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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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Full CD spectrum for the tandem ACP fragment obtained (A) before (B) during and (C) after thermal denaturation at 90°C.
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pone-0057859-g010: Full CD spectrum for the tandem ACP fragment obtained (A) before (B) during and (C) after thermal denaturation at 90°C.

Mentions: In order to assess whether the tandem arrangement of ACP domains resulted in the overall stabilization of the protein compared to the individual domains, the thermal denaturation of both tandem and individual ACP domains was carried out by circular dichroism (CD) spectroscopy. The loss of molar ellipticity at 222 nm was measured as a function of temperature between 20–90°C (Figure 9). The denaturation temperature (Tm) for the tandem ACP domains (59°C) was lower than for the individual ACP domain (71°C), indicating that the presence of multiple domains in tandem does not result in further stabilization of the protein (Figure 9). The unfolding of the tandem domains was not cooperative, speaking against stable domain-domain interactions. The thermal unfolding of tandem and individual ACP constructs was found to be non-cooperative and fully reversible as evidenced by the full recovery of the signal on successive temperature scans (Figure 10). Analysis of the CD data using the online program K2D2 for the determination of secondary structure composition, reveals a 38% helical content which is consistent with the 41% helical content encoded within the four-helix bundles of the ACP domains. Thus, the helical content of the tandem fragment can be fully accounted for by the ACP domains, with no contribution from the linker regions.


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)

Full CD spectrum for the tandem ACP fragment obtained (A) before (B) during and (C) after thermal denaturation at 90°C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057859-g010: Full CD spectrum for the tandem ACP fragment obtained (A) before (B) during and (C) after thermal denaturation at 90°C.
Mentions: In order to assess whether the tandem arrangement of ACP domains resulted in the overall stabilization of the protein compared to the individual domains, the thermal denaturation of both tandem and individual ACP domains was carried out by circular dichroism (CD) spectroscopy. The loss of molar ellipticity at 222 nm was measured as a function of temperature between 20–90°C (Figure 9). The denaturation temperature (Tm) for the tandem ACP domains (59°C) was lower than for the individual ACP domain (71°C), indicating that the presence of multiple domains in tandem does not result in further stabilization of the protein (Figure 9). The unfolding of the tandem domains was not cooperative, speaking against stable domain-domain interactions. The thermal unfolding of tandem and individual ACP constructs was found to be non-cooperative and fully reversible as evidenced by the full recovery of the signal on successive temperature scans (Figure 10). Analysis of the CD data using the online program K2D2 for the determination of secondary structure composition, reveals a 38% helical content which is consistent with the 41% helical content encoded within the four-helix bundles of the ACP domains. Thus, the helical content of the tandem fragment can be fully accounted for by the ACP domains, with no contribution from the linker regions.

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