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A phasin with many faces: structural insights on PhaP from Azotobacter sp. FA8.

Mezzina MP, Wetzler DE, Catone MV, Bucci H, Di Paola M, Pettinari MJ - PLoS ONE (2014)

Bottom Line: Phasins are a group of proteins associated to granules of polyhydroxyalkanoates (PHAs).Several experimental data support that it is a tetramer, probably due to interactions between coiled-coil regions.These structural features have also been detected in other phasins, and may be related to their functional diversity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IQUIBICEN-CONICET, Buenos Aires, Argentina.

ABSTRACT
Phasins are a group of proteins associated to granules of polyhydroxyalkanoates (PHAs). Apart from their structural role as part of the PHA granule cover, different structural and regulatory functions have been found associated to many of them, and several biotechnological applications have been developed using phasin protein fusions. Despite their remarkable functional diversity, the structure of these proteins has not been analyzed except in very few studies. PhaP from Azotobacter sp. FA8 (PhaPAz) is a representative of the prevailing type in the multifunctional phasin protein family. Previous work performed in our laboratory using this protein have demonstrated that it has some very peculiar characteristics, such as its stress protecting effects in recombinant Escherichia coli, both in the presence and absence of PHA. The aim of the present work was to perform a structural characterization of this protein, to shed light on its properties. Its aminoacid composition revealed that it lacks clear hydrophobic domains, a characteristic that appears to be common to most phasins, despite their lipid granule binding capacity. The secondary structure of this protein, consisting of α-helices and disordered regions, has a remarkable capacity to change according to its environment. Several experimental data support that it is a tetramer, probably due to interactions between coiled-coil regions. These structural features have also been detected in other phasins, and may be related to their functional diversity.

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Related in: MedlinePlus

PhaPAz thermal stability.(A) Denaturation was followed by measuring the ellipticity change in the far-UV region at 222 nm. (○) PhaPAz 3 µM (apparent Tm = 55°C) (•) PhaPAz 1.5 µM. (B) Far-UV CD Spectra of PhaPAz (3 µM) in 20 mM sodium phosphate buffer pH 7.3 containing NaCl 50 mM and 1 mM DTT at different temperatures (from 20°C to 80°C). Recovered spectra were registered upon immediate cooling down the scanned sample and after a 10 min waiting period at 20°C.
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pone-0103012-g007: PhaPAz thermal stability.(A) Denaturation was followed by measuring the ellipticity change in the far-UV region at 222 nm. (○) PhaPAz 3 µM (apparent Tm = 55°C) (•) PhaPAz 1.5 µM. (B) Far-UV CD Spectra of PhaPAz (3 µM) in 20 mM sodium phosphate buffer pH 7.3 containing NaCl 50 mM and 1 mM DTT at different temperatures (from 20°C to 80°C). Recovered spectra were registered upon immediate cooling down the scanned sample and after a 10 min waiting period at 20°C.

Mentions: The thermal denaturation of PhaPAz was monitored by CD. At 222 nm, where the largest change in ellipticity took place, a sharp symmetric transition could be observed (Fig. 7A). When using 3 µM protein, the cooperative transition started at 30°C, showed an apparent Tm of 55°C, and was completed at 65°C. The thermal unfolding process was not reversible, as above 65°C the protein tended to aggregate, and the native spectrum was not recovered completely upon cooling (Fig. 7B). This aggregation was likely caused by residual secondary structures at the end of the transition. The isodichroic point observed before protein aggregation (∼205 nm) indicates that the complete unfolding (between the folded oligomer and the unfolded state) was a two state process. The lower Tm and the decrease in cooperativity observed at lower protein concentrations are consistent with the monomerization process occurring during unfolding (Fig. 7A).


A phasin with many faces: structural insights on PhaP from Azotobacter sp. FA8.

Mezzina MP, Wetzler DE, Catone MV, Bucci H, Di Paola M, Pettinari MJ - PLoS ONE (2014)

PhaPAz thermal stability.(A) Denaturation was followed by measuring the ellipticity change in the far-UV region at 222 nm. (○) PhaPAz 3 µM (apparent Tm = 55°C) (•) PhaPAz 1.5 µM. (B) Far-UV CD Spectra of PhaPAz (3 µM) in 20 mM sodium phosphate buffer pH 7.3 containing NaCl 50 mM and 1 mM DTT at different temperatures (from 20°C to 80°C). Recovered spectra were registered upon immediate cooling down the scanned sample and after a 10 min waiting period at 20°C.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117528&req=5

pone-0103012-g007: PhaPAz thermal stability.(A) Denaturation was followed by measuring the ellipticity change in the far-UV region at 222 nm. (○) PhaPAz 3 µM (apparent Tm = 55°C) (•) PhaPAz 1.5 µM. (B) Far-UV CD Spectra of PhaPAz (3 µM) in 20 mM sodium phosphate buffer pH 7.3 containing NaCl 50 mM and 1 mM DTT at different temperatures (from 20°C to 80°C). Recovered spectra were registered upon immediate cooling down the scanned sample and after a 10 min waiting period at 20°C.
Mentions: The thermal denaturation of PhaPAz was monitored by CD. At 222 nm, where the largest change in ellipticity took place, a sharp symmetric transition could be observed (Fig. 7A). When using 3 µM protein, the cooperative transition started at 30°C, showed an apparent Tm of 55°C, and was completed at 65°C. The thermal unfolding process was not reversible, as above 65°C the protein tended to aggregate, and the native spectrum was not recovered completely upon cooling (Fig. 7B). This aggregation was likely caused by residual secondary structures at the end of the transition. The isodichroic point observed before protein aggregation (∼205 nm) indicates that the complete unfolding (between the folded oligomer and the unfolded state) was a two state process. The lower Tm and the decrease in cooperativity observed at lower protein concentrations are consistent with the monomerization process occurring during unfolding (Fig. 7A).

Bottom Line: Phasins are a group of proteins associated to granules of polyhydroxyalkanoates (PHAs).Several experimental data support that it is a tetramer, probably due to interactions between coiled-coil regions.These structural features have also been detected in other phasins, and may be related to their functional diversity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IQUIBICEN-CONICET, Buenos Aires, Argentina.

ABSTRACT
Phasins are a group of proteins associated to granules of polyhydroxyalkanoates (PHAs). Apart from their structural role as part of the PHA granule cover, different structural and regulatory functions have been found associated to many of them, and several biotechnological applications have been developed using phasin protein fusions. Despite their remarkable functional diversity, the structure of these proteins has not been analyzed except in very few studies. PhaP from Azotobacter sp. FA8 (PhaPAz) is a representative of the prevailing type in the multifunctional phasin protein family. Previous work performed in our laboratory using this protein have demonstrated that it has some very peculiar characteristics, such as its stress protecting effects in recombinant Escherichia coli, both in the presence and absence of PHA. The aim of the present work was to perform a structural characterization of this protein, to shed light on its properties. Its aminoacid composition revealed that it lacks clear hydrophobic domains, a characteristic that appears to be common to most phasins, despite their lipid granule binding capacity. The secondary structure of this protein, consisting of α-helices and disordered regions, has a remarkable capacity to change according to its environment. Several experimental data support that it is a tetramer, probably due to interactions between coiled-coil regions. These structural features have also been detected in other phasins, and may be related to their functional diversity.

Show MeSH
Related in: MedlinePlus