Changing the data representation from the classical time delay histogram to the phasor representation provides a global view of the fluorescence decay at each pixel of an image. In the phasor representation we can easily recognize the presence of different molecular species in a pixel or the occurrence of fluorescence resonance energy transfer. The analysis of the fluorescence lifetime imaging microscopy (FLIM) data in the phasor space is done observing clustering of pixels values in specific regions of the phasor plot rather than by fitting the fluorescence decay using exponentials. The analysis is instantaneous since is not based on calculations or nonlinear fitting. The phasor approach has the potential to simplify the way data are analyzed in FLIM, paving the way for the analysis of large data sets and, in general, making the FLIM technique accessible to the nonexpert in spectroscopy and data analysis.

High-mobility group box 1 protein (HMGB1) is a nuclear component, but extracellularly it serves as a signaling molecule involved in acute and chronic inflammation, for example in sepsis and arthritis. The identification of HMGB1 inhibitors is therefore of significant experimental and clinical interest. We show that glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, inhibits HMGB1 chemoattractant and mitogenic activities, and has a weak inhibitory effect on its intranuclear DNA-binding function. NMR and fluorescence studies indicate that glycyrrhizin binds directly to HMGB1 (K(d) approximately 150 microM), interacting with two shallow concave surfaces formed by the two arms of both HMG boxes. Our results explain in part the anti-inflammatory properties of glycyrrhizin, and might direct the design of new derivatives with improved HMGB1-binding properties.

Limited proteolysis or autolysis of thermolysin under different experimental conditions leads to fission of a small number of peptide bonds located in exposed surface segments of the polypeptide chain characterized by highest mobility, as given by the temperature factors (B values) determined crystallographically [Holmes, M.A., & Matthews, B.W. (1982) J. Mol. Biol. 160, 623-639]. Considering also similar findings observed previously with other protein systems, it is proposed that this correlation between segmental mobility and sites of limited proteolysis in globular proteins is quite general. Thus, flexibility of the polypeptide chain of a globular protein at the site of proteolytic attack promotes optimal binding and proper interaction with the active site of the protease. These findings emphasize that apparent thermal motion seen in protein crystals is relevant to motion in solution and appear to be of general significance in protein-protein recognition processes.

Correspondence to Francisco S á nchez-Madrid: fsanchez.hlpr@salud.madrid.org Abbreviations used in this paper: ACF, autocorrelation function; EAP, endothelial adhesive platform; FCS, fl uorescence correlation spectroscopy; FLIM, fl uorescence lifetime imaging microscopy; FN, fi bronectin; FRET, F ö rster resonance energy transfer; FRETeff, FRET effi ciency; GPI, glycosylphosphatidylinositol; HUVEC, human umbilical vein endothelial cell; knn, k nearest neighbor; LEL, large extracellular loop; mEGFP, monomeric EGFP; TEM, tetraspanin-enriched microdomain; VE-cadherin, vascular endothelial cadherin.The online version of this article contains supplemental material.