Single-Molecule Optical Probes of Protein Biophysics

Stanford University, Stanford CA

Investigators

Abstract

The objective of this project is to study protein and enzyme dynamics and function using optical single-molecule detection and spectroscopy with in vitro and in vivo techniques. The central goals of this research are to utilize diffraction-limited confocal, total internal reflection, and wide-field epifluorescence optical microscopy and spectroscopy to attack three primary thrust areas: (a) characterization and understanding of the photophysical behavior of several red-emitting autofluorescent proteins at the single-molecule level, proteins which are key elements in a wide array of in vivo cellular labeling and gene expression studies, (b) development of reliable and reproducible methods for specific surface attachment of functional proteins to transparent surfaces, so that unwanted proteins do not stick to the surface in single-molecule studies, and (c) extension of recent success in observing single transmembrane proteins in living cells to establish new methods and protocols for observing single molecules in vivo. In all of these studies, the fact that individual copies of proteins will be studied, one at a time, provides an additional critical source of information about the presence and extent of heterogeneity in these complex systems. To accomplish these ends, physical, chemical, and optical expertise will be combined with the molecular biological and biochemical expertise of several collaborators. Owing to its multidisciplinary organization, the fundamental research in this program will not only generate new knowledge about the biophysical properties of important protein systems, but, in addition the advances in instrumentation will provide novel groundwork for technology transfer to other relevant disciplines. Furthermore, the increased knowledge about both optically driven and spontaneous changes in the emissive state of fluorescent proteins may lead to design of mutants with improved properties for application in biology and other fields. At the same time, the broadly-based multidisciplinary environment which has been assembled to perform this research will provide excellent training and education opportunities for the next generation of biophysical scientists. This project is supported by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Divisions of Physics and Chemistry in the Mathematical and Physical Sciences Directorate.