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.
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.
|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|
|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|
|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|
|McDonald, Sarah K; Fleming, Karen G (2016) Negative Charge Neutralization in the Loops and Turns of Outer Membrane Phospholipase A Impacts Folding Hysteresis at Neutral pH. Biochemistry 55:6133-6137|
|Plummer, Ashlee M; Fleming, Karen G (2016) From Chaperones to the Membrane with a BAM! Trends Biochem Sci 41:872-82|
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