shown in a space-filling model. The figures in (B) and (C) were prepared using the GroES portion of the GroEL/GroES/ADP complex (PDB code: 1AON), and drawn by PyMOL (DeLano Scientific).</p