Affiliations

Dean M. Willard Chair, Chemistry and Biochemistry

Director, Advanced Light Microscopy/Spectroscopy Lab, Macro-Scale Imaging Lab

Lab Director, S. Weiss Lab

Professor, Chemistry and Biochemistry, Physical Chemistry, Physiology

Member, Biochemistry, Biophysics & Structural Biology GPB Home Area, California NanoSystems Institute, Cell & Developmental Biology GPB Home Area, Molecular Biology Institute, Molecular, Cellular & Integrative Physiology GPB Home Area

Researcher, Biophysics and Structural Biology, Nanoscience and Materials, Physical

Identifying and cataloging all gene sequences and their corresponding protein high-resolution static 3-dimensional structures is only a prelude to the real challenges of future biological inquiries. We need to understand the dynamic structural changes of isolated macromolecules undergoing biochemical reactions. We need to identify transient interactions between macromolecules. We need to decipher the cell circuitry and its detailed time-dependent responses to various stimuli. We need to decode intracellular and extracellular communication signals and the corresponding molecular-level responses of live cells, tissues and whole organs. Analytical tools will need to be developed in order to identify and measure these molecular conformations and interactions. High-sensitivity fluorescence methods will play a vital role in this endeavor because they offer many advantages for probing isolated molecules and molecules in live cells, tissues and organs. Fluorescence is non-invasive; it provides imaging and sectioning capabilities in three dimensions; it has high sensitivity, down to the single molecule level, and it allows the observation of molecular- and organelle-specific signals. Our group develops and applies ultrahigh-resolution, ultrahigh-sensitivity fluorescence imaging and spectroscopy tools to solving outstanding problems in biology. CONFORMATIONAL DYNAMICS OF BIOMOLECULES: Many of the molecular machines that are essential for cell function and survival (transcription, replication, translation, recombination, splicing, protein folding etc.) are asynchronous, have multiple kinetic pathways and intermediates, and are comprised of several interacting components. The ability to watch one molecule at a time helps us obtain unique information on distribution functions of relevant observables, resolve subpopulations in heterogeneous samples, and record asynchronous time trajectories of observables that would otherwise be hidden. For example, dynamic distance changes between two sites on a macromolecule (or between two different molecules) can be measured via single-pair fluorescence resonance energy transfer (spFRET) by following spectral changes in the emission of a single donor-acceptor pair. Orientational changes can be detected via single-molecule fluorescence polarization anisotropy (smFPA) by following changes in the dipole orientation of a rigidly-attached or tethered probe. Our group developed such tools and uses them to study protein folding and transcription on the single-molecule level. We study the folding reaction energy landscape, pathways, conformational distributions and folding intermediates. The same tools are used to answer 25-years-old outstanding questions about the mechanisms of DNA transcription by the enzyme RNA polymerase. We are studying the relative motions between the enzyme and its DNA "molecular track", the conformational motions in the enzyme itself, and conformational transitions in the DNA during the various steps of the transcription reaction. DEVELOPMENT OF SEMICONDUCTOR NANOCRYSTALS AS BIOLOGICAL PROBES: Single-molecule experiments are currently performed predominantly in in-vitro environments. The ultimate challenge is to image cellular substructures, determine the relationships and dynamics of vesicles and organelles, describe existing conformational dynamics and biomolecular interactions, and localization all of the above in-vivo with single molecule sensitivity and nanometer-accuracy. This will allow the study of enzymes and multi-component molecular machines in their natural environment, with the signaling and regulation circuitry all wired-up. Towards the accomplishment of this tremendously complex task, we are developing colloidal fluorescent semiconductor nanocrystals (a class of quantum dots) for biological labeling. Nanocrystals posses several properties that make them very attractive as fluorescent probes for in-vivo single-molecule experiments: broad excitation spectra, narrow emission spectra, precise-tunable emission peaks, long fluorescence lifetimes, and negligible photobleaching. We are developing organic coatings, bioconjugation schemes, targeting strategies, and unique instrumentation that take advantage of nanocrystals?\225 spectral properties. Recently, we have shown how these distinctive properties increase the resolution of fluorescence microscopy measurements down to the nanometer level using far-field optics. We have also shown that their long fluorescence lifetime can be used to observe molecules and organelles in live cells without interference from autofluorescence background (a pre-requisite for single molecule detectability). We are currently pursuing applications in genomics (physical mapping, genome rearrangement), vesicle trafficking, cargo selection and in-vivo motility assays of molecular motors. BIOMOLECULAR INTERACTIONS BY COINCIDENCE DETECTION: Understanding the intricate network of protein interactions that take place within the cell of an organism a llows the understanding of the mechanisms that control its growth, maintenance, and disease. Protein-protein interactions are most commonly identified using the yeast two-hybrid (Y2H) assay. However, Y2H has several limitations including complications associated with the use of protein fusions, incorrect protein folding, incorrect post-translational modification, potential for toxicity and incompatibility with DNA binding proteins. We are developing a new method for detecting static and transient macromolecular interactions with ultrahigh sensitivity, ba sed on two-color coincident detection of single molecules in a confocal detection volume. Towards this end, we are developing unique fluorescent reagents, site- directed labeling schemes and novel single-molecule detection and analysis tools for monitoring protein-protein and protein-DNA interactions initially in-vitro and at a later stage in-vivo. We envision applications in cellular signaling and bio-sensing.

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