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Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time.

ABSTRACT: Here we present a technique called single-molecule high-resolution colocalization (SHREC) of fluorescent dyes that allows the measurement of interfluorophore distances in macromolecules and macromolecular complexes with better than 10-nm resolution. By using two chromatically differing fluorescent molecules as probes, we are able to circumvent the Rayleigh criterion and measure distances much smaller than 250 nm. The probes are imaged separately and localized individually with high precision. The registration between the two imaging channels is measured by using fiduciary markers, and the centers of the two probes are mapped onto the same space. Multiple measurements can be made before the fluorophores photobleach, allowing intramolecular and intermolecular distances to be tracked through time. This technique's lower resolution limit lies at the upper resolution limit of single molecule FRET (smFRET) microscopy. The instrumentation and fluorophores used for SHREC can also be used for smFRET, allowing the two types of measurements to be made interchangeably, covering a wide range of interfluorophore distances. A dual-labeled duplex DNA molecule (30 bp) was used as a 10-nm molecular ruler to confirm the validity of the method. We also used SHREC to study the motion of myosin V. We directly observed myosin V's alternating heads while it walked hand-over-hand along an actin filament.

SUBMITTER: Churchman LS

PROVIDER: S-EPMC545495 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time.

Churchman L Stirling LS Okten Zeynep Z Rock Ronald S RS Dawson John F JF Spudich James A JA

Proceedings of the National Academy of Sciences of the United States of America 20050124 5

Here we present a technique called single-molecule high-resolution colocalization (SHREC) of fluorescent dyes that allows the measurement of interfluorophore distances in macromolecules and macromolecular complexes with better than 10-nm resolution. By using two chromatically differing fluorescent molecules as probes, we are able to circumvent the Rayleigh criterion and measure distances much smaller than 250 nm. The probes are imaged separately and localized individually with high precision. Th ...[more]

PMID: 15668396

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Project description:Organic fluorophores, such as Cy3 and Cy5, have been widely used as chemical labels to probe the structure and dynamics of membrane proteins. Although a number of previous studies have reported on the possibility of some of the water-soluble fluorophores to interact with lipid bilayers, detailed fluorophore-lipid interactions and, more importantly, the potential effect of such interactions on the natural dynamics of the labeled membrane proteins have not been well studied. We have performed a large set of all-atom molecular dynamics simulations employing the highly mobile membrane mimetic model to describe spontaneous partitioning of the fluorophores into lipid bilayers with different lipid compositions. Spontaneous membrane partitioning of Cy3 and Cy5 fluorophores captured in these simulations proceeds in two steps. Electrostatic interaction between the fluorophores and the lipid headgroups facilitates the initial, fast membrane association of the fluorophores, followed by slow insertion of hydrophobic moieties into the lipid bilayer core. After the conversion of the resulting membrane-bound systems to full-membrane representations, biased-exchange umbrella sampling simulations are performed for free energy calculations, revealing a higher energy barrier for partitioning into negatively charged (phosphatidylserine or phosphatidylcholine) membranes than purely zwitterionic (phosphatidylcholine or phosphatidylethanolamine) ones. Furthermore, the potential effect of fluorophore-lipid interactions on membrane proteins has been examined by covalently linking Cy5 to single- and multipass transmembrane helical proteins. Equilibrium simulations show strong position-dependent effects of Cy5-tagging on the structure and natural dynamics of membrane proteins. Interactions between the tagged protein and Cy5 were also observed. Our results suggest that fluorophore-lipid interactions can affect the structure and dynamics of membrane proteins to various extents, especially in systems with higher structural flexibility.

Dataset Information

Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time.

Publications

Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time.

Similar Datasets

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