Multiple sequence alignment of the five ACP domains.

<p>The ACP domains from the <i>Photobacterium profundum</i> PUFA synthase were aligned using ClustalW. The black bars denote the stretches of protein sequence predicted to be α-helices.</p

Expected and Measured Molecular Weights of ACP Constructs in Solution.

<p>Expected and Measured Molecular Weights of ACP Constructs in Solution.</p

Domain structure prediction.

<p>The UMA method was employed to identify the ACP domains. The UMA score, a measurement of the likelihood that an amino acid is located within a domain (as opposed to within an unstructured linker) was plotted as a function of the position in the amino acid sequence. Five areas of high UMA score were identified as potential ACP fragments.</p

Characterization of ACP domains by mass spectrometry.

<p>(A) An ESI-MS spectrum was generated for the tandem ACP (MW  = 59,116) before modification by attachment of the phosphopantetheine moiety of CoA. (B) After incubation with PPTase and CoA the protein was modified in four attachment sites, as evidenced by the ESI-MS mass spectrum.</p

Oligonucleotides used for the amplification of the different proteins in this study.

<p>Oligonucleotides used for the amplification of the different proteins in this study.</p

Full CD spectrum for the tandem ACP fragment obtained (A) before (B) during and (C) after thermal denaturation at 90°<b>C.</b>

<p>Full CD spectrum for the tandem ACP fragment obtained (A) before (B) during and (C) after thermal denaturation at 90°<b>C.</b></p

Solution structure of tandem ACP.

<p>(A) The three-dimensional bead model constructed by ‘dammif’ reveals a molecular volume of 96,600 ų. (B) Simulation of the scattering data based on structural models reveals that an extended and flexible overall configuration can sufficiently account for the observed data. Figures A and B are on a different scale.</p

The polyunsaturated fatty acid synthase domain structure.

<p>A total of five genes are required for the production of PUFAs: pfaA contains a beta-ketoacyl synthase (KS), an acyltransferase (AT), five tandem acyl carrier proteins (ACP) and a ketoreductase (KR) domain. pfaB consists of a single AT. pfaC contains two KS domains and two tandem dehydratase (DH) domains, pfaD consists of a single enoyl reductase (ER) domain. pfaE consists of a phosphopantetheinyl transferase (PPTase).</p

Input parameters for the UMA calculations.

<p>Input parameters for the UMA calculations.</p

Thermal denaturation of individual and tandem ACP.

<p>Thermal denaturation of (A) the tandem ACP and (B) the individual ACP1 was followed by the loss of molar ellipiticity at 222 nm on a circular dichroism (CD) spectropolarimeter. The denaturation temperature (T_m) for the tandem ACP was interpolated to be 59°C, whereas the individual ACP1 had a T_m of 71°C.</p