Pavel Leiderman

An alternative excited-state proton transfer pathway in green fluorescent protein variant S205V.

Wild‐type green fluorescent protein (wt‐GFP) has a prominent absorbance band centered at ∼395 nm, attributed to the neutral chromophore form. The green emission arising upon excitation of this band results from excited‐state proton transfer (ESPT) from the chromophore hydroxyl, through a hydrogen‐bond network proposed to consist of a water molecule and Ser205, to Glu222. Although evidence for Glu222 as a terminal proton acceptor has already been obtained, no evidence for the participation of Ser205 in the proton transfer process exists. To examine the role of Ser205 in the proton transfer, we mutated Ser205 to valine. However, the derived GFP variant S205V, upon excitation at 400 nm, still produces green fluorescence. Time‐resolved emission spectroscopy suggests that ESPT contributes to the green fluorescence, and that the proton transfer takes place ∼30 times more slowly than in wt‐GFP. The crystal structure of S205V reveals rearrangement of Glu222 and Thr203, forming a new hydrogen‐bonding network. We propose this network to be an alternative ESPT pathway with distinctive features that explain the significantly slowed rate of proton transfer. In support of this proposal, the double mutant S205V/T203V is shown to be a novel blue fluorescent protein containing a tyrosine‐based chromophore, yet is incapable of ESPT. The results have implications for the detailed mechanism of ESPT and the photocycle of wt‐GFP, in particular for the structures of spectroscopically identified intermediates in the cycle.

Effect of temperature on excited-state proton tunneling in wt-green fluorescent protein.

Steady-state emission and time-correlated single-photon counting (TCSPC) are used to measure the temperature dependence of the proton-transfer rate of wt-GFP in H2O and D2O. As the temperature decreases, the proton-transfer rate from the protonated form slows down. At about 80 K, the rate is about 10-fold slower than the rate at room temperature. At lower temperatures of 70 K down to 13 K (the lowest temperature studied), the rate of proton transfer is almost temperature independent. We explain the temperature dependence of the proton-transfer rate by an intermolecular vibration assisted tunneling mechanism. We attribute the specific intermolecular vibration to the oscillation of two oxygen atoms: the chromophores phenol ring and the nearby water molecule. The kinetic isotope effect is about 5 and is almost temperature independent.

Origin of the Nonexponential Dynamics of Excited-State Proton Transfer in wt-Green Fluorescent Protein

We used an inhomogeneous excited-state proton-transfer kinetics model to explain the origin of the non-exponential time-resolved emission of the A-band of wt-green fluorescence protein. The calculated fit is rather good for both H 2O and D 2O samples in a wide temperature range of 80-229 K. We attribute the inhomogeneous kinetics to the distance dependence of the excited-state proton-transfer rate between the proton donor (the hydroxyl group of the chromophore) and the oxygen of a nearby water molecule.

Effect of temperature and pressure on proton transfer rate from a photoacid to ethanol solution

Abstract The proton dissociation of photoacids is studied as a function of temperature and pressure in liquid ethanol. For this purpose we used a strong photoacid, 5,8-dicyano-2-naphthol (DCN2) ( pK a * ∼−4.5 in water ), capable of transferring a proton to alcohols. At high temperatures, the proton transfer rate is almost temperature independent, while at low temperatures the rate constant has strong temperature dependence. At relatively low pressures, the proton transfer rate increases with pressure while, at high pressures, the rate constant decreases as the pressure increases. The unusual temperature dependence is explained by a two coordinate stepwise mechanism. The two coordinates are the generalized solvent coordinate and the actual proton coordinate between two oxygen atoms. The pressure dependence is explained using the same model used for the temperature dependence. The decrease of the proton transfer rate at high-pressures denotes the solvent-control limited, while the increase in rate at low-pressures denotes the nonadiabatic limit.

Ultrafast intermolecular proton transfer from 10-hydroxy-camptothecin

Fluorescence probes are widely used for the investigation of biologically important systems. In most cases the steady-state spectral data is used for the characterization of properties of their microenvironment. Surprisingly, few synthetic probes are known to utilize their excited-state reactivity, such as excited-state proton transfer, as an additional tool for the investigation of complex systems. Proton transfer, in both the ground and excited states, is a fundamental process in chemistry and biology. For 50 years it has been known for 50 years that the acidity of various hydroxyaromatic compounds (ROH) increases significantly upon excitation, and, therefore, protolytic photodissociation (excited-state proton transfer to solvent or PTTS) has been studied intensively. Camptothecin (CPT) is a pentacyclic alkaloid, first isolated from extracts of the Chinese tree Camptotheca acuminata . This brightly fluorescent compound was found to be a potent inhibitor of the growth of leukemia cells by exhibiting a unique mechanism of action: inhibition of DNA topoisomerase I [l] . A more Figure options Download full-size image Download as PowerPoint slide potent water-soluble analog of CPT, 10− hydroxycamptothecin (10-CPT), has a subunit identical to 6- hydroxyquinoline (6HQ). Hydroxyquinoline derivatives are known to be both strong photoacids and strong photobases and, therefore, undergo efficient tautomerization in the very wide range of pH resulting in weak tautomer (zwitterion) emission [2] . In the only known report on 10-CPT spectral properties. Mi and Burke [3] considered 10-CPT dual fluorescence in methanol-water mixtures as solvent polarity-dependent without taking into account any possible PTTS. We report here the study of the ultrafast excited state intermolecular proton transfer processes of 10-CPT to a water-methanol mixture.