Phale, P SP SPhaleSchirmer, TTSchirmerPrilipov, AAPrilipovKUO-LONG LOUHardmeyer, AAHardmeyerRosenbusch, J PJ PRosenbusch2024-01-252024-01-251997-06-240027-8424https://scholars.lib.ntu.edu.tw/handle/123456789/638888In the homotrimeric OmpF porin from Escherichia coli, each channel is constricted by a loop protruding into the beta-barrel of the monomer about halfway through the membrane. The water-filled channels exist in open or closed states, depending on the transmembrane potential. For the transition between these conformations, two fundamentally different mechanisms may be envisaged: a bulk movement of the constriction loop L3 or a redistribution of charges in the channel lumen. To distinguish between these hypotheses, nine mutant proteins were constructed on the basis of the high-resolution x-ray structure of the wild-type protein. Functional changes were monitored by measuring single-channel conductance and critical voltage of channel closing. Structural alterations were determined by x-ray analysis to resolutions between 3.1 and 2.1 A. Tethering the tip of L3 to the barrel wall by a disulfide bridge (E117C/A333C), mobilizing L3 by perturbing its interaction with the barrel wall (D312N, S272A, E296L), or deleting residues at the tip of the loop (Delta116-120) did not alter appreciably the sensitivity of the channels to an external potential. A physical occlusion, due to a gross movement of L3, which would cause the channels to assume a closed conformation, can therefore be excluded.enβ-barrel | Outer membrane protein | Single- channel conductance | Site-directed mutagenesis | X-ray structurejournal article10.1073/pnas.94.13.674191926352-s2.0-0031001514https://api.elsevier.com/content/abstract/scopus_id/0031001514