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Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component.

Cai F, Dou Z, Bernstein SL, Leverenz R, Williams EB, Heinhorst S, Shively J, Cannon GC, Kerfeld CA - Life (Basel) (2015)

Bottom Line: Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins.Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome.Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. fcai@lbl.gov.

ABSTRACT
The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

No MeSH data available.


Biophysical characterizations of Prochlorococcus CsoS2. (a) SAXS analysis of MED4 CsoS2 and (b) the pair distribution function of the same SAXS data; (c) Near UV Circular Dichroism spectroscopy of the same protein; (d) The pair distribution functions of SAXS data measured on MIT9313 CsoS2 in isolation and after mixing with MIT9313 CsoS1 and MIT9313 RuBisCO.
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life-05-01141-f009: Biophysical characterizations of Prochlorococcus CsoS2. (a) SAXS analysis of MED4 CsoS2 and (b) the pair distribution function of the same SAXS data; (c) Near UV Circular Dichroism spectroscopy of the same protein; (d) The pair distribution functions of SAXS data measured on MIT9313 CsoS2 in isolation and after mixing with MIT9313 CsoS1 and MIT9313 RuBisCO.

Mentions: To characterize Prochlorococcus CsoS2, we expressed the MED4 and MIT9313 CsoS2 orthologs as full-length, as individual N-, M- or C-regions and as N- or C truncation (MC or NM, respectively) with a cleavable glutathione S-transferase (GST) tag fused to the N-terminus. Taking advantage of tandem purification (cation exchange chromatography followed by affinity chromatography) and on-column GST-tag cleavage, full-length or shorter versions of CsoS2 proteins were purified and concentrated to approximately 30 mg/mL in 20 mM HEPES pH 7.4 with 50 mM NaCl. Full-length MED4 CsoS2 was subjected to Small Angle X-ray Scattering (SAXS). The SAXS curve has an overall smooth shape without a local minimum or maximum (Figure 9a). Similar observations have also been reported for other highly flexible proteins, and smoothness is considered one of the characteristic features of SAXS data for intrinsically flexible proteins [33,34]. Calculation of the pair distribution function, P<r>, shows a shape compatible with an elongated conformation, with a radius of gyration of 69.3 ± 0.26 nm and a maximum diameter of 226 nm (Figure 9b), which does not support a “beads-on-a-string” structure under the testing (solution) conditions with protein concentrations up to 8.5 mg/mL. MED4 CsoS2 was also subjected to Circular Dichroism (CD) spectroscopy (Figure 9c). The dominant single minimum at 199 nm indicates a high percentage of random coil conformation [35], which is consistent with the structural prediction and the SAXS observations. Size exclusion chromatography (SEC) of MED4 CsoS2 results in an observed MW of ~250 kDa, approximately three times that of the theoretical monomer MW (82 kDa). However, the estimated MW by SEC is based on the assumption that the protein is globular, and considering that the predictions for disorder in CsoS2 this estimate may be spurious.


Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component.

Cai F, Dou Z, Bernstein SL, Leverenz R, Williams EB, Heinhorst S, Shively J, Cannon GC, Kerfeld CA - Life (Basel) (2015)

Biophysical characterizations of Prochlorococcus CsoS2. (a) SAXS analysis of MED4 CsoS2 and (b) the pair distribution function of the same SAXS data; (c) Near UV Circular Dichroism spectroscopy of the same protein; (d) The pair distribution functions of SAXS data measured on MIT9313 CsoS2 in isolation and after mixing with MIT9313 CsoS1 and MIT9313 RuBisCO.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499774&req=5

life-05-01141-f009: Biophysical characterizations of Prochlorococcus CsoS2. (a) SAXS analysis of MED4 CsoS2 and (b) the pair distribution function of the same SAXS data; (c) Near UV Circular Dichroism spectroscopy of the same protein; (d) The pair distribution functions of SAXS data measured on MIT9313 CsoS2 in isolation and after mixing with MIT9313 CsoS1 and MIT9313 RuBisCO.
Mentions: To characterize Prochlorococcus CsoS2, we expressed the MED4 and MIT9313 CsoS2 orthologs as full-length, as individual N-, M- or C-regions and as N- or C truncation (MC or NM, respectively) with a cleavable glutathione S-transferase (GST) tag fused to the N-terminus. Taking advantage of tandem purification (cation exchange chromatography followed by affinity chromatography) and on-column GST-tag cleavage, full-length or shorter versions of CsoS2 proteins were purified and concentrated to approximately 30 mg/mL in 20 mM HEPES pH 7.4 with 50 mM NaCl. Full-length MED4 CsoS2 was subjected to Small Angle X-ray Scattering (SAXS). The SAXS curve has an overall smooth shape without a local minimum or maximum (Figure 9a). Similar observations have also been reported for other highly flexible proteins, and smoothness is considered one of the characteristic features of SAXS data for intrinsically flexible proteins [33,34]. Calculation of the pair distribution function, P<r>, shows a shape compatible with an elongated conformation, with a radius of gyration of 69.3 ± 0.26 nm and a maximum diameter of 226 nm (Figure 9b), which does not support a “beads-on-a-string” structure under the testing (solution) conditions with protein concentrations up to 8.5 mg/mL. MED4 CsoS2 was also subjected to Circular Dichroism (CD) spectroscopy (Figure 9c). The dominant single minimum at 199 nm indicates a high percentage of random coil conformation [35], which is consistent with the structural prediction and the SAXS observations. Size exclusion chromatography (SEC) of MED4 CsoS2 results in an observed MW of ~250 kDa, approximately three times that of the theoretical monomer MW (82 kDa). However, the estimated MW by SEC is based on the assumption that the protein is globular, and considering that the predictions for disorder in CsoS2 this estimate may be spurious.

Bottom Line: Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins.Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome.Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. fcai@lbl.gov.

ABSTRACT
The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

No MeSH data available.