Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa.
A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D002942:Circular Dichroism; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D007473:Ion Channels; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D018272:Porins; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000032:analysis*
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D007473:Ion Channels; D046911:Macromolecular Substances; D018272:Porins; D010447:Peptide Hydrolases