Kinetics of folding of Escherichia coli OmpA from narrow to large pore conformation in a planar bilayer.
The outer membrane protein of Escherichia coli, OmpA, is currently alleged to adopt two native conformations: a major two-domain conformer in which 171 N-terminal residues form a narrow eight beta-barrel pore and 154 C-terminal residues are in the periplasm and a minor one-domain conformer in which all 325 residues create a large pore. However, recent studies in planar bilayers indicate the conformation of OmpA is temperature-sensitive and that increasing temperature converts narrow pores to large pores. Here we examine the reversibility and kinetics of this transition for single OmpA molecules in planar bilayers of diphytanoylphosphatidylcholine (DPhPC). We find that the transition is irreversible. When temperatures are decreased, large pores close down, and when temperatures are stabilized they reopen in the large pore conformation, with gradually increasing open time. Large pores are converted to narrow pores only by denaturing agents. The transition from narrow to large pores requires temperatures >or= 26 degrees C and is a biphasic process with rates that rise steeply with temperature. The first phase, a flickering stepwise transition from a low-conductance to a high-conductance state requires approximately 7 h at 26 degrees C but only approximately 13 min at 42 degrees C, signifying an activation energy of 139 +/- 12 kJ/mol. This is followed by a gradual increase in conductance and open probability, interpreted as optimization of the large pore structure. The results indicate that the two-domain structure is a partially folded intermediate that is kinetically stable at lower temperatures and that mature fully folded OmpA is a large pore.
Zakharian
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Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824, USA.
Reusch
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D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D008823:Micelles; D010713:Phosphatidylcholines / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008823:Micelles; D010713:Phosphatidylcholines; C078167:OMPA outer membrane proteins; C021016:1,2-diphytanoylphosphatidylcholine