Gene, 53 (1987) 287-292 Elsevier 287 CEN 01969 Sequence determinants in the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ZumB gene of Escherichia coli influencing the binding and pore selectivity of maltoporin (R~ombin~t DNA; m~tod~xt~n binding: 1: : Tn9 phage; ammo acid sequence; starch amity} Hans-Georg Heine, Joanna Kyngdon * and Thomas Ferenci Depffrt~e~t o~~~c~obiolo~, University of Sy dney , Sy dney , N.S. W . 2006 (A~tr~Zi~~ Received 12 September 1986 Revised 16 January 1987 Accepted 20 January 1987 SUMMARY Maltoporin (LamB protein) is a m~to-oligosacch~de-selective pore protein in the outer membrane of ~~c~en~~iu coli. The genetic basis of binding and transport specificity was investigated through cloning, mapping and sequencing lam3 genes from seven independent mutants with various changes in maltodextrin binding affinities; these mutants were unchanged in binding phage a. Single amino acid substitutions specifically resulting in maltodextrin affinity changes were as follows: Arg8 -+ His in two independent mutants resulted in much reduced affinity for all ligands and a smaller pore no longer selective for maltodextrins. A Trp74 + Arg substitution resulted in a lower affinity for starch, a slight increase in maltose affinity but no striking pore changes. An Arg82 + Ser resulted in lowered maltodextrin affinity, but increased a.fTinity for sucrose in both binding and pore function. A Tyr 118 + Phe resulted in a higher affinity for both starch and maltose, a slightly larger pore and increased transport of maltohexaose by the pores. Asp121 --t Gly in two independent isolates resulted in a higher affinity for large dextrins and a marginally larger pore. These results suggest that the m~todext~n-selective functions reside in the N-terminal sequence of m~topor~ and are separate from the phage ,I binding domains. C~~re~~o~~e~ce to: Dr. T. Ferenci (until 1 Aug. 1987): Depart- ment of Molecular Biology, Princeton University, Princeton, NJ 08540 (U.S.A.) Tel. (609)452-5900; (after 1 Aug. 1987): Depart- ment of Microbiology, University of Sydney, Sydney, N.S.W. 2006 (Australia) Tel. (02)692-4271. * Present address: Biotechnology (Australia) Ltd., Barcoo St., Rosevitle, N&W. 2069 (Australia) Tel. (02)407-0271. Abbreviations: aa, amino acid(s); Ap, ampicillin; bp, base pair(s); Cm, chlor~phenicol; A, deletion; LB, Luria broth; nt, nucleotide(s); p.f.u., plaque-forming units; R, resistance, resis- tam; ‘, sensitive; Tc, tetracycline; Tn, transposon; wt, wild-type; : : , novel joint. 0378-I 119/87/$03.50 Q 1987 El sevier Science Publishers B.V.(Biomedical Divisio n)