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.
|Zaccai, Nathan R; Sandlin, Clifford W; Hoopes, James T et al. (2016) Deuterium Labeling Together with Contrast Variation Small-Angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins. Methods Enzymol 566:159-210|
|Costello, Shawn M; Plummer, Ashlee M; Fleming, Patrick J et al. (2016) Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins. Proc Natl Acad Sci U S A 113:E4794-800|
|Plummer, Ashlee M; Fleming, Karen G (2016) From Chaperones to the Membrane with a BAM! Trends Biochem Sci 41:872-82|
|McDonald, Sarah K; Fleming, Karen G (2016) Aromatic Side Chain Water-to-Lipid Transfer Free Energies Show a Depth Dependence across the Membrane Normal. J Am Chem Soc 138:7946-50|
|Fleming, Patrick J; Patel, Dhilon S; Wu, Emilia L et al. (2016) BamA POTRA Domain Interacts with a Native Lipid Membrane Surface. Biophys J 110:2698-709|
|Sandlin, Clifford W; Zaccai, Nathan R; Fleming, Karen G (2015) Skp Trimer Formation Is Insensitive to Salts in the Physiological Range. Biochemistry 54:7059-62|
|Fleming, Karen G (2015) A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria. Philos Trans R Soc Lond B Biol Sci 370:|
|Danoff, Emily J; Fleming, Karen G (2015) Membrane defects accelerate outer membrane Î²-barrel protein folding. Biochemistry 54:97-9|
|Danoff, Emily J; Fleming, Karen G (2015) Aqueous, Unfolded OmpA Forms Amyloid-Like Fibrils upon Self-Association. PLoS One 10:e0132301|
|Plummer, Ashlee M; Gessmann, Dennis; Fleming, Karen G (2015) The Role of a Destabilized Membrane for OMP Insertion. Methods Mol Biol 1329:57-65|
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