Membrane proteins play essential roles in many cellular processes. The overall goal of the proposed research is to understand the physical basis of 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079440-07
Application #
8970707
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2009-06-01
Project End
2017-11-30
Budget Start
2015-12-01
Budget End
2016-11-30
Support Year
7
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21205
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
Plummer, Ashlee M; Fleming, Karen G (2016) From Chaperones to the Membrane with a BAM! Trends Biochem Sci 41:872-882
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
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

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