Structural and Energetic Principles of Helical Membrane Proteins
Genome sequencing efforts are revealing that perhaps as many as 20–40% of open reading frames in complex organisms may encode proteins containing at least one helical transmembrane segment. Contrasting with the approaching tidal wave of helical membrane proteins is the fact that our understanding of the sequence-structure-function relationships for membrane proteins lags far behind that of soluble proteins. This paradox emphasizes the extensive biophysical and structural work that remains to be done in the field of helical membrane proteins.
Our research is aimed at elucidating structural and energetic principles of membrane proteins. We are especially focused on understanding the structural and energetic basis of transmembrane helix-helix recognition. Our lab takes a multidisciplinary approach to address scientific questions employing molecular biology techniques, structural studies, thermodynamic investigations and computational analysis and modeling.
Currently our research projects include:
- Structural and energetic dissection of the glycophorin A transmembrane dimer. A paradigm for transmembrane helix-helix interactions, we are probing the structural and energetic effects on dimerization of single and multiple point mutants. Our interaction studies on glycophorin A suggest the idea that specificity in helix-helix interactions for this protein may be independent of the hydrophobic environment.
- Transmembrane interactions in SNARE fusion proteins. We are using computational modeling and thermodynamics to probe the transmembrane helix-helix interactions of synaptobrevin and other SNARE proteins. Our studies on the dimerization of synaptobrevin offer an opportunity for protein engineering and design of a membrane protein as a means to understanding its structural stability.
- 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.