Regulation of the Chloroplast ATP Synthase Rotary Motor

University Of Kansas Center For Research Inc, Lawrence KS

Investigators

Abstract

The F1-ATPase is a tiny molecular rotary motor driven by binding and hydrolysis of ATP in one direction and by trans-membrane proton flux in the other direction. Preliminary studies using single molecule fluorescence microspectroscopy demonstrate that a hybrid ATPase assembled from recombinant F1 subunits from photosynthetic organisms is capable of generating sufficient torque to propel large (1-2 micrometer) actin filaments through solution in a manner similar in most respects to its bacterial counterpart. Remarkably, rotation can be driven either by hydrolysis of CaATP or MgATP yet calcium, unlike magnesium, is known to be an ineffective co-substrate for proton coupled ATP hydrolysis or ATP synthesis. The fact that CaATP hydrolysis can drive the fully cooperative catalytic process without being coupled to proton movement has suggested a new catalytic model in which MgATP/ADP binding to the enzyme induces a special conformation of the enzyme which is required for proton coupling but not ATP hydrolysis. This project has the following two specific aims to test the new model and to identify the dynamic events leading to torque generation: 1. To identify the specific interactions between the gamma and epsilon subunits and the core hexameric alpha3 beta3 structure of the F1-ATPase that define the intermediate states of the catalytic sites involved in the catalytic cycle and its regulation. This will be addressed using a combination of site-directed mutagenesis and biochemical approaches; 2. To determine the magnitude and direction of relative movements of the gamma and epsilon subunits during catalysis and regulation of the F1-ATPase using real-time single molecule fluorescence microspectroscopy. The photosynthetic ATP synthase has several unique properties which separate it from its mitochondrial and bacterial counterparts and that offer new inroads to examine the remarkable rotary mechanism of the enzyme. One such property is the presence of a special regulatory segment located in the gamma subunit that has evolved in higher plant species to provide a molecular "switch" mechanism that tightly controls the catalytic activity of the enzyme. The switch is designed to block futile energy loss during photosynthesis. A major goal of this research is to identify the productive binding interactions between the gamma subunit and other subunits within the enzyme structure that are involved in the molecular switch mechanism. The information to be gained from this work is likely to prove seminal in understanding natural processes that have evolved to regulate the rotary motor, identifying different intermediate structural states that occur during rotation and the molecular steps involved in achieving these structural states. This in turn will provide the template to design gated "nanomachines" for a wide variety of important future practical applications. The research will also provide training in critical research skills for two doctoral students and at least two semesters of laboratory research experience for a minimum of eight undergraduate students, most of whom are likely to pursue graduate studies and to develop into independent scientists.