Membrane proteins play essential roles in many cellular processes. The overall goal of the proposed research is to understand the physical basis the protein folding process and the biophysical basis of membrane protein structures. At present, neither of these is well understood for membrane proteins. Thermodynamically, our work addresses the critical role that hydrophobicity plays in membrane protein folds. In the previous granting period, we developed a novel hydrophobicity scale that measures side-chain transfer free energies from water to the membrane center using a real bilayer and a real, folded membrane protein. Based on this achievement, we now propose to test the generality of this scale (1) By measuring side-chain transfer free energies using distinct membrane protein scaffolds;(2) By determining how extent-of-burial in the bilayer modulates water to bilayer transfer free energies;and (3) By engineering of our protein scaffold for measurements as a function of pH to address how the energetic consequences of ionizable group mutations vary with charge state. Kinetically, we discovered in the previous grant period that E. coli lipid head groups may act as energetic potentials that sort membrane proteins away from the wrong (inner) membranes and towards the correct (outer) membrane locations. We propose in a fourth aim to dissect the biophysical basis for this sorting by determining the kinetic lifetimes and conformations and activation energies to folding induced by E. coli-containing lipid head groups.

Public Health Relevance

Membrane protein misfolding causes many diseases that are difficult to cure. To find better drugs for these conditions, this basic science project aims at a better understanding of the dynamical process that a membrane protein takes to fold to its native conformation and of the physical forces essential for maintaining its structue. The knowledge gained from this research will eventually lead to the design of therapeutics to combat protein misfolding diseases, will be useful in the computational modeling of protein structures and of drug-binding to protein structures, and in the design of proteins with novel functions.

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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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

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