Features of native recombinant <i>Hs</i>DH protein and its five clinically interesting patient mutation variants.

<p>The activities of human dehydrogenase and its variants were measured by detecting spectrophotometrically the formation of magnesium complex of 3R-hydroxydecanoyl-CoA at 303 nm from the 2E-decenoyl-CoA substrate in concentration range from 0.5 to 30 µM. Kinetic parameters are calculated by using GraFit 5.0 program (Erithacus Software). Main features characterizing the observed structural changes in the variant proteins and the patients are also given (and discussed in more details in the text).</p><p>n.d. = not determined (recording reliable and comparable data would have necessitated using a large surplus of NAD⁺).</p

On the Molecular Basis of D-Bifunctional Protein Deficiency Type III

<div><p>Molecular basis of D-bifunctional protein (D-BP) deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2) protein. Complementation analysis <em>in vivo</em> in yeast and <em>in vitro</em> enzyme kinetic and stability determinants as well as <em>in silico</em> stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K_m, V_max and k_cat) of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.</p> </div

The hydrogen bonds analyzed in MD simulations.

<p>In total 18 hydrogen bonds, which were found to either be stable in the native protein or to form when the variation was present, were analyzed. The symbol (+) indicates the stable existence of the hydrogen bond and the symbol (–) clear fluctuation.</p

Growth of the BY4741 <i>Δfox2</i> cells on oleic acid transformed with pYE352::<i>ScMFE-2,</i> pYE352::<i>CTA1</i>, pYE352::<i>HsMFE-2</i>, and its five clinically interesting patient variants.

<p>The dilution series were done on YPD (0.2% glucose)- oleic acid (0.125%) plates and the BY4741 <i>Δfox2</i> cells were grown at +30°C for one week indicated by: (1) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>, (2) BY4741 <i>Δfox2</i>+ pYE352::<i>ScMFE-2</i>, (3) BY4741 <i>Δfox2</i> strain, (4) BY4741 <i>Δfox2</i>+ pYE352::<i>CTA1</i>, (5) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(T15A), (6) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(N158D), (7) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(E232K), (8) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(R248C), (9) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(W249G). When the yeast is able to utilize the oleic acid as a sole source of carbon, there will appear a clear zone around (samples 1, 2, 5, 6, 7, 8 & 9), but if the functional <i>fox2</i> gene is missing the environment remains opaque (samples 3 & 4).</p

Coomassie-stained SDS-PAGE gel of purified <i>Hs</i>DH recombinant protein and its five clinically interesting patient variants under reducing conditions.

<p>The first line represents Low molecular weight protein standard (Bio-Rad) and lines 2–7 protein samples purified by Ni-NTA and Superdex 200 gel filtration columns as follows: 2) wild type <i>Hs</i>DH, 3) T15A variant, 4) N158D variant, 5) E232K variant, 6) R248C variant and 7) W249G variant. The molecular masses of all the recombinant protein monomers (∼36 kDa) correspond to the monomer mass (36.14 kDa) estimated from the aa sequence of the wild type <i>Hs</i>DH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053688#pone.0053688-Gasteiger1" target="_blank">[29]</a>.</p

Five biologically interesting variations located in the dehydrogenase dimer of human MFE-2.

<p>The two dehydrogenase monomers in the middle of the figure are colored in green and blue, amino acids T15, N158, E232, R248 and W249 are shaded grey and shown in stick presentations, NAD⁺ are colored in yellow and shown also in stick presentation. The rectangles indicate parts of the structure that are represented in larger details in small figures. The figures were done using the program PyMol (Schrödinger) and human 3R-hydroxyacyl-CoA dehydrogenase structure (PDB ID 1ZBQ; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053688#pone.0053688-Lukacik1" target="_blank">[7]</a>).</p

Detection of channel-forming activities in subcellular fractions.

<p>Fractions 2–4 (glycosomes), 8–11 (fragments of flagella), and 15–18 (mitochondria) from Optiprep density gradients (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g001" target="_blank">Figure 1A</a>) were combined and treated with Genapol X-080 to solubilize membrane proteins (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#s4" target="_blank">Materials and methods</a> section). After sedimentation of insoluble material, aliquots of the resulting supernatants were used for MCR (<b>A</b>–<b>C</b>) or SCA (<b>D</b>). (<b>A</b>) Traces of the current monitoring in the presence of glycosomal (upper panel) or mitochondrial (lower panel) preparations. The middle trace represents a timescale-expanded current recording of the upper trace. The bath solution contained 3 M KCl and the applied voltage was +10 mV. (<b>B</b>) Histograms of insertion events registered in subcellular fractions (see panel <b>A</b>). Bin size is 4.0 pA. The total number of insertion events (I.e.) is indicated. Here and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figure 3</a> C (upper panel) all insertion events with current increments over 180 pA (for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figure 3C</a>, lower panel −90 pA) are combined in one bin (180 pA or 90 pA, respectively). Note that the amount of insertion events in the flagella fraction (see <b>B</b>, middle panel) is lower than that observed in other fractions. This is mainly due to low channel-forming activity (per protein content) in the preparations of this fraction. For the sake of compatibility we used the same amounts of protein for measurements in different fractions. (<b>C</b>) Histograms of insertion events detected for glycosomal preparations using NH₄Cl as the electrolyte. Bin size: 4 pA (upper panel) or 2 pA (lower panel). See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figures 3A and 3B</a> for other details. (<b>D</b>) Trace of the current monitoring using the glycosomal fraction (initial holding potential +10 mV) indicating the insertion (marked by one asterisk) of a large-conductance channel that spontaneously closed (marked by two asterisks) after stepwise (each step is +10 mV) increase in the holding potential up to 50 mV.</p

SCA of a very-low-conductance channel.

<p>(<b>A</b>) Current recording of a single very-low-conductance channel. The bath solution (panels <b>A</b>, <b>B</b>, and <b>C</b>) contained 3 M KCl. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>A</b></a> for other details. (<b>B</b>) Current trace of the channel in response to the shown voltage-ramp protocol. Dotted line indicates the current level at zero holding potential. Note the near linear dependence of the current on the applied voltage. (<b>C</b>) Current traces of a single channel in response to the indicated voltage-step protocol. (<b>D</b>) Ion-selectivity of the channel. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>D</b></a> for details.</p

Electron microscopy of cellular organelles separated by Optiprep gradient centrifugation.

<p>Fractions enriched in glycosomes (fractions 2–5, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g001" target="_blank">Figure 1A</a>), fragments of flagella (fractions 8–11) or mitochondria and other organelles (fractions 15–18) were combined and processed for EM examination (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#s4" target="_blank">Materials and methods</a> section). (<b>A</b> and <b>B</b>) Isolated glycosomes shown at lower (<b>A</b>) and higher (<b>B</b>) magnifications. The fraction consists mostly of glycosomes. Some contamination by fragments of flagella is also visible. Importantly, fragments of flagella (paraflagellar rods and axonemes) show no sign of attachment to the flagellar membrane. Note the presence of intact glycosomes as electron-dense vesicles surrounded by a single membrane (marked by arrows in panel <b>B</b>). (<b>C</b> and <b>D</b>) Fractions enriched in flagella at low (<b>C</b>) and high (<b>D</b>) magnifications. One can see many paraflagellar rods in longitudinal section (<b>C</b>) and recognize flagellar axonemes (marked by arrows in panel <b>D</b>). Some glycosomes are also visible in panel <b>C</b>. (<b>E</b> and <b>F</b>) Composition of the fraction from the top of the Optiprep gradient that is enriched with mitochondria. Several types of organelles – mitochondria, lysosomes, lipid droplets, clathrin-coated vesicles, and components from the flagellar apparatus – can be observed. Note the shrinking of the mitochondrial inner membrane (see panel <b>F</b>) apparently due to osmotic misbalance. Scale bars: 2 µm (<b>C</b> and <b>E</b>); 1 µm (<b>A</b>); 0.5 µm (<b>D</b> and <b>F</b>), and 0.1 µm (<b>B</b>).</p

SCA of a high-conductance channel.

<p>(<b>A</b>) Current trace of a single high-conductance channel. The insertion event (marked by an asterisk) was registered at +10 mV and the applied voltage was then switched to −10 mV. The dashed line indicates the current level (zero) before insertion of the channel. The data in panels <b>A</b>, <b>B</b>, and <b>C</b> were collected using 3 M KCl as the electrolyte. (<b>B</b>) Current trace of the channel in response to the indicated voltage-ramp protocol. Note the near linear dependence of the current on the applied voltage. (<b>C</b>) Single channel currents in response to the indicated voltage-step protocol. (<b>D</b>) Dependence of the single channel conductance on the KCl concentration. After detection of a single channel insertion using 3 M KCl as bath solution (holding potential +10 mV), the electrolyte was diluted and registration of the current amplitudes of the same channel was conducted at 2.0 M and 1.0 M KCl, respectively. Data points are mean±SD for at least 4 independent measurements. (<b>E</b>) Current traces of a single channel in response to a low-speed linear increase (upper trace) or decrease (lower trace) of the holding potential. The bath solution contained 1.0 M NH₄Cl, 20 mM Tris-Cl, pH 7.8, and 2 mM DTT at both sides of the membrane. Note that the channel was still open even at hyperpolarizing holding potentials of ±150 mV. (<b>F</b>) Current-voltage relationship of the high-conductance channel under asymmetric salt conditions: 3.0 M KCl <i>trans</i>/1.5 M KCl <i>cis</i> compartment. The insertion of a single channel was detected at 3 M KCl at both sides of the membrane and at a voltage of +10 mV, then the electrolyte concentration in the <i>cis</i> compartment was decreased by dilution and an initial current recording was conducted at zero potential followed by stepwise (±10 mV) change of the applied voltage. Data points are mean±SD, n = 4–5. Bars in some cases are smaller than symbols.</p