Determination of picomolar concentrations of metal ions using fluorescence anisotropy: biosensing with a "reagentless" enzyme transducer.
AuthorsThompson RB, Maliwal BP, Feliccia VL, Fierke CA, McCall K
JournalAnal Chem
PubMed ID9844569
Because of their high affinity and selectivity, metalloproteins can be used as transducers in novel sensors, i.e., biosensors, for the determination of trace levels of metal ions in solution. Here, we exploit carbonic anhydrase to determine picomolar to nanomolar concentrations of free transition metal ions by fluorescence anisotropy (polarization) in ... More
Selectivity and sensitivity of fluorescence lifetime-based metal ion biosensing using a carbonic anhydrase transducer.
AuthorsThompson RB, Maliwal BP, Fierke CA
JournalAnal Biochem
PubMed ID9918670
'A key performance criterion for metal ion determinations in complex media like serum, cytoplasm of the cell, and sea water is selectivity: the ability to determine the analyte(s) of interest, in the presence of relatively high concentrations of interferents. Cu(II), Zn(II), Cd(II), Co(II), and Ni(II) may be determined by changes ... More
Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel.
AuthorsVaid M, Claydon TW, Rezazadeh S, Fedida D,
JournalJ Gen Physiol
PubMed ID18625849
'Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study ... More
Fluorescent peptide probes for high-throughput measurement of protein phosphatases.
AuthorsNoble JE, Ganju P, Cass AE
JournalAnal Chem
PubMed ID12720338
'A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore ... More
The voltage-clamp fluorometry technique.
AuthorsGandhi CS, Olcese R,
JournalMethods Mol Biol
PubMed ID18998096
Ion channels are the cell's gatekeepers. These proteins selectively allow ionic current to flow down its electrochemical gradient. In some cases, specialized chemical or voltage sensing domains respond to environmental changes and signal the cell to adjust its internal chemistry in response to its surroundings. Because of their importance in ... More
Voltage-dependent conformational changes in human Ca(2+)- and voltage-activated K(+) channel, revealed by voltage-clamp fluorometry.
AuthorsSavalli N, Kondratiev A, Toro L, Olcese R
JournalProc Natl Acad Sci U S A
PubMed ID16895996
Large conductance voltage- and Ca(2+)-activated K(+) (BK(Ca)) channels regulate important physiological processes such as neurotransmitter release and vascular tone. BK(Ca) channels possess a voltage sensor mainly represented by the S4 transmembrane domain. Changes in membrane potential displace the voltage sensor, producing a conformational change that leads to channel opening. By ... More
Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex.
AuthorsKreimer DI, Chai KP, Ferro-Luzzi Ames G
JournalBiochemistry
PubMed ID11087367
The membrane-bound complex of the Salmonella typhimurium histidine permease, an ABC transporter (or traffic ATPase), is composed of two membrane proteins, HisQ and HisM, and two identical copies of an ATP-hydrolyzing protein, HisP. We have developed a technique that monitors quantitatively the sulfhydryl modification levels within the intact complex, and ... More
Conformational changes in rhodopsin. Movement of helix f detected by site-specific chemical labeling and fluorescence spectroscopy.
AuthorsDunham TD, Farrens DL
JournalJ Biol Chem
PubMed ID9880548
A recent proposal for the formation of functionally active rhodopsin has placed critical importance on a movement of one of its transmembrane helices (Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L., and Khorana, H. G. (1996) Science 274, 768-770). We investigated this hypothesis using a series of eight ... More