Membrane proteins are important pharmaceutical targets and play essential roles in cells, and misfolding of membrane proteins is associated with human diseases, such as cystic fibrosis. The ability to develop therapies for membrane protein misfolding diseases is substantially limited by the lack of data on membrane protein folding and stability. In addition to the native states, we need to understand the conformations and stabilities of unfolded, partially folded, and misfolded membrane proteins. Compared to soluble proteins, whose folding has been studied for decades, the database of thermodynamic and mechanistic information on membrane protein folding is minute. Since biophysical folding studies can provide detailed access to sequence-structure- function relationships that no in vivo studies or crystal structures can provide, there is a need for additional quantitative studies of membrane proteins to better understand their physical origins. In this proposal we address this lack of information on membrane protein folding. We will study 8 outer membrane proteins, OmpX, OmpW, OmpA, PagP, OmpT, OmpLa, FadL and Omp85. Our work will double the number of unique membrane proteins whose folding has been interrogated in lipid bilayers. In the first aim, we will establish in vitro conditions under which they fold into membranes prepared from native lipid extracts. In a second aim, we will use kinetic and thermodynamic experiments employing SDS-PAGE, circular dichroism, fluorescence spectroscopy and analytical ultracentrifugation to determine the steps involved in folding and to ascertain why membrane proteins differ in their folding propensities. In the final aim we address how membrane proteins accommodate the introduction of ionizable mutations on the lipid facing surfaces of their membrane spanning regions. These experiments will provide insight into the mechanisms of how genetically-occurring ionizable group mutations cause malfunctions in human proteins.
Little is known about the dynamical process of membrane protein folding, and human diseases occur when membrane proteins misfold. An understanding of the factors that influence membrane protein stability and membrane protein folding will find practical utility in rationalizing the effects of genetic mutations that occur in membrane proteins. This knowledge will ultimately be useful in the design of therapeutic agents to combat disease.
|Gessmann, Dennis; Chung, Yong Hee; Danoff, Emily J et al. (2014) Outer membrane ?-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA. Proc Natl Acad Sci U S A 111:5878-83|
|Wu, Emilia L; Fleming, Patrick J; Yeom, Min Sun et al. (2014) E. coli outer membrane and interactions with OmpLA. Biophys J 106:2493-502|
|Fleming, Karen G (2014) Energetics of membrane protein folding. Annu Rev Biophys 43:233-55|
|Moon, C Preston; Zaccai, Nathan R; Fleming, Patrick J et al. (2013) Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm. Proc Natl Acad Sci U S A 110:4285-90|
|O'Neill, Maura J; Bhakta, Mehul N; Fleming, Karen G et al. (2012) Induced fit on heme binding to the Pseudomonas aeruginosa cytoplasmic protein (PhuS) drives interaction with heme oxygenase (HemO). Proc Natl Acad Sci U S A 109:5639-44|
|Fleming, Patrick J; Freites, J Alfredo; Moon, C Preston et al. (2012) Outer membrane phospholipase A in phospholipid bilayers: a model system for concerted computational and experimental investigations of amino acid side chain partitioning into lipid bilayers. Biochim Biophys Acta 1818:126-34|
|Moon, C Preston; Fleming, Karen G (2011) Using tryptophan fluorescence to measure the stability of membrane proteins folded in liposomes. Methods Enzymol 492:189-211|
|Moon, C Preston; Kwon, Sarah; Fleming, Karen G (2011) Overcoming hysteresis to attain reversible equilibrium folding for outer membrane phospholipase A in phospholipid bilayers. J Mol Biol 413:484-94|
|Moon, C Preston; Fleming, Karen G (2011) Side-chain hydrophobicity scale derived from transmembrane protein folding into lipid bilayers. Proc Natl Acad Sci U S A 108:10174-7|
|Danoff, Emily J; Fleming, Karen G (2011) The soluble, periplasmic domain of OmpA folds as an independent unit and displays chaperone activity by reducing the self-association propensity of the unfolded OmpA transmembrane ?-barrel. Biophys Chem 159:194-204|
Showing the most recent 10 out of 11 publications