F1Fo-ATP synthase is the smallest known biological motor, found from bacteria to man. It 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). A typical 70 kg human with relatively sedentary life style will generate around 2.0 million kg of ATP in a 75-year life span. In its simplest form, Escherichia coli contains eight different subunits. It can be separated into two sectors F1 and Fo. F1 has five subunits, namely 1323345, and Fo three subunits, namely ab2c10. ATP hydrolysis and synthesis occur on three catalytic sites in the F1 sector, whereas proton transport occurs through membrane embedded Fo sector. The generation of ATP depends on the phosphate binding. Pi binding in the catalytic sites is linked to 3- subunit rotation. In order to understand the molecular function of this nanomotor, it is paramount to identify and characterize the role of phosphate binding amino acids and their interactions. So far, six amino acids from the catalytic sites have been characterized. Four of them, namely 2Lys-155, 2Arg-182, 2Arg-246, and 1Arg-376, are involved in phosphate binding while two, 2Asn-243 and 1Phe-291, are not. Bovine X-ray crystal structure of catalytic sites indicates the presence of some other residues which seem to be involved in Pi binding and need to be characterized. Moreover, the identification and characterization of the genetic revertants for the above mentioned phosphate binding amino acid mutants, especially 2R246A and 1F291E, will help in improving the motor function of this enzyme. Identification of genetic suppressors will also contribute in fully understanding the molecular mechanism of ATP synthase. The long term goal is to be able to use ATP synthase as a base model for the development of nanomotors in nanomedicine. This enzyme is widely distributed in nature and is involved in many disease states. Some polyphenols are known to inhibit ATP synthesis and/or hydrolysis consequently depriving the tumor cells for energy thereby causing cell death or apoptosis. Thus, identification and characterization of more potent and specific polyphenol compounds will have a broad impact in biology and medicine.
It is evident from our recent findings that the successful modulation of Pi binding is possible and will be an asset in understanding the molecular mechanism ATP synthase. From six catalytic site residues, which were identified and characterized based on the X-ray crystal structure of bovine ATP synthase, four amino acids, namely 2Lys-155, 2Arg-182, 2Arg-246, and 1Arg-376, were found to be involved in phosphate binding while two, 2Asn-243 and 1Phe-291, were not. This is a great success story. Now we would also like to characterize other amino acids present in the catalytic site, namely 1Ser-347, 1Ile-348, 1Thr-349, 1Asp-350, and 1Gly-351. These amino acids are positioned on top of the Pi binding triangle and are definitely playing a vital role there. We would also like to introduce positive charge, in the form of Arg, in incremental order so as to create 1S347R, 1I348R, 1T349R, 1D350R, and 1G351R. The idea behind this insertion is to provide more opportunity for the Pi to bind leading to a more efficient nanomotor. The identification of genetic suppressors for Pi binding mutant residues, especially 2R246A and 1F291E, as proposed, will help us to achieve our goal of improving the motor and catalytic function. Screening of these mutants on the non-fermentable carbon source succinate will give rise to a wide variety of enzymes with varying activities. Some of which would be more active than wild-type. Creation of a six catalytic site enzyme from a three catalytic site enzyme is a novel idea which will require much time and input. All six sites of ATP synthase bind nucleotides but only three of them are catalytic. There is no doubt that ATP synthase with six catalytic sites will be a catalytically superior nanomotor enzyme. Current knowledge of the triangular arrangement of Pi binding site residues is an asset in achieving the above goal. The use of the 2Y331W fluorescence probe in the past has greatly enhanced our understanding of the nucleotide occupancy in catalytic sites. New molecular probes, like 2Y331W, are required to detect and report the Pi binding and release as opposed to nucleotide binding and release. Consequently, development of a new Pi binding probe will aid our NBD-Cl inhibition assay. Four perfectly positioned candidates for the fluorescence approach are 2R323W, 2Y245W, 1R282W, and 1F340W. 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. A better understanding of this enzyme has the potential to greatly aid patients with these diseases. Inhibition of ATP synthase by some polyphenols have been attributed to the inhibition of ATP synthesis thus depriving the tumor cell of energy and causing apoptosis. Accordingly, a better understanding of the inhibitory effects of polyphenols will have an immense impact on biology and medicine. We have this system in working order which puts us in a unique position to successfully carry out this work. I was trained in Professor Alan Senior's laboratory, and as should be evident from my published work I bring a large amount of expertise to this project. Professor Senior is retired leaving no conflict of interest. E. coli is easily amenable to molecular biology, biophysical, biochemical, and genetic techniques. Intact F1Fo and soluble F1ATPase can be obtained in pure form. F1-ATPase yield is high and the enzyme can be stored in -70 oC for a long period of time with excellent retention of ATPase activity.
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