Equipment Setup for Fluorescence Imaging with One-Nanometer Accuracy (FIONA).

INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This article describes procedures for setting up the equipment necessary for FIONA and achieving total internal reflection (TIR).

Data Analysis Methods for Fluorescence Imaging with One-Nanometer Accuracy (FIONA).

INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. Data analysis of a FIONA experiment requires the use of several software programs. Data must be acquired and exported into a proper format before analysis can take place. This article describes various options for data analysis.

Constructing Sample Chambers for Fluorescence Imaging with One-Nanometer Accuracy (FIONA).

INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This protocol provides details on constructing an inexpensive sample chamber for use in single-molecule FIONA experiments and two methods for cleaning slides and coverslips.

Fluorescence Imaging with One-Nanometer Accuracy (FIONA) of Cy3-DNA under Deoxygenation Conditions.

INTRODUCTIONFIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This protocol provides the steps necessary to run a control experiment, using DNA labeled with Cy3, to assess the efficacy of the FIONA setup and the level of attainable resolution. The Cy3-DNA is immobilized on a coverslip and imaged under deoxygenation conditions. It is important that the fluorophores remain photostable throughout the experiment. This requires an oxygen-scavenging system (e.g., glucose oxidase and catalase) in the medium.

Fluorescence Imaging with One-Nanometer Accuracy (FIONA).

INTRODUCTIONFluorescence imaging with one-nanometer accuracy (FIONA) is a technique for localizing a single dye, or a single group of dyes, to within ~1-nm accuracy. This high degree of precision is achieved using total internal reflection fluorescence microscopy, deoxygenation agents, and a high quantum yield, low-noise detector. There are several variations of FIONA, including some capable of better than 10-nm resolution. One such variant is single-molecule high-resolution imaging with photobleaching (SHRIMP), which requires only one type of dye, e.g., two green fluorescent proteins (GFPs), or two rhodamines. However, SHRIMP can only achieve high resolution on static systems. Single-molecule high-resolution colocalization (SHREC), on the other hand, is a FIONA variant that is capable of high resolution with dynamic systems. Defocused orientation and positional imaging (DOPI) enables the three-dimensional orientation to be determined, and either by itself or in combination with FIONA can localize the dye-bound molecules to within a few nanometers. Finally, bright-field imaging with one-nanometer accuracy (bFIONA) achieves the temporal and spectral localization of FIONA but with bright-field microscopy, thus avoiding the use of fluorescence.

Photomodulation of ionic current through hemithioindigo-modified gramicidin channels.

Incorporation of photo-switchable amino acids into peptides and proteins offers prospects for the control of complex biochemical processes using light. Currently, only a few photo-switchable amino acids are known. We report the design and synthesis of a novel hemithioindigo-based amino acid and its incorporation into the model ion channel gramicidin. Photoisomerization of the hemithioindigo moiety between E and Z isomeric forms is shown to modulate ionic current through the channel in a predictable way. This new amino acid thus expands the possibilities for photo-control in diverse systems.

Engineering charge selectivity in model ion channels.

Most ion channel proteins exhibit some degree of charge selectivity, that is, an ability to conduct ions of one charge more efficiently than ions of the opposite charge. The structural origins of charge selectivity remain incompletely understood despite recent advances in the determination of cation-selective and anion-selective channel protein structures. Helix bundle channels formed via self-assembly of the peptide alamethicin provide a tractable model system for exploring the structural basis of charge selectivity. We synthesized covalently-linked alamethicin dimers, with amino acid substitutions at position 18 [lysine (Lys), arginine (Arg), glutamine (Gln), 2,3-diaminopropionic acid (Dpr)] in each helix, to assess the role of this position as a charge-selectivity determinant in alamethicin channels. Of the position 18 substitutions investigated, the Lys derivative exhibited the greatest degree of anion selectivity. Arg-containing channels were slightly less anion-selective than Lys. Interestingly, Dpr channels showed cation selectivity nearly equivalent to that exhibited by the neutral Gln derivative. We suggest that this result is due to a wider pore diameter that permits a greater number of counter-ions leading to enhanced charge screening and a lower effective side-chain positive charge.

Modeling ion channel regulation.

Remarkable recent successes in structure determinations of voltage-gated channels, ligand-gated channels, mechanosensitive channels and proton channels have advanced our understanding of the molecular basis of ion channel gating substantially. Models have helped to clarify aspects of this process and are now being designed as sophisticated biomimetics for various technological applications.