Karen Fleming is a professor in the Thomas C. Jenkins Department of Biophysics.
We study the dynamical process of membrane protein folding.
Nearly one third of open reading frames encode proteins that live in membranes. These membrane proteins are essential for many biological functions including ion transport, molecular sorting, energy transduction, bacterial pathogenesis, and cell signaling. Over half of the drugs on the market today are thought to target membrane proteins, emphasizing their medical importance. Paradoxically, very little is known about how membrane proteins attain their native folds and how membrane proteins assemble into molecular complexes.
Research in my laboratory addresses fundamental biological questions concerning the formation of native structures in membrane proteins:
- How are outer membrane proteins sorted through the periplasm to the outer membranes?
- How does the sequence for a membrane protein specify the fold?
- What are the physical principles dictating membrane protein folding and interactions?
- What is the role of the lipid bilayer environment?
- How are mutations tolerated in membrane proteins?
- What principles for protein folding are similar between soluble and membrane proteins?
To address these questions our research efforts are focused on developing a physical understanding of membrane proteins, their folding and interactions, their specificity, their stability, their regulation, and their evolution. We carry out experiments that probe the chemistry of both helical and beta-barrel transmembrane proteins.
Our tools include all atom molecular simulations of membrane proteins in membranes, systems modeling using ordinary differential equations and an array of biophysical methods (sedimentation equilibrium, sedimentation velocity, small angle neutron scattering, light scattering, fluorescence spectroscopy, circular dichroism). Through these biophysical studies on a variety of membrane proteins with a diversity of folds we aim to elucidate governing principles for membrane protein folding and interactions.
Our most recent work has resulted in a novel hydrophobicity scale that describes the free energy of transfer of amino acid side chains from water to the bilayer in the context of a natively folded protein. We are currently determining the bilayer-depth dependence of these free energy changes, and we are exploring the generality of our results by experiments on a variety of different membrane proteins.
We are also interested in membrane protein folding kinetics and how membrane proteins are able to insert and fold into membranes and use biophysical tools to investigate folding time courses and conformations and the influences that cellular chaperones have on the kinetic pathways of membrane protein folding.
Fleming PJ & KG Fleming (2018) Hullrad: “Fast Calculations of Folded and Disordered Protein and Nucleic Acid Hydrodynamic Properties” Biophys J114: 856-869. PMID: 29490246.
KG Fleming(2018) “Taking Deterministic Control of Membrane Protein Monomer-dimer Measurements” J Gen Physiol 150: 181-183. PMID: 29343502.
Peterson JH, Plummer AM, Fleming KG& HD Bernstein (2017) “Selective Pressure for Rapid Membrane Integration Constrains the Sequence of Bacterial Outer Membrane Proteins” Mol Microbiol.106: 777-792. PMID: 28941249
Marx DC & KG Fleming (2017) “Influence of Protein Scaffold on Side-Chain Transfer Free Energies”Biophys J113: 597-604.PMID: 28793214
Mo GC, Ross B, Hertel F, Manna P, Yang X, Greenwald E, Booth C, Plummer AM, Tenner B, Chen Z, Wang Y, Kennedy EJ, Cole PA, Fleming KG, Palmer A, Jimenez R, Xiao J, Dedecker P & J Zhang (2017) “Genetically Encoded Biosensors for Visualizing Live-Cell Biochemical Activity at Super-resolution” Nat Methods 14: 427-434. PMID: 28288122
Fleming KG (2015) “A kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria” Phil Trans R Soc Lond B Biol Sci 370: PMID: 26370938
Danoff, E.J., Fleming, K.G. (2015) Aqueous, unfolded OmpA forms amyloid-like fibrils upon self-association PLoS One 10:e0132301.
Danoff, E.J., Fleming, K.G. (2015) Membrane defects accelerate outer membrane beta-barrel protein folding Biochemistry 54: 97-99.
Gessmann, D., Y.H. Chung, E.J. Danoff, A.M. Plummer, C.W. Sandlin, N.R. Zaccai and K.G. Fleming (2014) Outer Membrane beta-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA. Proc. Natl. Acad. Sci. USA 111:5878-83.
Wu, E.L., P.J. Fleming, M.S. Yeom, G. Widmalm, J.B. Klauda, K.G. Fleming and W. Im (2014) E coli Outer Membrane and Interactions with OmpLA Biophys. J 106:2493-502.
Fleming, K.G. (2014) Energetics of Membrane Protein Folding Ann Rev Biophys. Invited Article 43:233-55.
Moon, C.P., N.R. Zaccai, P.J. Fleming, D. Gessmann and K.G. Fleming. (2013) Membrane protein thermodynamic stability may be the energy sink for sorting in the periplasm. Proc. Natl. Acad. Sci. USA 110:4285.
Buchanan, S.K., Y. Yamashita, and K.G. Fleming. (2012) Structure and folding of outer membrane proteins. in Comprehensive Biophysics, eds. E.H. Engelman and L.K. Tamm, Oxford: Academic Press Vol. 5: 139-163.
Fleming, P.J., J.A. Freites, C.P. Moon, D.J. Tobias, and K.G. Fleming. (2011) 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. BBA Biomembranes 1818:126-134.
Danoff, E.J., and K.G. Fleming. (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 b-barrel. Biophys. Chem. (in press)
Moon, C.P., and K.G. Fleming. (2011) From the Cover: Side-chain hydrophobicity scale derived from transmembrane protein folding in lipid bilayers. Proc. Natl. Acad. Sci. USA 108:10174-10177.
Moon, C.P., S. Kwon, and K.G. Fleming. (2011) Overcoming hysteresis to attain reversible equilibrium folding for outer membrane phospholipase A in phospholipid bilayers. J. Mol. Biol. 413:484-494.
Moon, C.P., and K.G. Fleming. (2011) Using tryptophan fluorescence to measure the stability of membrane proteins folded in liposomes. Methods Enzymol. 492:189-211.
Ebie Tan, A., N.K. Burgess, J.D. Marold, D.S. DiAndrade, and K.G. Fleming. (2010) Self association of unfolded outer membrane proteins. Macromol. Biosci. 10:763-767.
Pang, T., C.G. Savva, K.G. Fleming, D.K. Struck, and R. Young. (2009) Structure of the lethal phage pinhole. Proc. Natl. Acad. Sci. 106:18966-18971.
Burgess, N.K., T.P. Dao, A.M. Stanley, and K.G. Fleming. (2008) Beta-barrel proteins that reside in the E. coli outer membrane in vivo demonstrate varied folding behavior in vitro. J. Biol. Chem. 283:26748-26758.
Mackenzie, K.R., and K.G. Fleming. (2008) Association energetics of membrane spanning alpha-helices. Curr. Opin. Struct. Biol. 18:412-419.
Stanley, A.M., and K.G. Fleming. (2008) The process of folding proteins into membranes: challenges and progress. Arch. Biochem. Biophys. 469:46-66.
Burgess, N.K., A.M. Stanley, and K.G. Fleming. (2008) Determination of membrane protein molecular weights and association equilibrium constants using sedimentation equilibrium and sedimentation velocity. Methods Cell Biol. 84:181-211.
Duong, M.T., T.M. Jaszewski, K.G. Fleming, and K.R. Mackenzie. (2007) Changes in apparent free energy of helix-helix dimerization in a biological membrane due to point mutations. J. Mol. Biol. May 18 [Epub ahead of print]
Stanley, A.M., and K.G. Fleming. (2007) The role of a hydrogen bonding network in the transmembrane beta-barrel OMPLA. J. Mol. Biol. 370:912-924.
Stanley, A.M., A.M. Treubrodt, P. Chuawong, T.L. Hendrickson, and K.G. Fleming. (2007) Lipid chain selectivity by outer membrane phospholipase A. J. Mol. Biol. 366:461-468.
Ebie, A.Z., and K.G. Fleming. (2007) Dimerization of the erythropoietin receptor transmembrane domain in micelles. J. Mol. Biol. 366:517-524.
Stanley, A.M., P. Chuawong, T.L. Hendrickson, and K.G. Fleming. (2006) Energetics of outer membrane phospholipase A (OMPLA) dimerization. J. Mol. Biol. 358:120-131.
Kroch, A.E., and K.G. Fleming. (2006) Alternate interfaces may mediate homomeric and heteromeric assembly in the transmembrane domains of SNARE proteins. J. Mol. Biol. 357:184-94.
Fleming, K.G. (2005) Analysis of membrane proteins using analytical ultracentrifugation. (Invited book chapter) Analytical Ultracentrifugation, Techniques and Methods, (Scott DJ, Harding SE, & Rowe AJ, Eds.) Royal Society of Chemistry Publishing, Cambridge, UK.
Stanley, A.M. and K.G. Fleming. (2005) The transmembrane domains of the ErbB receptors do not dimerize strongly in micelles. J. Mol. Biol. 347:759-772.
Kobus, F.J. and K.G. Fleming. (2005) The GxxxG-containing transmembrane domain of the CCK4 oncogene does not encode preferential self-interactions. Biochemistry 44:1464-1470.
Doura, A.K. and K.G. Fleming. (2004) Complex interactions at the helix-helix interface stabilize the glycophorin A transmembrane dimer. J. Mol. Biol. 343:1487-1497.
Raasi, S., I. Orlov, K.G. Fleming and C.M. Pickart. (2004) Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A. J. Mol. Biol. 341:1367-1379.
Doura, A.K., F.J. Kobus, L. Dubrovsky, E. Hibbard and K.G. Fleming. (2004) Sequence context modulates the stability of a GxxxG mediated transmembrane helix-helix dimer. J. Mol. Biol. 341:991-998.
Fleming, K.G., C.C. Ren, A.K. Doura, F.J. Kobus, M.E. Eisley and A.M. Stanley. (2004) Thermodynamics of glycophorin A transmembrane helix-helix association in C14 betaine micelles. Biophys. Chem. 108:43-49.
Fleming, K.G. (2002) Standardizing the free energy change of transmembrane helix-helix interactions. J. Mol. Biol. 323:563-571.
Vergis, J.M., K.G. Bulock, K.G. Fleming and G.P. Beardsley. (2001) Human AICAR transformylase/IMP cyclohydrolase: A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity. J. Biol. Chem. 276:7727-7733.
Trombetta, E.S., K.G. Fleming and A. Helenius. (2001) Quaternary and domain structure of glycoprotein processing glucosidase II. Biochemistry 40:10717-10722.
Fleming, K.G. and D.M. Engelman. (2001) Specificity in transmembrane helix-helix interactions defines a hierarchy of stability for sequence variants. Proc. Natl. Acad. Sci. USA 98:14340-14344.
Fleming, K.G., and D.M. Engelman. (2001) Computation and mutagenesis suggest a right-handed structure for the synaptobrevin transmembrane dimer. Proteins 45:313-317.