Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

Nature of interaction between basic fibroblast growth factor and the antiangiogenic drug 7,7-(Carbonyl-bis[imino-N-methyl-4, 2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino] )bis-(1, 3-naphthalene disulfonate).

PNU145156E (7,7-(carbonyl-bis[imino-N-methyl-4, 2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]) -bis-(1, 3-naphthalene disulfonate)) is a naphthalene sulfonic distamycin A derivative that interacts with heparin-binding growth factors. Because PNU145156E inhibits tumor angiogenesis, it was selected for clinical development. Picosecond time-resolved fluorescence emission and anisotropy were used to characterize the binding of PNU145156E to the basic fibroblast growth factor (a protein associated with tumor angiogenesis). A decrease in PNU145156E fluorescence lifetime was observed as a function of human basic fibroblast growth factor (bFGF) concentration. Nonlinear least-squares fitting of the binding isotherm yielded Kd = 145 nM for a single class of binding sites. Time-resolved anisotropy gave Kd = 174 nM. Kd = 150 nM was independently verified by quantitative high-performance affinity chromatography. The displaced volume of the complex, calculated from its rotational correlation time, fitted a sphere of 1:1 stoichiometry. These results account for the formation of a tight yet reversible PNU145156E:bFGF complex. An evaluation of PNU145156E fluorescence lifetimes in various solvents has highlighted the forces involved in stabilizing the complex. These are mostly electrostatic-hydrophobic in nature, with a relatively low contribution from hydrogen bonding. Both polar and nonpolar groups are involved on the protein-binding site within a largely hydrophobic cleft. A potential binding trajectory, based on a combination of these results with site-directed chemical modification and known bFGF x-ray structure, is suggested