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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
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Johns Hopkins University
Schools of Arts and Sciences
United States
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Fleming, Patrick J; Fleming, Karen G (2018) HullRad: Fast Calculations of Folded and Disordered Protein and Nucleic Acid Hydrodynamic Properties. Biophys J 114:856-869
Fleming, Karen G (2018) Taking deterministic control of membrane protein monomer-dimer measurements. J Gen Physiol 150:181-183
Lessen, Henry J; Fleming, Patrick J; Fleming, Karen G et al. (2018) Building Blocks of the Outer Membrane: Calculating a General Elastic Energy Model for ?-Barrel Membrane Proteins. J Chem Theory Comput 14:4487-4497
Danoff, Emily J; Fleming, Karen G (2017) Novel Kinetic Intermediates Populated along the Folding Pathway of the Transmembrane ?-Barrel OmpA. Biochemistry 56:47-60
Mo, Gary C H; Ross, Brian; Hertel, Fabian et al. (2017) Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution. Nat Methods 14:427-434
Peterson, Janine H; Plummer, Ashlee M; Fleming, Karen G et al. (2017) Selective pressure for rapid membrane integration constrains the sequence of bacterial outer membrane proteins. Mol Microbiol 106:777-792
Marx, Dagen C; Fleming, Karen G (2017) Influence of Protein Scaffold on Side-Chain Transfer Free Energies. Biophys J 113:597-604
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
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
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

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