RESEARCH INTERESTS - ADITI DAS

OVERVIEW 

One of the key challenges in biochemistry is the study of membrane-associated macromolecules, which mainly involves studies of proteins, small molecules and lipids. Membrane proteins, which constitute about 30% of the entire protein content of the cell, are very important pharmaceutical targets. Despite their importance, only a few membrane proteins have been investigated using detailed biophysical techniques. The main barrier to such investigations has been the tendency of membrane proteins to aggregate (due to their hydrophobic nature), in aqueous solution as well as on surface.

The Sligar laboratory, where I have conducted my post-doctoral research, has developed a simple and robust system, called “Nanodisc”, to circumvent the above problem. ¹ This system enables the self-assembly of membrane proteins into phospholipid bilayers that mimic the membrane, in a controlled manner. Nanodiscs provide stability and full functionality to a variety of membrane proteins in solution and on surfaces. ² The ability to control the stoichiometry, membrane lipid compositions and the number of proteins in a complex makes this system ideally suited to study the effect of lipids and oligomerization on membrane protein activity. Additionally, these nanostructures can be coupled to highly sensitive surface-based detection modalities for high throughput screening of human therapeutics and the discovery of new targets for intervention. ^3-4  

CURRENT RESEARCH

  The focus of my current research work is on (A) understanding redox regulation in human membrane-bound P450s, which play a critical role in drug metabolism, (B) using ultra-sensitive detection modalities based on localized surface plasmon resonance (LSPR) to monitor membrane protein molecular recognition events, and more recently, (C) identifying the key central nervous system (CNS) receptors involved in Alzheimer disease. The common theme in these research problems is the mechanistic study and manipulation of membrane-bound proteins.

A. REDOX REGULATION IN HUMAN MEMBRANE ASSOCIATED P450s: 

Cytochrome P450s are ubiquitous heme proteins that catalyze the mono-oxygenation of bound organic substrates. Hepatic cytochrome P450s and in particular cytochrome P450 3A4 (CYP3A4) are membrane bound and play a critical role in drug metabolism. My research showed that drug binding to CYP3A4 changes its redox potential leading to onset of drug metabolism, thus avoiding unwanted production of toxic, reduced oxygen species. ⁵

Microsomal cytochrome P450s need a redox partner, cytochrome P450 reductase (CPR) to metabolize drugs. I showed that the redox potential of CPR is altered by the composition of the lipid bilayer, to make electron transfer to P450 thermodynamically feasible. ⁶ In vivo, the redox partners of cytochrome P450s work in tandem. Currently, the linked redox equilibria between the redox partners are being studied by co-incorporating them into Nanodiscs.

B. ULTRA-SENSITIVE ASSAYS FOR MEMBRANE PROTEINS WITH CHROMOPHORES: 

In the field of drug development, there is an intense search for novel label-free techniques that can be used for analysis of biomolecular interactions in membrane-bound proteins. Human membrane-bound cytochrome P450s are key players in drug−drug interactions that can lead to drug toxicity. In collaboration with Van Duyne and Schatz group at Northwestern University, we have developed a prototype nanoparticle biosensor to detect drug binding to cytochrome P450 3A4 (CYP3A4) ³ , which, as noted above, is one of the most important enzymes in drug and xenobiotic metabolism in the human body. The new, ultra-sensitive detection mechanism is based on localized surface plasmon resonance (LSPR), and exploits a strong coupling between the molecular resonances of chromophores and the nanoparticle LSPR. Our studies have revealed that the LSPR shift from resonant analyte binding is highly dependent on the spectral overlap between the molecular resonances and the nanoparticle LSPR, a finding that is at the heart of our detection method. ^7-8 This work opens up a way to detect small molecule binding to functionally stable membrane proteins with femto-molar sensitivity.     

C. NOVEL ALZHEIMER DISEASE RECEPTOR TARGETS:  

As research in human neurodegeneration has moved from descriptive phenomenology to mechanistic analysis, it is important to understand the biochemical mechanism by which soluble oligomers of Aβ (ADDLs) ⁹ bind to synaptic plasma membranes and cause the early memory loss in Alzheimer Disease. ¹⁰ Currently it is unknown whether ADDLs bind to specific receptors or bind non-specifically to various receptors and channel proteins. We are collaborating with the Klein laboratory at Northwestern University to determine the synaptosomal receptor targets of ADDLs. (Further details on this finding are protected with an invention disclosure at the Northwestern University and University of Illinois-Urbana Champaign.) 

REFERENCES

(1)            Nath, A.; Atkins, W. M.; Sligar, S. G. Biochem. 2007, 46, 2059-2069.

(2)           Borch, J.; Hamann, T. Biol. Chem. 2009, 390, 805-814.

(3)           Das, A.; Zhao, J.; Schatz, G. C.; Sligar, S. G.; Van Duyne, R. P. Anal. Chem. 2009, 81, 3754-3759.

(4)           Shaw, A. W.; Pureza, V. S.; Sligar, S. G.; Morrissey, J. H. J. Biol. Chem. 2007, 282, 6556-6563.

(5)           Das, A.; Grinkova, Y. V.; Sligar, S. G. J. Am. Chem. Soc. 2007, 129, 13778-13779.

(6)           Das, A. Sligar,  S.G. Biochem. 2009, Submitted

(7)           Zhao, J.; Das, A.; Zhang, X.; Schatz, G. C.; Sligar, S. G.; Van Duyne, R. P. J. Am. Chem. Soc. 2006, 128, 11004-11005.

(8)           Zhao, J.; Das, A.; Schatz, G. C.; Sligar, S. G.; Van Duyne, R. P. J. Phys. Chem. C 2008, 112, 13084-13088.

(9)           Lambert, M. P.; Barlow, A. K.; Chromy, B. A.; Edwards, C.; Freed, R.; Liosatos, M.; Morgan, T. E.; Rozovsky, I.; Trommer, B.; Viola, K.L.;   Wals, P.; Zhang, C.; Finch, C. E.; Krafft, G. A.; Klein, W. L. Proc. Nat. Acad. Sci (USA), 1998, 95, 6448-6453.