Ongoing Projects
For the ongoing projects we use a variety of methodologies, including molecular biological (mutagenesis), biochemical (protein purification and reconstitution, production of nanodiscs), physiological (transport assays), biophysical (electrophysiology, photolysis kinetics, fluorescence, luminescence resonance energy transfer, light scattering), and structural (X-ray crystallography, cryo-electron microscopy) techniques.
Structure/function/inhibition of connexin hemichannels
Our connexin-related projects focus on hemichannels. We are interested in the structure of the hemichannels, their mechanism of regulation, and the discovery of new inhibitors for treatment of disorders associated with abnormal hemichannel activation.
Selected Publications:
| Fiori MC, Cuello LG, Altenberg GA. A Simple assay to evaluate the function of human connexin hemichannels expressed in Escherichia coli that can be used for drug discovery and mutant analysis. Curr Protoc Pharmacol 87(1):e68, 2019.doi: 10.1002/cpph.68. PubMed PMID: 31756040; PubMed Central PMCID: PMC6876698. |
| AlFindee MN, Subedi YP, Fiori MC, Krishnan S, Kjellgren A, Altenberg GA, Chang CT. Inhibition of connexin hemichannels by new amphiphilic aminoglycosides without antibiotic activity. ACS Med Chem Lett 9(7):697-701, 2018.doi: 10.1021/acsmedchemlett.8b00158. PubMed PMID: 30034603; PubMed Central PMCID: PMC6047172. |
| Fiori MC, Figueroa V, Zoghbi ME, Saéz JC, Reuss L, Altenberg GA. Permeation of calcium through purified connexin 26 hemichannels. J Biol Chem 287(48):40826-34, 2012.doi: 10.1074/jbc.M112.383281. PubMed PMID: 23048025; PubMed Central PMCID: PMC3504794. |
| Bao X, Lee SC, Reuss L, Altenberg GA. Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC. Proc Natl Acad Sci USA 104(12):4919-24, 2007.PubMed PMID: 17360407; PubMed Central PMCID: PMC1817834. |
| Chen Y, Deng Y, Bao X, Reuss L, Altenberg GA. Mechanism of the defect in gap-junctional communication by expression of a connexin 26 mutant associated with dominant deafness. FASEB J 19(11):1516-8, 2005.PubMed PMID: 16009703. |
| Bao X, Reuss L, Altenberg GA. Regulation of purified and reconstituted connexin 43 hemichannels by protein kinase C-mediated phosphorylation of serine 368. J Biol Chem 279(19):20058-66, 2004.PubMed PMID: 14973142. |
| Bao X, Altenberg GA, Reuss L. Mechanism of regulation of the gap junction protein connexin 43 by protein kinase C-mediated phosphorylation. Am J Physiol Cell Physiol 286(3):C647-54, 2004.PubMed PMID: 14602580. |
Molecular mechanism of ATP-binding cassette (ABC) exporters
Our general goal is to elucidate the conformational changes of ABC exporters at the molecular level during the transport cycle. In particular, our focus is on the movements of different domains in real time with Angstrom resolution with the proteins reconstituted in nanodiscs membranes.
Selected Publications:
| Arana MR, Fiori MC, Altenberg GA. Functional and structural comparison of the ABC exporter MsbA studied in detergent and reconstituted in nanodiscs. Biochem Biophys Res Commun 512(3):448-52, 2019.doi: 10.1016/j.bbrc.2019.03.069. Epub 2019 Mar 20. PubMed PMID: 30902387. |
| Zoghbi ME, Mok L, Swartz DJ, Singh A, Fendley GA, Urbatsch IL, Altenberg GA. Substrate-induced conformational changes in the nucleotide-binding domains of lipid bilayer-associated P-glycoprotein during ATP hydrolysis. J Biol Chem 292(50):20412-24, 2017.doi: 10.1074/jbc.M117.814186. PubMed PMID: 29018094; PubMed Central PMCID: PMC5733581. |
| Zoghbi ME, Altenberg GA. Luminescence resonance energy transfer spectroscopy of ATP-binding cassette proteins. Biochim Biophys Acta Biomembr 1860(4):854-67, 2018.doi: 10.1016/j.bbamem.2017.08.005. PubMed PMID: 28801111. |
| Zoghbi ME, Cooper RS, Altenberg GA. The lipid bilayer modulates the structure and function of an ATP-binding cassette exporter. J Biol Chem 291(9):4453-61, 2016.doi: 10.1074/jbc.M115.698498. PubMed PMID: 26725230; PubMed Central PMCID: PMC4813473. |
| Swartz DJ, Mok L, Botta SK, Singh A, Altenberg GA, Urbatsch IL. Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance. Biosci Rep 34(3). pii: e00116, 2014.doi: 10.1042/BSR20140062. PubMed PMID: 24825346; PubMed Central PMCID: PMC4069687. |
| Zoghbi ME, Altenberg GA. Hydrolysis at one of the two nucleotide-binding sites drives the dissociation of ATP-binding cassette nucleotide-binding domain dimers. J Biol Chem 288(47):34259-65, 2013.doi: 10.1074/jbc.M113.500371. PubMed PMID: 24129575; PubMed Central PMCID: PMC3837166. |
| Zoghbi ME, Fuson KL, Sutton RB, Altenberg GA. Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain. J Biol Chem 287(6):4157-64, 2012.doi: 10.1074/jbc.M111.318378. PubMed PMID: 22158619; PubMed Central PMCID: PMC3281709. |
Design, production and characterization of polymer nanodiscs
In recent years, nanodiscs have been introduced as a new approach for the study of membrane proteins in a lipid bilayer. They consist of two molecules of a membrane scaffold protein that encase a small patch of lipid bilayer. Our goal is to design and produce new types of nanodiscs where the membrane scaffold proteins and/or the lipid bilayer are replaced with synthetic copolymers, yielding new nanostructures with increased stability and chemical versatility.
Selected Publications:
| Fiori MC, Jiang Y, Zheng W, Anzaldua M, Borgnia MJ, Altenberg GA, Liang H. Polymer nanodiscs: Discoidal amphiphilic block copolymer membranes as a new platform for membrane proteins. Sci Rep 7(1):15227, 2017.doi: 10.1038/s41598-017-15151-9. PubMed PMID: 29123151; PubMed Central PMCID: PMC5680229. |
| Fiori MC, Jiang Y, Altenberg GA, Liang H. Polymer-encased nanodiscs with improved buffer compatibility. Sci Rep 7(1):7432, 2017.doi: 10.1038/s41598-017-07110-1. PubMed PMID: 28785023; PubMed Central PMCID: PMC5547149. |