Journal ArticleDOI
Binding Kinetics and Affinities of Heterodimeric versus Homodimeric HIV-1 Reverse Transcriptase on DNA–DNA Substrates at the Single-Molecule Level
Ryan A Marko,Hsiao-Wei Liu,Christopher J. Ablenas,Maryam Ehteshami,Matthias Götte,Gonzalo Cosa +5 more
TLDR
This work investigates the binding of the three RT isoforms to a fluorescently labeled 19/50-nucleotide primer/template DNA duplex by exploiting single-molecule protein-induced fluorescence enhancement (SM-PIFE), which allows us to directly visualize the binding/unbinding of RT to a double-stranded DNA substrate.Abstract:
During viral replication, HIV-1 reverse transcriptase (RT) plays a pivotal role in converting genomic RNA into proviral DNA. While the biologically relevant form of RT is the p66-p51 heterodimer, two recombinant homodimer forms of RT, p66-p66 and p51-p51, are also catalytically active. Here we investigate the binding of the three RT isoforms to a fluorescently labeled 19/50-nucleotide primer/template DNA duplex by exploiting single-molecule protein-induced fluorescence enhancement (SM-PIFE). PIFE, which does not require labeling of the protein, allows us to directly visualize the binding/unbinding of RT to a double-stranded DNA substrate. We provide values for the association and dissociation rate constants of the RT homodimers p66-p66 and p51-p51 with a double-stranded DNA substrate and compare those to the values recorded for the RT heterodimer p66-p51. We also report values for the equilibrium dissociation constant for the three isoforms. Our data reveal great similarities in the intrinsic binding affinities of p66-p51 and p66-p66, with characteristic Kd values in the nanomolar range, much smaller (50-100-fold) than that of p51-p51. Our data also show discrepancies in the association/dissociation dynamics among the three dimeric RT isoforms. Our results further show that the apparent binding affinity of p51-p51 for its DNA substrate is to a great extent time-dependent when compared to that of p66-p66 and p66-p51, and is more likely determined by the dimer dissociation into its constituent monomers rather than the intrinsic binding affinity of dimeric RT.read more
Citations
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Journal ArticleDOI
Demystifying PIFE: The Photophysics Behind the Protein-Induced Fluorescence Enhancement Phenomenon in Cy3
TL;DR: It is proved that the enhancement of Cy3 fluorescence correlates with a decrease in the efficiency of photoisomerization, and occurs in conditions where the dye is sterically constrained by the protein.
Journal Article
Single-Molecule Measurements of Synthesis by DNA Polymerase with Base-Pair Resolution
TL;DR: A single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution observes a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymer enzyme.
Journal ArticleDOI
A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET).
TL;DR: A theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET is provided, which allows the extraction of the fold-decrease in isomerization mobility from experimental data, and how these results provide information on changes in the accessible volume of Cy3 is demonstrated.
Journal ArticleDOI
Mechanism of allosteric inhibition of HIV-1 reverse transcriptase revealed by single-molecule and ensemble fluorescence
TL;DR: It is found that NNRTI binding to RT induces opening of the fingers and thumb subdomains, which increases the dynamic sliding motion of the enzyme on the T/P and reduces dNTP binding affinity, and efavirenz promotes formation of the E138-K101 salt bridge between the p51 and p66 subunits of RT.
Journal ArticleDOI
Simple Design for DNA Nanotubes from a Minimal Set of Unmodified Strands: Rapid, Room-Temperature Assembly and Readily Tunable Structure
Graham D. Hamblin,Amani A. Hariri,Karina M. M. Carneiro,Kai L. Lau,Gonzalo Cosa,Hanadi F. Sleiman +5 more
TL;DR: This work presents a design strategy for DNA nanotubes that uses an alternative, more controlled growth mechanism, while using just five unmodified component strands and a long enzymatically produced backbone.
References
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Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments
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