Faculty Bibliography

Viral infection begins with the binding of a virus to a specific target on the surface of the host cell, followed by viral genome delivery into the host and a continuation of the infection process. Before binding occurs, the virus must first find its receptor by a process whose details are largely unknown. We applied high-resolution fluorescence microscopy and single-particle tracking to elucidate the target-finding process in bacteriophage lambda as it infects an Escherichia coli cell. By monitoring the motion of individual viruses through the early stages of infection, we identified a unique spatial focusing process that allows a virus to arrive from its initial random landing site to its destination at the cell pole. The search process is governed by the interaction between the virus and the LamB receptors, and by the spatial organization of the receptor network on the cell surface. Our findings allowed us to develop a theoretical model for the target-finding process that reproduces the key features observed in experiment. We discuss the possible implications of our findings for the process of viral receptor-finding in higher systems

Rad52 promotes the annealing of complementary strands of DNA bound by replication protein A (RPA) during discrete repair pathways. Here, we used a fluorescence resonance energy transfer (FRET) between two fluorescent dyes incorporated into DNA substrates to probe the mechanism by which human Rad52 (hRad52) interacts with and mediates annealing of ssDNA-hRPA complexes. Human Rad52 bound ssDNA or ssDNA-hRPA complex in two, concentration-dependent modes. At low hRad52 concentrations, ssDNA was wrapped around the circumference of the protein ring, while at higher protein concentrations, ssDNA was stretched between multiple hRad52 rings. Annealing by hRad52 occurred most efficiently when each complementary DNA strand or each ssDNA-hRPA complex was bound by hRad52 in a wrapped configuration, suggesting homology search and annealing occur via two hRad52-ssDNA complexes. In contrast to the wild type protein, hRad52(RQK/AAA) and hRad52(1-212) mutants with impaired ability to bind hRPA protein competed with hRPA for binding to ssDNA and failed to counteract hRPA-mediated duplex destabilization highlighting the importance of hRad52-hRPA interactions in promoting efficient DNA annealing

In recent years, advancements in single-biomolecule probing techniques have provided critical information on and greater insight into the nature of biomolecules. Of significance is the application of single-molecule fluorescence resonance energy transfer (smFRET) to probe isolated events and changes at the nanometer scales. In particular, the study of helicases using smFRET has supplied much information regarding the nature and dynamics of these enzymes and provided a toolbox for further investigations. In this chapter we provide a general guide for the construction and execution of single-molecule FRET assays for the study of helicase properties and functionalities

The Rad52 protein has critical functions in distinct pathways of the homology-directed DNA repair, one of which is to promote the annealing of complementary strands of DNA. Both yeast and human Rad52 proteins organize into ring-shaped oligomers with the predominant form being a heptamer. Despite the wealth of information obtained in previous investigations, how Rad52 mediates homology search and annealing remains unclear. Here, we developed single-molecule fluorescence resonance energy transfer approaches to probe hRad52-mediated DNA annealing events in real time. We found that annealing proceeds in successive steps involving rearrangements of the ssDNA-hRad52 complex. Moreover, after initial pairing, further search for extended homology occurs without dissociation. This search process is driven by an interaction between 2 overlapping nucleoprotein complexes. In light of these observations we propose a model for hRad52-mediated DNA annealing where ssDNA release and dsDNA zippering are coordinated through successive rearrangement of overlapping nucleoprotein complexes

The archaeal minichromosome maintenance protein MCM forms a homohexameric complex that functions as the DNA replicative helicase and serves as a model system for its eukaryotic counterpart. Here, we applied single molecule fluorescence resonance energy transfer methods to probe the substrate specificity and binding mechanism of MCM from the hyperthermophilic Archaea Sulfolobus solfataricus on various DNA substrates. S. solfataricus MCM displays a binding preference for forked substrates relative to partial or full duplex substrates. Moreover, the nature of MCM binding to Y-shaped substrates is distinct in that MCM loads on the 3'-tail while interacting with the 5'-tail likely via the MCM surface. These results provide the first elucidation of a dynamic nature of interaction between a ring-shaped helicase interacting with an opposing single-stranded DNA tail. This interaction contributes to substrate selectivity and increases the stability of the forked DNA-MCM complex, with possible implications for the MCM unwinding mechanism

A synthesis of soluble III-V semiconductor quantum rods using gold nanoparticles to direct and catalyze one-dimensional growth is developed. The growth takes place via the solution-liquid-solid (SLS) mechanism where proper precursors are injected into a coordinating solvent. We report the synthesis of InP nanorods using indium acetate and myristic acid with gold nanoparticles as the catalysts in the SLS growth mode. A similar route was successfully developed for the growth of InAs nanorods. We find that the amount of Au catalyst in the reaction is an important parameter to achieve shape control. Transmission electron microscope (TEM) images of InP and InAs nanocrystals revealed that the crystals are mostly rod-shaped, and provide strong evidence for Au presence in one edge. The rods were characterized structurally using X-ray diffraction and high-resolution TEM and optically by absorption and photoluminescence. [All rights reserved Elsevier]

The exceptional fluorescence properties of single CdSe quantum rods (QRs) arising from internal and external electric fields are studied. Reversible external field induced switching of the emission in single QRs is reported for the first time. This effect was correlated with local field induced emission intensity reduction and newly observed darkening mechanism. Bimodal spectral jumps under a zero field were also observed and assigned to charged exciton emission, a phenomenon that was likewise directly controlled through an external field. These phenomena point to the use of single QRs as spectrally tunable charge sensitive fluorophores with polarized emission in fluorescence tagging and optical switching applications

We show the anisotropic selective growth of gold tips onto semiconductor (cadmium selenide) nanorods and tetrapods by a simple reaction. The size of the gold tips can be controlled by the concentration of the starting materials. The new nanostructures display modified optical properties caused by the strong coupling between the gold and semiconductor parts. The gold tips show increased conductivity as well as selective chemical affinity for forming self-assembled chains of rods. Such gold-tipped nanostructures provide natural contact points for self-assembly and for electrical devices and can solve the difficult problem of contacting colloidal nanorods and tetrapods to the external world

Two-photon polarization fluorescence microscopy is used to study the nature of the emission and nonlinear absorption dipole of single CdSe/ZnS quantum rods. Rods showed strongly polarized nonlinear excitation with sharp angular dependence, following a cos(4)(phi) functional form, in agreement with the predicted two-photon absorption process. The two-photon absorption is parallel to the emission polarization and allows high orientation selectivity in excitation to be achieved. This further demonstrates the role of single molecule measurements in unraveling basic principles of light-matter interactions otherwise masked by ensemble averaging. $$: