Date of Award


Degree Type


Degree Name

Master of Science (MS)


Biological Science

Committee Chair

Jue Chen

Committee Member 1

Frederick S. Gimble

Committee Member 2

Christine A. Hrycyna

Committee Member 3

Cynthia V. Stauffacher

Committee Member 5

David M. Umulis


The ATP-binding cassette (ABC) transporter superfamily is one of the largest families of transport proteins. The ABC transporters are responsible for selective permeability of solute across membranes energized by ATP hydrolysis, which occurs in all domains of life. Maltose transporter is an ABC importer that mediates maltose/maltodextrin uptake in bacteria and archaea. It is identified as an essential virulence factor in pathogenic species of Streptococcis pyogenes and Vibrio cholera (1, 2). Escherichia coli maltose transporter is a well-characterized system with crystal structures and exclusive biochemical studies available. Knowledge of the E. coli maltose transport mechanism will lead to a better understanding of maltose transporters in the other species as well as other classes of ABC importers.

The goal of this study was to characterize the mechanism by which the substrate binding protein stimulates the transport cycle. To study how MBP initiates the transport process, MBP was spin-labeled at a single residue of the N-lobe and the mobility surrounding the labeling site was detected by Electron Paramagnetic Resonance. The

second periplasmic loop of MalF, P2, was found to bind with MBP N-lobe throughout the catalytic cycle, suggesting that P2 is involved in the recruitment of MBP, as corroborated by previous studies. In addition, a lack of mobility change at the interface of the MBP N-lobe and P2 suggested that the 5 Å decrease in the distance between MBP and MalF-P2 upon addition of ATP or maltose from previous crosslink study is likely due to a rotation between MBP and P2. In comparison, maltose decreased the mobility between MalG P3 and MBP at residue 41, suggesting that P3s' conformation may be sensitive to maltose binding.

Among the periplasmic loops of the transmembrane domains, the MalF-P2 loop shares an extraordinarily large interface with MBP N-lobe. It is possible that P2 has essential roles other than recruiting MBP. However, this is questioned by the fact that P2 is missing in gram-positive and archaea maltose transporters. To test the functional roles of this region, P2 was truncated and the maltose transporter mutant without P2 was tested through in vivo maltose transport assays and then in vitro ATPase activity assay in nanodiscs. Results demonstrate that the interaction between P2 and MBP is essential to maltose transport. To further investigate the functions of P2, a P2-truncated mutant of a binding-protein independent mutant (F500) was constructed. The MacConkey assay result shows that this mutant can transport maltose in vivo, indicating that P2 is likely to be involved only in recruiting MBP.