A Promiscuous DNA Packaging Machine from Bacteriophage T4

Packaged viral genome can be removed from bacteriophage T4 capsid and the capsid refilled with any double-stranded DNA, single or multiple molecules, using a powerful ATP-fueled DNA packaging machine

Single molecule fluorescence studies of biomolecular interactions

Single molecule fluorescent techniques have become standard approaches to study protein-DNA interactions. However, these techniques have largely been confined by limitations in assays to studying the interaction between simple DNA substrates and a single protein. During my PhD I developed several novel assays to study a long-standing controversial biophysics question (flexibility of short dsDNA), genome packaging in viruses (Influenza and T4) and dynamics of challenging protein complexes (membrane proteins). The classical view of DNA posits that DNA must be stiff below the persistence length (<150 base pair) but recent studies addressing this have yielded contradictory results. We developed a fluorescence-based, protein-free, assay for studying the cyclization of single DNA molecules in real time. The looping rate for short DNA molecules has remarkably weak length dependence between 67 and 106 bps, deviating significantly from the worm-like chain model. We propose that many biologically significant protein-DNA interactions that involve looping and bending of DNA below 100 bp likely use this intrinsic bendability of DNA. One of the critical aspects of a virus life cycle is packaging of the viral genome. Different viruses have devised intelligent mechanisms to perform this task. I studied packaging mechanism in Bacteriophage T4 and Influenza. Influenza A virus possesses a segmented genome of eight, single-stranded RNAs. However, the exact copy number of each viral RNA segment per individual virus particles has been controversial for the past 50 years. To address this question we combined single molecule TIRF microscopy and multi-color fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Our results showed that a high percentage of virus particles package a single copy of each segment of viral RNAs. Our findings support a model that the packaging of influenza genome is a selective and robust process. Finally we developed a single molecule fluorescence assay to study initiation and re-initiation of dsDNA packaging in the T4 bacteriophage. Using this assay we quantified the details of T4 “packasome” assembly. Also, we showed that the T4 packaging machine can package multiple DNA into the same head in burst-like fashion

Conformational dynamics of a class C G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors in eukaryotes. Crystal structures have provided insight into GPCR interactions with ligands and G proteins, but our understanding of the conformational dynamics of activation is incomplete. Metabotropic glutamate receptors (mGluRs) are dimeric class C GPCRs that modulate neuronal excitability, synaptic plasticity, and serve as drug targets for neurological disorders. A 'clamshell' ligand-binding domain (LBD), which contains the ligand-binding site, is coupled to the transmembrane domain via a cysteine-rich domain, and LBD closure seems to be the first step in activation. Crystal structures of isolated mGluR LBD dimers led to the suggestion that activation also involves a reorientation of the dimer interface from a 'relaxed' to an 'active' state, but the relationship between ligand binding, LBD closure and dimer interface rearrangement in activation remains unclear. Here we use single-molecule fluorescence resonance energy transfer to probe the activation mechanism of full-length mammalian group II mGluRs. We show that the LBDs interconvert between three conformations: resting, activated and a short-lived intermediate state. Orthosteric agonists induce transitions between these conformational states, with efficacy determined by occupancy of the active conformation. Unlike mGluR2, mGluR3 displays basal dynamics, which are Ca(2+)-dependent and lead to basal protein activation. Our results support a general mechanism for the activation of mGluRs in which agonist binding induces closure of the LBDs, followed by dimer interface reorientation. Our experimental strategy should be widely applicable to study conformational dynamics in GPCRs and other membrane proteins