Lawrence W. Miller

Assistant Professor

Biochemistry

We develop and apply chemical methods to label proteins and study their function in living mammalian cells. Our experimental approach incorporates synthetic organic chemistry, molecular and cell biology, and microscopy. Currently, our primary efforts include: 1) the simultaneous and specific labeling of multiple proteins in a single cell with functional small molecules; and 2) the labeling of proteins in cells with long-lifetime luminophores for the purpose of time-resolved microscopy.

Fluorescent protein fusions enable microscopic visualization of protein localization and dynamics in living cells. Now, chemical methods are needed to label proteins in vivo with a wider variety of functionalities so that mechanistic questions about protein function in the complex cellular environment can be addressed. With chemical labeling, the protein of interest is expressed as a fusion to a polypeptide tag that does not form a fluorophore itself, but interacts specifically with a synthetic fluorophore or other chemical probe (Figure 1). This method can be used to develop labels with significant advantages over fluorescent proteins, such as superior brightness, longer fluorescence lifetime, higher photostability or red-shifted emission. Synthetic probes can also impart proteins with properties that are not easily encoded genetically, for example environmentally sensitivity, photocrosslinking ability or high electron density.

Figure 1: Schematic of selective, multiplexed labeling of fusion proteins in living cells. Different target proteins of biological interest are genetically encoded as fusions to receptor proteins and expressed in mammalian cells. Cell-permeable ligands with the desired optical functionality are added to the cell culture medium, where they enter the cells and bind selectively to the receptor. After washing excess ligands out of the cells, the fluorescently labeled fusion proteins can be detected microscopically. The success of this approach depends on identifying or developing ligand-receptor pairs that are orthogonal to mammalian cells, as well as engineering cell permeable ligands with suitable pharmacokinetic properties.

Multiplexed chemical labeling of proteins in living cells: We are currently synthesizing and characterizing antifolate ligand-probe conjugates that bind tightly and specifically to different forms of the enzyme, dihydrofolate reductase. Here, the overall goal is to have two or more mutually orthogonal ligand-receptor pairs that can be used to simultaneously label multiple proteins in a single cell.

Dynamic visualization of protein-protein interactions: Transient protein-protein interactions control cell growth and function. We are developing lanthanide-based, protein labels with long (msec) luminescent lifetimes. These probes will facilitate in vivo, time-resolved imaging of resonance energy transfer between interacting proteins. By analyzing the luminescent decay curves of long-lifetime donors, it will be possible to determine the stoichiometry of the interaction, as well as the dynamic localization within the cell. Furthermore, it will be possible to time-gate out any background fluorescence from endogenous biomolecules, allowing luminescent live cell imaging with unprecedented signal-to-noise ratio.

Recent Publications:

L. W. Miller, J. Sable, P. Goelet, M. P. Sheetz, V. W. Cornish, Angew Chem Int Ed Engl 2004, 43, 1672.

L. W. Miller, V. W. Cornish, Curr Opin Chem Biol 2005, 9, 56.

L. W. Miller, Y. Cai, M. P. Sheetz, V. W. Cornish, Nat Methods 2005, 2, 255.

N.T. Calloway, M. Choob, A. Sanz, M.P. Sheetz, L.W. Miller, V.W. Cornish Chembiochem2007, 8, 767.

L.W. Miller (Ed.) “Probes and Tags to Study Biomolecular Function” Wiley-VCH, Weinheim, 2008.