The resolution limit of fluorescence correlation spectroscopy for two-component solutions is investigated theoretically and experimentally. The autocorrelation function for two different particles in solution were computed, statistical noise was added, and the resulting curve was fitted with a least squares fit. These simulations show that the ability to distinguish between two different molecular species in solution depends strongly on the number of photons detected from each particle, their difference in size, and the concentration of each component in solution. To distinguish two components, their diffusion times must differ by at least a factor of 1.6 for comparable quantum yields and a high fluorescence signal. Experiments were conducted with Rhodamine 6G and Rhodamine-labeled bovine serum albumin. The experimental results support the simulations. In addition, they show that even with a high fluorescence signal but significantly different quantum yields, the diffusion times must differ by a factor much bigger than 1.6 to distinguish the two components. Depending on the quantum yields and the difference in size, there exists a concentration threshold for the less abundant component below which it is not possible to determine with statistical means alone that two particles are in solution.
A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligandreceptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants containing an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calculated by the theory of Fö rster, permit to fix the ligand in space and define the structure of the receptor in a molecular model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of experimental distances between a G proteincoupled receptor and its ligand by FRET. The method presented here can be generally applied to the analysis of spatial relationships in integral membrane proteins such as receptors or channels.
Lateral force microscopy in the wearless regime was used to study the friction behavior of a lipid monolayer on mica. In the monolayer, condensed domains with long-range orientational order of the lipid molecules were present. The domains revealed unexpectedly strong friction anisotropies and non-negligible friction asymmetries. The angular dependency of these effects correlated well with the tilt direction of the alkyl chains of the monolayer, as determined by electron diffraction and Brewster angle microscopy. The molecular tilt causing these frictional effects was less than 15 degrees, demonstrating that even small molecular tilts can make a major contribution to friction.
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