Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa (original) (raw)

The ~racfion ~f phospha~ ~ns wi|h the Pseudomonas aerug~osa anOn-specific prote~ P channd was probe~ The single<hannd conduc~nce of proton P incorpor~ed into ~anar lipid bilayer membranes in the presence of ~3 M H2PO ~ was shown to be ~0 pS, demons~afing ~at proton P channds ~wed the permeation of phosph~e. When large numbe~ of proton P channds were incorporated into lipid ~y~r membranes in ~e presence of 40 mM Cl-, ad~tion of small concen~afions of phosph~e res~d in reduction of macrosco~c Cl-conductance ~ a dose-(and pH-) dependent fas~o~ Th~ allowed c~culation of an I~ v~ue of e.~ ~46 mM at pH %~ suggesting that the affini~ of proton P for i~ norm~ subs~a~ phosphate was at least 60-100~d gre~er than the affini~ of the channd for other ~ns such as c~odde. Pyrophospha~ and the phospha~ an~ogu~ arsenate, ~so ~d macrosco~c CI-conductance through protein P with I~ v~ues ~ pH ~0 of ~9 mM and 1~ raM, respectivdy. To probe the nature ~f ~e phosph~e ~n~ng ~te, the ~amino groups of availa~e ~ne re,dues of ~rotein P were chem~ modified. Ace~lafion and carbam~ation w~ch produced uncharge~ mottled ~nes des~oyed both the a~on ~.g. CI-) ~n~ng ~ and the phosph~e ~n~ng ~ as de~rmined by ~n~channd expedmen~ and macrosco~c conduc~nce ~fion expedments respectively. Neve~hde~, the modified proteins still ret~ned thor Wimefic configuration and thor a~liff ~o recon~itu~ sin~e channds ~ lipid bil~y~r membranes. M~hy~tio~ whkh allowed re~nfion of the charge on the mottled ~sine res~ue~ increased the K d of the channd for CI-33-f~d and the I~ for phosph~e inhibition of macrosco~c CI-conductance Z5-4-f~& A m~ec~ar model for ~e phosph~e ~n~ng si~ of the protein P channd is presente~