Instrument Development for Imaging and Manipulation of Single Biomolecules

University Of Illinois At Urbana-Champaign, Urbana IL

Investigators

Abstract

A grant has been awarded to the University of Illinois, Urbana-Champaign, under the direction of Drs. Paul Selvin and Taekjip Ha to develop novel single molecule microscopes that combine the ability to both watch and manipulate individual biomolecules under conditions that simulate the native environment in a cell. The molecules are tagged with a fluorescent dye or dyes that emit light after excitation. Information about the position and/or shape of the molecule can be gathered by monitoring this fluorescence. At the same time, the molecules are put under a variety of environmentally realistic stresses - including mechanical forces and torques, electrical and chemical forces, and temperature changes. How the molecules react to these stresses can then be monitored. Specifically, Selvin and Ha propose to build two microscope stations. One will examine one molecule at a time with very high spatial and temporal resolution. This uses what is called confocal optics, where a single molecule is interrogated with an intense, tightly focussed laser beam, while being manipulated. The other microscope will look at many individual molecules simultaneously, by placing the molecules in an array and interrogating them in parallel with more diffusely focused light. This method can acquire statistical data much more rapidly. Such pParallel detection is particularly important for monitoring irreversible reactions. High-resolution imaging will also be developed on this microscope station. These microscopes will be used to examine a variety of specific biological systems. One is a class of proteins called molecular motors. These biomolecules uses the chemical energy of small energy-rich molecules (such as adenosine triphosphate, ATP) and convert it into mechanical (or other forms) of energy. Membrane proteins are another class of biomolecules central to life yet in many cases poorly understood. Selvin and Ha will study how several classes of ion channels - membrane proteins that enable the cell to control its salt (ion) composition - open and close in response to small molecules (ligand) and transmembrane voltage. Folding reactions of proteins, DNA, and RNA are also essential to life. These will be studied by inducing folding via rapid changes in temperature or chemical concentrations. Single molecule studies have revolutionized our understanding of how biomolecules work. The ability to watch one molecule at a time reveals not only the average properties detected in ensemble measurements, but can yield the entire distribution of relevant properties. Single molecule studies also reveal the time evolution of biochemical reactions, which, if asynchronous, are not observable via ensemble measurements. At the same time, there is an ongoing revolution in the use of fluorescence, which, via specific labeling to parts of the biomolecules, enables scientists to dissect how each part of the biomolecule is moving. The combination of single molecule manipulation and fluorescence techniques has the potential for revolutionizing our understanding of how these tiny biomolecules work.