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Abstract
Escherichia coli accumulates maltooligosaccharides via the maltose transport system. Five proteins are involved in transport: LamB, an outer membrane diffusion pore; the maltose binding protein (MBP), a periplasmic protein that binds substrate and interacts with the inner membrane complex, and the MalF, MalG and MalK proteins that form the inner membrane complex (MalFGK$\sb2$) that facilitates the transport of substrate into the cytoplasm. The energy for the transport process is derived from the binding and hydrolysis of ATP.
In the first chapter of this thesis, a vesicle transport system is used to test the predictions made by a mathematical model composed of equations that represent individual steps in the transport process. By comparing the known behavior of the maltose transport system with a mathematical model, it is proposed that both unliganded and liganded MBP must interact with the inner membrane complex. Experimental results support this hypothesis. It was also possible to estimate the affinities of both forms of MBP for the inner membrane complex.
In the last two chapters of this thesis, two mutants of the maltose transport system that transport lactose are characterized. The first mutant, MalF515, contains a leucine to tryptophan alteration at codon 334 of MalF, implicating this residue as playing a role in determining binding specificity. Surprisingly, this mutant requires MBP for the transport of lactose although MBP is incapable of binding lactose. Thus, this mutant supports the model that unliganded MBP interacts with the inner membrane complex and indicates that MBP plays a role in triggering transport. The second mutant, MalF540, has a glutamine at codon 99 of MalF changed to an amber stop codon. This mutant displays constitutive ATPase activity and cannot transport maltose. Transport data obtained with this mutant as well as other amber mutants of MalF suggest that the transmembrane helices of MalG are sufficient to form a transport path for substrate and that regions distal to codon 99 of MalF are important for regulating ATPase activity.
The implications of the data on the function of the wild-type maltose transporter, particularly the contributions of the transmembrane helices in forming a transport path for substrate and the possible roles MBP plays in the transport cycle, are discussed.
