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Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

Trujillo U, Vázquez-Rosa E, Oyola-Robles D, Stagg LJ, Vassallo DA, Vega IE, Arold ST, Baerga-Ortiz A - PLoS ONE (2013)

Bottom Line: However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit.Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein.Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico.

ABSTRACT
The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.

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Size exclusion chromatography of tandem ACP.The tandem ACP analyzed on the same gel filtration column had the retention time of a protein three times its size (MWest  = 177,000).
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pone-0057859-g008: Size exclusion chromatography of tandem ACP.The tandem ACP analyzed on the same gel filtration column had the retention time of a protein three times its size (MWest  = 177,000).

Mentions: When the tandem ACP (MW  = 59,116) was analyzed by gel filtration chromatography the estimated molecular weight (MWest ≈ 177,000) based on its retention time, was more consistent with a trimeric domain than with a monomeric one (Figure 8). Similarly, when individual ACP domains were purified and analyzed by gel filtration, their estimated molecular weights were found to be more consistent with dimeric rather than monomeric domains (Table 1). However the Stokes Radius (Rs) of the tandem arrangement (48.6 Å), derived from analytical gel filtration, was very similar to the average Rs of the EOM ensemble determined by SAXS (47 Å). Thus the apparent trimeric state of tandem ACP can be explained by a shortened retention time of the monomer due to its extended conformation. Similarly, the apparent dimeric states of the individual ACPs are explained by their greater apparent size which is possibly due to the presence of the flexible linkers flanking each individual ACP.


Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

Trujillo U, Vázquez-Rosa E, Oyola-Robles D, Stagg LJ, Vassallo DA, Vega IE, Arold ST, Baerga-Ortiz A - PLoS ONE (2013)

Size exclusion chromatography of tandem ACP.The tandem ACP analyzed on the same gel filtration column had the retention time of a protein three times its size (MWest  = 177,000).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585217&req=5

pone-0057859-g008: Size exclusion chromatography of tandem ACP.The tandem ACP analyzed on the same gel filtration column had the retention time of a protein three times its size (MWest  = 177,000).
Mentions: When the tandem ACP (MW  = 59,116) was analyzed by gel filtration chromatography the estimated molecular weight (MWest ≈ 177,000) based on its retention time, was more consistent with a trimeric domain than with a monomeric one (Figure 8). Similarly, when individual ACP domains were purified and analyzed by gel filtration, their estimated molecular weights were found to be more consistent with dimeric rather than monomeric domains (Table 1). However the Stokes Radius (Rs) of the tandem arrangement (48.6 Å), derived from analytical gel filtration, was very similar to the average Rs of the EOM ensemble determined by SAXS (47 Å). Thus the apparent trimeric state of tandem ACP can be explained by a shortened retention time of the monomer due to its extended conformation. Similarly, the apparent dimeric states of the individual ACPs are explained by their greater apparent size which is possibly due to the presence of the flexible linkers flanking each individual ACP.

Bottom Line: However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit.Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein.Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico.

ABSTRACT
The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.

Show MeSH