Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP -anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale. Femtosecond spectroscopy revealed that the protonated excited-state (YFPH*) decayed predominantly by a radiationless mechanism, but emission at 460 nm was detected within the first picosecond. Limited excited-state proton transfer leads to 527 nm emission characteristic of the YFP -* anion. Prolonged continuous wave illumination at the peak of YFP -absorbance (514 nm) yields, irreversibly, a weakly fluorescent product that absorbs at 390 nm. This "photobleaching" process also gives a different species (YFPHrb) that absorbs at 350/430 nm and spontaneously regenerates YFP -in the dark on the time scale of hours but can be photoactivated by UV light to regenerate YFP -within seconds, via a ground-state protonated intermediate. Using a pulsed laser for photobleaching resulted in decarboxylation of YFP as indicated by the mass spectrum. These observations are accounted for in a unifying kinetic scheme.Green fluorescent protein (GFP) 1 and its color variants have found wide use in biology to probe protein dynamics in vitro and within cells (1). They have also attracted the attention of spectroscopists owing to their complex photochemistry (2-5). In particular, those in the YFP class (i.e. containing a T203Y or T203F mutation to shift the emission to 527 nm) have been shown to undergo fluctuations of emission intensity over a wide range of times scales and reversible photobleaching (6-8). Fluctuations in emission were observed by wide field microscopy at the single molecule level by immobilizing individual YFP molecules in acrylamide or agarose gels and using TIRF excitation (6, 9). Molecules were found to blink on the seconds time scale at low laser powers (<500 W cm -2 ). Increasing laser power reduced the lifetime of the emitting state, but not in direct proportion to the laser power. Initial studies discussed the possibility that the fluctuations were related to the protonation of the phenolate moiety of the fluorophore derived from Y66 (6), but later studies found little pH dependence of the onand off-times when monitored at a laser power of 5000 W cm -2 (9).Members of the YFP class also showed a photochromic switching behavior. Molecules that were bleached by 488 nm irradiation, could be induced to fluoresce by illumination with near UV or blue light (6-8). This reversible photobleaching reaction of YFP was observed in FRET measurements (10). Illumination of YFP at 514 nm resulted in bleaching of the YFP acceptor and dequenching of a donor CFP molecule. Once the YFP was bleached, illumina...

The conserved switch 1 loop of P-loop NTPases is implicated as a central element that transmits information between the nucleotide-binding pocket and the binding site of the partner proteins. Recent structural studies have identified two states of switch 1 in G-proteins and myosin, but their role in the transduction mechanism has yet to be clarified. Single tryptophan residues were introduced into the switch 1 region of myosin II motor domain and studied by rapid reaction methods. We found that in the presence of MgADP, two states of switch 1 exist in dynamic equilibrium. Actin binding shifts the equilibrium towards one of the MgADP states, whereas ATP strongly favors the other. In the light of electron cryo-microscopic and X-ray crystallographic results, these findings lead to a specific structural model in which the equilibrium constant between the two states of switch 1 is coupled to the strength of the actin-myosin interaction. This has implications for the enzymatic mechanism of G-proteins and possibly P-loop NTPases in general.

Chromosomal abnormalities and genomic instability are common features of, and possible driving forces in, tumorigenesis. Recently, several mitotic proteins that are critical to proper chromosome segregation have been identified. Members of the Aurora kinase family have been identified as having important roles in mitosis; overexpression induces multicellularity and fosters polyploidy. As aneuploidy is a common feature of malignant gliomas, particularly glioblastomas (GBMs), we examined 25 prospectively collected GBMs to assess the role that overexpression of one member of this family, Aurora B, might have in the clinical behaviour of GBMs. Aurora B expression levels were markedly correlated with a shortened survival. Aurora B expression was not directly related to age, tumour proliferation status or to several common molecular changes found in GBMs. These results suggest that Aurora B may be a prognostic feature of impaired survival and a novel therapeutic target in some patients.

Hemangioblastomas are central nervous system (CNS) tumors of unknown histogenesis, which can occur sporadically or in von Hippel-Lindau disease. Hemangioblastomas are composed of neoplastic ''stromal'' cells of unknown origin, accompanied by intensive reactive angiogenesis. Failure to specify the cytologic origin of the stromal cell has precluded the development of nonsurgical therapies and limits understanding of its basic biology. We report that the stromal cells express proteins (Scl, brachyury, Csf-1R, Gata-1, Flk-1, and Tie-2) that characterize embryonic progenitor cells with hemangioblastic differentiation potential and conclude that embryonic progenitors with hemangioblast potential represent a possible cytologic equivalent of the stromal cell. We also identified a new autocrine/ paracrine stimulatory loop between the receptor Tie-2 and the hypoxia-inducible factor target Ang-1, which, combined with previous observations, suggests that a variety of autocrine loops may be initiated in hemangioblastomas, depending on the differentiation status of the tumor cells and the extent of HIF downstream activation. Finally, the consistent identification of Scl in the stromal cells may help explain the unique and characteristic topographical distribution of hemangioblastomas within the CNS. (Cancer Res 2006; 66(8): 4167-72)