Robust Gd3+ -based spin labels for structural studies of membrane proteins

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Intellectual Merit: The border between the interior and exterior of a living cell is a membrane that is only a few nanometers thick. Proteins embedded in cell membranes are the molecular machines that control the flow of matter, energy and information across the border of the cell, and understanding their structures helps to understand how they function. Unfortunately, structural techniques like crystallography and NMR don't work as well for membrane proteins, so new methods are needed. This research project will develop new molecular probes and experimental methods that will provide information on protein structure. The new probes take advantage of the remarkable magnetic properties of ions of the element gadolinium (Gd3+), which are already used to enhance contrast in magnetic resonance imaging (MRI). When excited by a laser inside a very high magnetic field, gadolinium ions precess (wobble) like tops at a frequency of 240 billion cycles per second (240 gigahertz); this occurs about 20,000 times before relaxing most of the way to thermal equilibrium. When a Gd3+ ion is close to another, it relaxes more quickly. Moreover, when the two are less than 10 nanometers apart, the precession amplitude oscillates between the two ions at a "beat frequency" that varies sensitively with the distance between the ions. Together, the relaxation times, precession frequencies, and beat frequency give distance (and ultimately structural) information. These parameters will be measured using newly developed electron paramagnetic resonance (EPR) spectrometers at UC Santa Barbara (240 gigahertz) and the Weizmann Institute of Science in Israel (95 gigahertz). To calibrate the new methodologies, measurements of the distance between gadolinium ions will be performed on "ruler" compounds consisting of Gd3+ ions separated by a stiff, linear polymer to known distances between 2 and 12 nm. Distance measurements will then be performed between carefully-chosen sites on proteorhodopsin. Proteorhodopsin is a light-activated proton pump isolated from a marine bacterium and is similar in structure to membrane proteins that occur in all other organisms. Broader impacts: The project is a new interdisciplinary international collaboration between the U. S. (Song-I Han and Mark Sherwin, UC Santa Barbara), Israel (Daniella Goldfarb, Weizmann Institute of Science), and Germany (Adelheidt Godt, University of Bielefeld, probe synthesis). The collaborators and co-PI are female physicists with considerable expertise in developing new technology. This proposal will support two graduate students and one undergraduate. These students are cross-trained by shuttling between the Han lab (physical chemistry and biochemistry) and the Sherwin lab (experimental physics), and are thus immersed in the science of "soft matter," magnetic resonance, proteins, microwave electronics, terahertz science and technology, and visible optics. Students also will perform experiments in the remarkable magnetic resonance community at the Weizmann Institute of Science in Israel, greatly enriching their training with an international scientific experience. The Gd3+ spin labels and high-field electron paramagnetic resonance techniques that the students develop will enable new studies of membrane proteins in life-like environments by many other scientists. These labels are stable enough to be used inside the complex environment of living cells, where measurements of protein structure and dynamics are most interesting but also most challenging.