Bacterial Outer Membrane Proteins: Porins
Omp32 is a homotrimeric beta-barrel protein with a particularly narrow pore constriction. Here, a cluster of three arginines dominates the properties of the channel. Arginine clusters are typical for bacterial porins built from 16 beta-strands; their charges are stabilised by salt bridges (Arg–Glu or Arg–Asp). But in Omp32, the arginine charges are not attenuated or overcompensated by additional glutamate or aspartate residues. The narrow constriction zone forces passing molecules to interact intimately with the arginines and selects chloride ions (Cl–) by a factor of about 20 compared to potassium ions (K+).
Omp32 shows nonlinear and asymmetric voltage-current-curves in planar lipid membranes at low, i.e. physiological, salt concentrations. Asymmetric ion current is due to unidirectional incorporation of porins into lipid membranes and vectorial features of the pore. Nonlinearity is a functional effect of the open channel and reflects the influence of electrostatic properties of the protein and the interaction between ions and charged amino acid residues.
Porins show voltage-dependent 'closing' and 'opening', i.e. stepwise break down and recurrence of ion current. Closing already occurs at relatively low voltages in Omp32 and Omp34 (porin of the related species Acidovorax delafieldii ). The closing phenomenon is completely different from nonlinearity of open channel conductance. Analysis of the relaxation (closing) kinetics revealed a complex behaviour. We derived a new model for pore relaxation kinetics which appears applicable to bacterial porins in general.
Molecular dynamics (MD) simulations illustrate that chloride ions are not simply diffusing through the water-filled pore but that they are attracted and guided by the electrostatic field of the protein. The anions are frequently immobilised (bound) at arginines and 'jump' from one charged residue to the next on their way through the channel. Since the amino acid residues are not fixed in place but dynamic the chloride ion is often 'handed over' to the next arginine in the channel. The ions are translocated rather than freely diffusing.
The bacterium D. acidovorans prefers organic acids as the major carbon source and grows particularly well on malate. It is thus conceivable that the porin is functionally adapted to translocate organic acids efficiently. Omp32 possesses a binding site for malate that co-crystallises with the porin. X-ray crystallography revealed the bound ligand in the constriction zone in coordination with the clustered arginines and a threonine residue. MD simulations show the dynamics of malate attraction from the outer and the periplasmic side, desolvation and the first steps of binding.
The studies on Omp32 illustrate that 16-stranded porins cannot generally be classified as "general diffusion pores" as often suggested.
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