This work is aimed at developing greater understanding of the dynamic molecular properties, as well as the structure, function, and association, of model and biological membranes, bioactive peptides, and membrane proteins that underlie important biological processes or are implicated in health disorders, and the latest one and two-dimensional electron-spin resonance (ESR) technologies will be employed to better address these issues. Specific projects include the following: The study of the dynamic domain structure of the plasma membrane in live RBL-2H3 (and other related cells) and plasma membrane vesicles (PMV) will be employed to correlate the change in domain structure with signaling by the IgE receptor after activation by antigen to test the hypothesis that receptor activation is modulated by the domain structure of the surrounding lipids. This study will be based on recent results, which showed the existence of Liquid-ordered and Liquid-disordered domains. The effects of fusion peptides, such as from hemagglutinin of influenza virus (wt20), and curvature- inducing proteins on membrane ordering will be studied to test the hypotheses that increased bilayer ordering is associated with more robust membrane fusion and that membrane curvature-inducing proteins will induce changes in the head group ordering of negatively charged lipids, based on the observation that wt20 increases the ordering of the lipid headgroups. By means of the powerful pulsed-dipolar ESR spectroscopy (PDS) cultivated by the Freed group for studying membrane protein structure and aggregation, the aggregation number and structural changes of spin-labeled wt20 peptide as a function of wt20 concentration will be determined, such as structural changes will be correlated with the changes in lipid ordering profile in the membrane. By means of multi-frequency ESR and PDS, the effects of hydrophobic mismatch between peptide length and lipid bilayer thickness, such as tilting of trans-membrane helices and peptide aggregation, will be studied for synthetic WALP and KALP peptides, based on previously developed methods and results. Additional studies by PDS will be directed to the determination of structures of large membrane proteins and their complexes. This includes spin-labeled BAR domain-containing proteins to determine the conformational changes that occur upon membrane binding. A second study is that of membrane-bound conformations of human synucleins including their Parkinson's disease (PD) linked-mutants to test our hypothesis that alpha- synuclein (aS) may exist in vivo in both extended helix and U-shaped form, each of which have already been demonstrated in model systems. A third study is to determine the structure of intact chemoreceptors and the conformational changes they undergo upon activation. The focus will be on the complex that the four protein unit, CheA/CheW, forms with the receptor. These studies will involve extensive collaborations with leading research groups. Possible clinical applications include detection of membrane changes during immune response, prevention of viral entry, an neurological disorders (including PD).

Public Health Relevance

We are studying structures of proteins and cell membranes, as well as the way proteins interact with other components of cell membranes, such as lipids and cholesterols. Our study will focus on understanding mechanisms by which cells communicate with each other through exchange of cellular components. Disorder in the structure of the proteins and the manner of cell communication may lead to allergic conditions, arteriosclerosis, Parkinson's and other diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB003150-30
Application #
8212422
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Sastre, Antonio
Project Start
1978-12-01
Project End
2013-04-30
Budget Start
2012-02-01
Budget End
2013-04-30
Support Year
30
Fiscal Year
2012
Total Cost
$412,448
Indirect Cost
$153,047
Name
Cornell University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Lai, Alex L; Freed, Jack H (2014) HIV gp41 fusion peptide increases membrane ordering in a cholesterol-dependent fashion. Biophys J 106:172-81
Georgieva, Elka R; Xiao, Shifeng; Borbat, Peter P et al. (2014) Tau binds to lipid membrane surfaces via short amphipathic helices located in its microtubule-binding repeats. Biophys J 107:1441-52
Airola, Michael V; Sukomon, Nattakan; Samanta, Dipanjan et al. (2013) HAMP domain conformers that propagate opposite signals in bacterial chemoreceptors. PLoS Biol 11:e1001479
Georgieva, Elka R; Borbat, Peter P; Ginter, Christopher et al. (2013) Conformational ensemble of the sodium-coupled aspartate transporter. Nat Struct Mol Biol 20:215-21
Yu, Long-Xi; Dzikovski, Boris G; Freed, Jack H (2012) A protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke. J Vis Exp :e3406
Chiang, Yun-Wei; Costa-Filho, Antonio J; Baird, Barbara et al. (2011) 2D-ELDOR study of heterogeneity and domain structure changes in plasma membrane vesicles upon cross-linking of receptors. J Phys Chem B 115:10462-9
Chiang, Yun-Wei; Freed, Jack H (2011) A new Lanczos-based algorithm for simulating high-frequency two-dimensional electron spin resonance spectra. J Chem Phys 134:034112
Ge, Mingtao; Freed, Jack H (2011) Two conserved residues are important for inducing highly ordered membrane domains by the transmembrane domain of influenza hemagglutinin. Biophys J 100:90-7
Dzikovski, Boris G; Borbat, Peter P; Freed, Jack H (2011) Channel and nonchannel forms of spin-labeled gramicidin in membranes and their equilibria. J Phys Chem B 115:176-85
Bhatnagar, Jaya; Borbat, Peter P; Pollard, Abiola M et al. (2010) Structure of the ternary complex formed by a chemotaxis receptor signaling domain, the CheA histidine kinase, and the coupling protein CheW as determined by pulsed dipolar ESR spectroscopy. Biochemistry 49:3824-41

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