Expression, two-dimensional crystallization, and three-dimensional reconstruction of the beta8 outer membrane protein Omp21 from Comamonas acidovorans.
The Omp21 protein from the proteobacterium Comamonas (Delftia) acidovorans belongs to the recently described beta8 family of outer membrane proteins, characterized by eight antiparallel beta-strands which form a beta-barrel. This family includes virulence proteins, OmpA and OmpX from Escherichia coli, and other related molecules. After we established an expression system, recombinant Omp21 was purified by Ni(2+) chelation affinity chromatography and refolded in situ while bound to resin. The native state of refolded protein was proven by FTIR spectroscopy and monitored with denaturing PAGE (heat modification). Both native and recombinant Omp21 were reconstituted in lipid membranes and crystallized two-dimensionally by controlled dialysis. Recombinant Omp21 crystallized as dimer and formed a p22(1)2(1) lattice with constants of a = 11.1 nm, b = 12.2 nm, gamma = 89.5 degrees. The 3-D structure of negatively stained, recombinant Omp21 was determined at a resolution of 1.8 nm by means of electron crystallography. Comparison with 3-D maps of OmpX and the transmembrane domain of OmpA revealed a high similarity between the mass distribution of exoplasmic loops of Omp21 and OmpA.
Baldermann
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C
Molekulare Strukturbiologie, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, Martinsried, D-82152, Germany.
Engelhardt
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H
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure*; D003001:Cloning, Molecular; D003460:Crystallization; D003461:Crystallography; D020605:Delftia acidovorans / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D011487:Protein Conformation; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D017550:Spectroscopy, Fourier Transform Infrared
D001425:Bacterial Outer Membrane Proteins; C113718:Omp21 protein, Delftia acidovorans; D011994:Recombinant Proteins; C078167:OMPA outer membrane proteins