All living cells must transport essential solutes across a semi-permeable membrane. A central pathway for the uptake of many different types of hydrophilic solutes is via membrane bound proteins that function as cation/solute cotransporters or symporters. These include uptake systems for sugars, amino acids, inorganic ions, and other small molecules. Human genetic diseases, such as type I cystinuria, involve defects in Na+/solute cotransporters. The broad aim of the proposed research is to understand the relationship between the biochemical structure of the H+/lactose permease found in Escherichia coli and its molecular mechanism of cotransporting H+ and lactose across the membrane. Our work utilizes FT-IR spectroscopy, site-directed mutagenesis, suppressor mutants, truncated versions of the lactose permease, and biochemical labeling methods. The first two aims use difference FT-IR spectroscopy to understand the mechanism of H+/lactose coupling and the nature of conformational changes associated with transport.
Aims three and four complement this spectroscopy work by the analysis of site-directed mutations and suppressor mutations. A fifth aim will determine if two halves of the lactose permease work together to facilitate lactose transport. And finally a sixth aim will determine if a conserved motif plays a role in the positioning of transmembrane segments in the lactose permease. Ultimately, it is hoped that the molecular features of the lactose permease will be of general significance so that our results can be extended to provide a better understanding of other cation/solute cotransport systems in bacteria, fungi, plant, and animal cells.
|Johnson, J L; Brooker, R J (2004) Control of H+/lactose coupling by ionic interactions in the lactose permease of Escherichia coli. J Membr Biol 198:135-46|
|Green, Aileen L; Hrodey, Heather A; Brooker, Robert J (2003) Evidence for structural symmetry and functional asymmetry in the lactose permease of Escherichia coli. Biochemistry 42:11226-33|
|Patzlaff, Jason S; Zhang, Jingyan; Brooker, Robert J et al. (2002) An isotope-edited FT-IR study of a symporter, the lactose permease. Biochemistry 41:7366-72|
|Green, A L; Brooker, R J (2001) A face on transmembrane segment 8 of the lactose permease is important for transport activity. Biochemistry 40:12220-9|
|Pazdernik, N J; Matzke, E A; Jessen-Marshall, A E et al. (2000) Roles of charged residues in the conserved motif, G-X-X-X-D/E-R/K-X-G-[X]-R/K-R/K, of the lactose permease of Escherichia coli. J Membr Biol 174:31-40|
|Patzlaff, J S; Brooker, R J; Barry, B A (2000) A reaction-induced fourier transform-infrared spectroscopic study of the lactose permease. A transmembrane potential perturbs carboxylic acid residues. J Biol Chem 275:28695-700|
|Green, A L; Anderson, E J; Brooker, R J (2000) A revised model for the structure and function of the lactose permease. Evidence that a face on transmembrane segment 2 is important for conformational changes. J Biol Chem 275:23240-6|
|Johnson, J L; Brooker, R J (1999) A K319N/E325Q double mutant of the lactose permease cotransports H+ with lactose. Implications for a proposed mechanism of H+/lactose symport. J Biol Chem 274:4074-81|
|Patzlaff, J S; Moeller, J A; Barry, B A et al. (1998) Fourier transform infrared analysis of purified lactose permease: a monodisperse lactose permease preparation is stably folded, alpha-helical, and highly accessible to deuterium exchange. Biochemistry 37:15363-75|
|Pazdernik, N J; Cain, S M; Brooker, R J (1997) An analysis of suppressor mutations suggests that the two halves of the lactose permease function in a symmetrical manner. J Biol Chem 272:26110-6|
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