Role of solvent, pH, and molecular size in excited-state deactivation of key eumelanin building blocks: implications for melanin pigment photostability.

Ultrafast time-resolved fluorescence spectroscopy has been used to investigate the excited-state dynamics of the basic eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA), its acetylated, methylated, and carboxylic ester derivatives, and two oligomers, a dimer and a trimer in the O-acetylated forms. The results show that (1) excited-state decays are faster for the trimer relative to the monomer; (2) for parent DHICA, excited-state lifetimes are much shorter in aqueous acidic medium (380 ps) as compared to organic solvent (acetonitrile, 2.6 ns); and (3) variation of fluorescence spectra and excited-state dynamics can be understood as a result of excited-state intramolecular proton transfer (ESIPT). The dependence on the DHICA oligomer size of the excited-state deactivation and its ESIPT mechanism provides important insight into the photostability and the photoprotective function of eumelanin. Mechanistic analogies with the corresponding processes in DNA and other biomolecules are recognized.

UV light blocks EGFR signalling in human cancer cell lines.

UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both overexpress the EGF receptor, leads to arrest of the EGFR signaling pathway. The phosphorylation status of the receptor and the level of phosphorylated downstream signaling molecules i.e. AKT and the mitogen activated protein kinases (MAPKs) ERK1 and 2 is detected by Western blotting using phosphospecific antibodies. There was a threshold level, below which the receptor could not be blocked. In addition, illumination caused the cells to upregulate the cyclin-dependent kinase inhibitor p21WAF1, irrespective of the p53 status. Since the EGF receptor is often overexpressed in cancers and other proliferative skin disorders, it might be possible to significantly reduce the proliferative potential of these cells making them good targets for laser-pulsed UV light treatment.

Quenchers induce wavelength dependence on protein fluorescence lifetimes.

We have analysed the picosecond resolved fluorescence emission decay of horseradish peroxidase A2 and of HEW lysozyme acquired with a streak camera. Analyses of the fluorescence decay data of both proteins revealed that the dynamics of the decay is dependent on the emission wavelength. Our data strongly indicates that resonance energy transfer occurring between aromatic residues and different protein fluorescence quencher groups, and the nature of the quencher groups, are the causes of the observed wavelength dependent mean lifetime distribution. Using the global analysis data to calculate the fluorescence mean lifetime at each wavelength revealed that for lysozyme, the mean fluorescence lifetime increased with observation wavelength, whereas the opposite was the case for peroxidase. Both proteins contain strong fluorescence quencher groups located in close spatial proximity to the protein's aromatic residues. Lysozyme contains disulfide bridges as the main fluorescence quencher whereas peroxidase contains a heme group. Both for lysozyme and horseradish peroxidase there is a clear correlation between the observed fluorescence mean lifetime of the protein at a particular emission wavelength and the respective quencher's extinction coefficient at the respective wavelength. Furthermore, our study also reports a comparison of the analyses of the fluorescence data done with three different methods. Analyses of the fluorescence decay at 10 different fluorescence emission wavelengths revealed significant differences in both fluorescence lifetimes and the pre-exponential factor distributions. Such values differed from the values recovered from the integrated decay curves and from global analyse.

Engineering the pH-optimum of a triglyceride lipase: from predictions based on electrostatic computations to experimental results.

The optimisation of enzymes for particular purposes or conditions remains an important target in virtually all protein engineering endeavours. Here, we present a successful strategy for altering the pH-optimum of the triglyceride lipase cutinase from Fusarium solani pisi. The computed electrostatic pH-dependent potentials in the active site environment are correlated with the experimentally observed enzymatic activities. At pH-optimum a distinct negative potential is present in all the lipases and esterases that we studied so far. This has prompted us to propose the "The Electrostatic Catapult Model" as a model for product release after cleavage of the ester bond. The origin of the negative potential is associated with the titration status of specific residues in the vicinity of the active site cleft. In the case of cutinase, the role of Glu44 was systematically investigated by mutations into Ala and Lys. Also, the neighbouring Thr45 was mutated into Proline, with the aim of shifting the spatial location of Glu44. All the charge mutants displayed altered titration behaviour of active site electrostatic potentials. Typically, the substitution of the residue Glu44 pushes the onset of the active site negative potential towards more alkaline conditions. We, therefore, predicted more alkaline pH optima, and this was indeed the experimentally observed. Finally, it was found that the pH-dependent computed Coulombic energy displayed a strong correlation with the observed melting temperatures of native cutinase.

How do lipases and esterases work: the electrostatic contribution.

This work explores the role of one of the factors explaining lipase/esterase activity: the contribution of electrostatic interactions to lipase/esterase activity. The electrostatic potential distribution on the molecular surface of an enzyme as a function of pH determines, to a large extent, the enzyme's pH activity profile. Other important factors include the presence and distribution of polar and hydrophobic residues in the active cleft. We have mapped the electrostatic potential distribution as a function of pH on the molecular surface of nine lipases/esterases for which the 3D structure is experimentally known. A comparison of these potential maps at different pH values with the corresponding pH-activity profile, pH optimum or pH range where the activity displayed by the enzyme is maximum, has revealed a considerable correlation. A negative potential in the active site appears correlated with maximum activity towards triglycerides, which has prompted us to propose a model for product release ('The electrostatic catapult model') after cleavage of an ester bond. At the same time as the bottom of the active site cleft becomes negatively charged, other nearby regions also titrate and become negatively charged when pH becomes more alkaline, for some of the studied lipases. If such lipases also show phospholipase activity (such as guinea pig lipase-related proteins 2 chimera) we raise the hypothesis that such other titratable regions after becoming negatively charged might stabilise the positive charge present in the polar head of phospholipids, such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The distribution of polar, weak polar and non-polar residues on the molecular surface of each studied lipase, in particular the active site region, was compared for all the lipases studied. The combination of graphical visualisation of the electrostatic potential maps and the polarity maps combined with knowledge about the location of key residues on the protein surface allows us to envision atomic models for lipolytic activity.