STABILITY OF TRIMERIC OMPF PORIN - THE CONTRIBUTIONS OF THE LATCHING LOOP L2

The channel-forming protein OmpF porin from Escherichia coli spans the bacterial outer membrane. Each of the three monomers comprises a holl ow, 16-stranded beta-barrel. These are associated to homotrimers which are unusually stable, due mostly to hydrophobic interactions between the beta-barrels. In addition, a loop, L2 connects one subunit to its neighbor by latching into its channel. Residue E71 on loop 2 is integr ated into an ionic network and forms salt bridges and hydrogen bonds w ith R100 and R132 on the channel wall in the adjacent subunit. To exam ine these contributions quantitatively, six single-site, two double, a nd one deletion mutant were constructed on the basis of the atomic coo rdinates of the protein. Differential scanning calorimetric analysis s howed that the salt-bridge, E71-R100, contributes significantly to tri mer stability: the substitution E71Q causes a decrease of the transiti on temperature from 72 to 48 degrees C, with Delta H-cal diminishing f rom 430 to 201 kcal mol(-1). A nearby substitution in the loop, D74N, has lesser effects on thermal stability, while the deletion in L2 (Del ta 69-77) has an effect comparable to that of E71Q. X-ray structure an alysis to 3.0 Angstrom resolution revealed only local structural diffe rences in the mutants except for the substitution R100A, where another residue, R132, is found to fill the gap left by the truncated side ch ain of A100. Functional assays in planar lipid bilayers show significa ntly increased cation selectivities if the charge distribution was aff ected.