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

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

<i>In Vitro</i> Selection of Anti-Akt2 Thioether-Macrocyclic Peptides Leading to Isoform-Selective Inhibitors

The Akt kinase family, consisting of three isoforms in humans, is a well-validated class of drug target. Through various screening campaigns in academics and pharmaceutical industries, several promising inhibitors have been developed to date. However, due to the mechanistic and structural similarities of Akt kinases, it is yet a challenging task to discover selective inhibitors against a specific Akt isoform. We here report Akt-selective and also Akt2 isoform-selective inhibitors based on a thioether-macrocyclic peptide scaffold. Several anti-Akt2 peptides have been selected from a library by means of an <i>in vitro</i> display system, referred to as the RaPID (Random nonstandard Peptide Integrated Discovery) system. Remarkably, the majority of these “binding-active” anti-Akt2 peptides turned out to be “inhibitory active”, exhibiting IC₅₀ values of approximately 100 nM. Moreover, these peptides are not only selective to the Akt kinase family but also isoform-selective to Akt2. Particularly, one referred to as Pakti-L1 is able to discriminate Akt2 250- and 40-fold over Akt1 and Akt3, respectively. This proof-of-concept case study suggests that the RaPID system has a tremendous potential for the discovery of unique inhibitors with high family- and isoform-selectivity

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

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

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

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

Time Series of Fitness and Amino Acid Sequence in <i>in vitro</i> Evolution.

<p>(A) Each bar represents the fitness of the fittest mutant in each generation. All generations except the 8′th generation, which was derived from the 7th, are of a single lineage. The error bar is the standard deviation for experiments repeated three times. The fd-tet phage is the wild-type, which possesses the native D2 domain. The solid curves through the 1st-8′th and 8th-13th generations are the theoretical curves fitted with Eqn. (13). For each generation, the average of the synonymous mutations between the initial sequence RP3-42 and selected clones at each generation are shown by the filled squares. (B) The whole sequences of the wild-type D2 domain and the initial random sequence are listed. Substitutions accumulated with generations are indicated in single-amino acid code.</p

Semi-quantitative Fitness Landscape Plotted from the Experimental Data.

<p>The number of sequences decreases with increasing relative fitness. There are many local peaks at fitness values of ≥0.4. Note that the landscape is not shown over a fitness of 0.6 due to the low frequency of the sequence.</p

Schematic Representation of <i>n-k</i> Fitness Landscape in the Sequence Space.

<p>Dashed and solid lines indicate the mean descending slopes of local mountains and ridges between local optima, respectively. Notations of parameters are described in the text.</p