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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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Small-angle X-ray Scattering Data.(A) Small X-ray Scattering (SAXS) Data for the tandem ACP fragment. (B) Calculation of the pair distribution function P(r) shows a shape compatible with an elongated structure, a radius of gyration (Rg) of 5.2±0.1 nm and a maximum diameter of 20±2 nm.
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pone-0057859-g006: Small-angle X-ray Scattering Data.(A) Small X-ray Scattering (SAXS) Data for the tandem ACP fragment. (B) Calculation of the pair distribution function P(r) shows a shape compatible with an elongated structure, a radius of gyration (Rg) of 5.2±0.1 nm and a maximum diameter of 20±2 nm.

Mentions: Small-angle X-ray scattering (SAXS) was used to generate a model for the solution structure of the tandem ACP. Analysis of the distance distribution (P(r)) of the signal resulted in an estimated radius of gyration of ∼5.2 nm and a maximum diameter (Dm) of ∼20 nm. The shape of P(r) is compatible with a rod-like elongated particle (Figure 6). The three-dimensional bead model constructed by ‘dammif’ reveals a molecular volume of 96,600 Å3, whereas the molecular (unhydrated) volume estimated from the three-dimensional models of the individual ACP domains is 90,400 Å3 (Figure 7A). This correspondence between the SAXS-derived volume and the volume calculated from the molecular models of the individual ACP domains strongly suggests that the tandem ACP is monomeric. In order to better understand the configurational variability or flexibility of the tandem ACP due to domain-domain interactions, we also used the ensemble optimization method (EOM) in which a mixture of different states was considered (Figure 7B). We obtained χ values of 1.144, 0.984, 0.971, 0.971, 0.968, 0.968 for ensembles constituted of 1-2-3-4-5 and 20 models respectively. The decrease of the χ value up to ensembles with 5 or more models is indicative of a flexible tandem arrangement, and speaks strongly against a compact quaternary arrangement of the ACP domains. The average Radius of gyration (Rg) of the selected 20-model ensemble (54 Å) was significantly larger than the average Rg of the 8000 generated models with random linker conformations (43 Å) indicating a tendency of the tandem construct to adopt an elongated arrangement. All of these data suggest that there are only a few interactions between the ACP domains consistent with a beads-on-a-string model in which the linkers are flexible and the domains are seemingly autonomous.


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)

Small-angle X-ray Scattering Data.(A) Small X-ray Scattering (SAXS) Data for the tandem ACP fragment. (B) Calculation of the pair distribution function P(r) shows a shape compatible with an elongated structure, a radius of gyration (Rg) of 5.2±0.1 nm and a maximum diameter of 20±2 nm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585217&req=5

pone-0057859-g006: Small-angle X-ray Scattering Data.(A) Small X-ray Scattering (SAXS) Data for the tandem ACP fragment. (B) Calculation of the pair distribution function P(r) shows a shape compatible with an elongated structure, a radius of gyration (Rg) of 5.2±0.1 nm and a maximum diameter of 20±2 nm.
Mentions: Small-angle X-ray scattering (SAXS) was used to generate a model for the solution structure of the tandem ACP. Analysis of the distance distribution (P(r)) of the signal resulted in an estimated radius of gyration of ∼5.2 nm and a maximum diameter (Dm) of ∼20 nm. The shape of P(r) is compatible with a rod-like elongated particle (Figure 6). The three-dimensional bead model constructed by ‘dammif’ reveals a molecular volume of 96,600 Å3, whereas the molecular (unhydrated) volume estimated from the three-dimensional models of the individual ACP domains is 90,400 Å3 (Figure 7A). This correspondence between the SAXS-derived volume and the volume calculated from the molecular models of the individual ACP domains strongly suggests that the tandem ACP is monomeric. In order to better understand the configurational variability or flexibility of the tandem ACP due to domain-domain interactions, we also used the ensemble optimization method (EOM) in which a mixture of different states was considered (Figure 7B). We obtained χ values of 1.144, 0.984, 0.971, 0.971, 0.968, 0.968 for ensembles constituted of 1-2-3-4-5 and 20 models respectively. The decrease of the χ value up to ensembles with 5 or more models is indicative of a flexible tandem arrangement, and speaks strongly against a compact quaternary arrangement of the ACP domains. The average Radius of gyration (Rg) of the selected 20-model ensemble (54 Å) was significantly larger than the average Rg of the 8000 generated models with random linker conformations (43 Å) indicating a tendency of the tandem construct to adopt an elongated arrangement. All of these data suggest that there are only a few interactions between the ACP domains consistent with a beads-on-a-string model in which the linkers are flexible and the domains are seemingly autonomous.

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