Biomedical subjects

Steven E Mansoor

Publications and source records attributed to Steven E Mansoor.

3 recordsLinked to original sources

PubMed2006

Rhodopsin self-associates in asolectin liposomes.

We show that the photoreceptor rhodopsin (Rh) can exist in the membrane as a dimer or multimer using luminescence resonance energy transfer and FRET methods. Our approach looked for interactions between Rh molecules reconstituted into asolectin liposomes. The low receptor density used in the measurements ensured minimal receptor crowding and artifactual association. The fluorescently labeled Rh molecules were fully functional, as measured by their ability to activate the G protein transducin. The luminescence resonance energy transfer measurements revealed a distance of 47-50 Angstroms between Rh molecules. The measured efficiency of FRET between receptors was close to the theoretical maximum possible, indicating nearly quantitative Rh-Rh association. Together, these results provide compelling evidence that Rh spontaneously self-associates in membranes.

Fluorescence Resonance Energy Transfer↗

PubMed2004

High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane.

We present a site-directed fluorescence labeling (SDFL) study of 25 different T4 lysozyme protein samples labeled with the thiol-cleavable fluorophore, (2-pyridyl)dithiobimane (PDT-Bimane). Our results demonstrate PDT-Bimane can be used in cysteine-scanning studies to detect protein secondary structure, and to map proximity between sites in proteins by monitoring tryptophan quenching of bimane fluorescence. In addition, the reducible nature of PDT-Bimane can be exploited to resolve problems often faced in SDFL studies: ensuring specific labeling of cysteine residues, determining the extent of free label contamination, and accurately determining labeling efficiency even at low concentrations. The ability to cleave PDT-Bimane off the protein enables rapid determination of these parameters, and positions it as an ideal fluorophore for automated, high-throughput structural studies of protein folding, the detection of protein-protein interactions, and the monitoring of real-time conformational changes.

Bridged Bicyclo Compounds, Heterocyclic↗

PubMed2002

Mapping proximity within proteins using fluorescence spectroscopy. A study of T4 lysozyme showing that tryptophan residues quench bimane fluorescence.

We present a novel method for mapping proximity within proteins. The method exploits the quenching of the fluorescent label bimane by nearby Trp residues. In studies of T4 lysozyme we show that this effect appears to be distance dependent and orientation specific. Specifically, we show that a proximal Trp residue can reduce bimane fluorescence intensity by up to 500% and induce complicated fluorescence decay kinetics. Replacing the neighboring Trp residue with phenylalanine removes these spectral perturbations. The advantages of using the Trp quenching of bimane fluorescence for protein structural studies include the low amount of protein required and the substantial simplification of labeling strategies. We anticipate this method will prove suitable for a wide array of high-throughput protein studies such as protein folding, the detection of protein-protein interactions, and, most importantly, the dynamic monitoring of conformational changes.

Bacteriophage T4↗