Dr. Zulfiqar Ahmad, Ph.D
Department of Biological Sciences
East Tennessee State University
Johnson City, TN 37614
Phone: (423) 439-6931
Molecular modulation of ATP Synthase- F1Fo-ATP Synthase is the smallest known biological nanomotor, found from bacteria to man. This is the fundamental means of cell energy production in animals, plants, and almost all microorganisms in the form of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). In order to synthesize ATP, the cell’s energy currency, a mechanical rotation mechanism is used in which subunits rotate at approximately 100 times per second in order to convert foodstuffs into energy by oxidation. A typical 70 kg human with relatively sedentary lifestyle will generate around 2.0 million kg of ATP from ADP and Pi in a 75-year lifespan. In its simplest form Escherichia coli ATP synthase contains eight different subunits which are divided into two sectors, F1 (a3b3dγe) and F0 (ab2c10).
Currently, our research is focused on three aspects of ATP synthase (1) Characterization/modulation of the phosphate binding subdomain in the catalytic sites. (2) Inhibitory effects of natural and modified polyphenol compounds on E. coli ATP synthase. (3) Identification molecular probes for Pi binding/release. (4) Characterization of the folding/unfolding profiles of E. coli ATP synthase subunits.
So far, using biochemical, biophysical, molecular biology and genetic techniques, we have identified the following five Pi binding residues: bLys-155, bArg-182, bArg-246, aArg-376, and aSer-347. It was shown in our recent work that the Pi binding residues appear as a triangle with ßLys-155 being at the apex and αArg-376, ßArg-182, ßArg-246, and aSer-347 being at the base (see figure above).
Significant aspects are: (1). ATP synthase is the smallest known molecular motor; this has brought it to the forefront of nanotechnology and nanomedicine. Nanomedicine, an offshoot of nanotechnology, refers to highly specific medical interventions on the molecular scale for curing disease or repairing damaged tissues. Questions that scientists and researchers are grappling with are “how many?”, “how big?”, and “how fast?” do nanomotors need to be? These questions must be addressed in order to build “nano machines” that are compatible with living tissues and can safely operate inside the body. We feel that one approach to learn more about these three basic “hows” is to generate a catalytically controllable ATP Synthase.
(2). ATP synthase is critical to human health. Malfunction of this complex has been implicated in a wide variety of diseases including Alzheimer’s, Parkinson’s, and the class of severely debilitating diseases known collectively as mitochondrial myopathies. In addition to the above-mentioned conditions, ATP synthase also plays a vital role in antimicrobial activity. Streptococcus mutans is a primary microbial agent in the pathogenesis of dental caries through biofilm formation and acid production. Inhibition of S. mutans ATP synthase inhibits biofilm formation and acid production. In Mycobacterium, mutations in the c-subunit confer resistance to the new tuberculosis drug diarylquinoline. Another study showing ATP synthase binding to angiostatin on the surface of human endothelial cells is of great value. This makes ATP synthase a potential model in anti-microbial and anti-tumor research. A better understanding of this enzyme will greatly aid patients with these diseases and will have a broad impact on biology and medicine.
(B) A wide range of health related beneficial effects such as protection against cardiovascular disease, cancer, aging, etc have been credited to the ingestion of polyphenols. The beneficial effects of polyphenols may be derived in part by preventing mitochondrial ATP synthesis in tumor cells, thereby inducing apoptosis. Polyphenols have been shown to prevent both the synthetic and hydrolytic activities of bovine ATP synthase by blocking both clockwise and anti-clockwise rotation of the γ-subunit. Thus we are interested in understanding the inhibitory effects of natural and modified polyphenols on E. coli ATP synthase.
Relevance of Work
|This research will provide extensive opportunities for students. As discussed below, undergraduates and graduates (MS) will have ample opportunities to learn a wide variety of molecular biology, genetic, biochemical, and biophysical techniques. Some of which are mutagenesis, cloning, protein purification, spectroscopy, etc. This will also bring fervor among our colleagues and in our institute as ETSU is not a major recipient of NIH funding. A total of eleven students, four graduates and seven undergraduates, are currently being trained in my lab; four of them have already published or submitted research papers as coauthors in peer-reviewed journals with me.|
Ahmad, Z. and Laughlin, T. F. (2010) Medicinal Chemistry of ATP synthase: a potential drug target of dietary polyphenols and amphibian antimicrobial peptides, Current Medicinal Chemistry (In Press).
Chinnam, N., Dadi, P.K., Sabri, S.A., Ahmad, M., and Ahmad, Z. (2010) Dietary bioflavonoids inhibit Escherichia coli ATP synthase in a differential manner Int. J. Biol. Macromol. 46, 478-486.
Laughlin, F. and Ahmad, Z. (2010) Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides. Int. J. Biol. Macromol 46, 367-374.
Li, W., Brudecki, L.E., Senior, A.E., and Ahmad, Z. (2009) Role of a-subunit VISIT-DG sequence residues Ser-347 and Gly-351 in the catalytic sites of Escherichia coli ATP synthase. J. Biol. Chem. 284, 10747-10754.
Ahmad, S. and Ahmad, Z. (2008) “ ATP-binding site as a further application of neural network to residue level prediction” In Proceedings of International Joint Conference on Neural Networks (IJCNN), World Conference on Computational Intelligence (WCCI), June 1-8, Hong Kong, IEEE. pp. 2431-2435.
Ahmad A, Ahmad Z and MA Baig (2003) Hepatic Sulfite oxidase: effect of anions on its activity. Trends Clinical. Biochem. Lab Medicine. 1, 751-755.
Ahmad Z, Salim M & Maines MD (2002) Human biliverdin reductase is a leucine zipper like DNA-binding protein and functions in transcriptional activation of heme oxygenase-1 by oxidative stress J. Biol. Chem. 277, 9226-9232.
Ahmad Z & Ahmad F (1994) Physicochemical characterization of products of unfolding of cytochrome c by calcium chloride. Biochem. Biophys. Acta. 1207, 223-230.
Current Lab Members:
Laura E. Brudecki, Laura became a graduate student in the spring of 2009. She initially joined my lab as an undergraduate in the fall 2006. She has been working on the characterization of Pi binging site residues of E. coli ATP synthase. Her current work involves the characterization of the a-subunit VISIT-DG sequence residues in the Pi binding subdomain of the catalytic sites.
Nagababu Chinnam, NBC joined our lab in January 2009 as a graduate student. His is joining the project with PKD on the inhibitory effects of polyphenols/bioflavonoids.
Sneha Reddy, Sneha joined our lab in January 2009 as a graduate student. She has joined the project on characterization of the a-subunit VISITDG sequence residues in the Pi binding subdomain of the catalytic sites and will specifically focus on the DG residues.
Chao Zhao , Chao joined us in August 2008. He will be working on characterization of the a-subunit VISITDG sequence residues in the Pi binding subdomain of the catalytic sites and will focus on the aI346 and aI348 residues.
Junior Tayou, Junior also joined us in August 2008. He is working on the inhibitory effects of peptides and their binding site on ATP synthse.
Each of these students is undertaking their own specific project or has been coupled with a graduate student.