Irina Petkova

An induced fit mechanism regulates p53 DNA binding kinetics to confer sequence specificity.

The p53 tumour suppressor gene, the most frequently mutated gene in human cancer, encodes a transcription factor that contains sequence‐specific DNA binding and homo‐tetramerization domains. Interestingly, the affinities of p53 for specific and non‐specific DNA sites differ by only one order of magnitude, making it hard to understand how this protein recognizes its specific DNA targets in vivo. We describe here the structure of a p53 polypeptide containing both the DNA binding and oligomerization domains in complex with DNA. The structure reveals that sequence‐specific DNA binding proceeds via an induced fit mechanism that involves a conformational switch in loop L1 of the p53 DNA binding domain. Analysis of loop L1 mutants demonstrated that the conformational switch allows DNA binding off‐rates to be regulated independently of affinities. These results may explain the universal prevalence of conformational switching in sequence‐specific DNA binding proteins and suggest that proteins like p53 rely more on differences in binding off‐rates, than on differences in affinities, to recognize their specific DNA sites.

Tuning the Excited-State Dynamics of GFP-Inspired Imidazolone Derivatives

The excited-state dynamics of five derivatives of the GFP-chromophore, which differ by the position and nature of their substituents, has been investigated in solvents of various viscosity and polarity and in rigid media using femtosecond-resolved spectroscopy. In polar solvents of low viscosity, like acetonitrile or methanol, the fluorescence decays of all compounds are multiexponential, with average lifetimes of the order of a few picoseconds, whereas in rigid matrices (polymer films and low temperature glasses), they are single exponential with lifetimes of the order of a few nanoseconds and fluorescence quantum yields close to unity. Global analysis of the fluorescence decays recorded at several wavelengths and of the transient absorption spectra reveals the presence of several excited-state populations with slightly different fluorescence and absorption spectra and with distinct lifetimes. These populations are attributed to the existence of multiple ground-state conformers. From the analysis of the dependence of the excited-state dynamics on the solvent and on the nature of the substituents, it follows that the nonradiative deactivation of all these excited chromophores involves an intramolecular coordinate with large amplitude motion. However, depending on the solvent and substituent, additional channels, namely, inter- and intramolecular hydrogen bond assisted nonradiative deactivation, are operative. This allows tuning of the excited-state lifetime of the chromophore. Finally, an ultrafast photoinduced intramolecular charge transfer is observed in polar solvents with one derivative bearing a dimethylaminophenyl substituent.

Targeting π-Conjugated Multiple Donor-Acceptor Motifs Exemplified by Tetrathiafulvalene-Linked Quinoxalines and Tetrabenz[bc,ef,hi,uv]ovalenes: Synthesis, Spectroscopic, Electrochemical, and Theoretical Characterization

An efficient synthetic approach to a symmetrically functionalized tetrathiafulvalene (TTF) derivative with two diamine moieties, 2-[5,6-diamino-4,7-bis(4-pentylphenoxy)-1,3-benzodithiol-2-ylidene]-4,7-bis(4-pentylphenoxy)-1,3-benzodithiole-5,6-diamine (2), is reported. The subsequent Schiff-base reactions of 2 afford large π-conjugated multiple donor-acceptor (D-A) arrays, for example, the triad 2-[4,9-bis(4-pentylphenoxy)-1,3-dithiolo[4,5-g]quinoxalin-2-ylidene]-4,9-bis(4-pentylphenoxy)-1,3-dithiolo[4,5-g]quinoxaline (8) and the corresponding tetrabenz[bc,ef,hi,uv]ovalene-fused pentad 1, in good yields and high purity. The novel redox-active nanographene 1 is so far the largest known TTF-functionalized polycyclic aromatic hydrocarbon (PAH) with a well-resolved (1)H NMR spectrum. The electrochemically highly amphoteric pentad 1 and triad 8 exhibit various electronically excited charge-transfer states in different oxidation states, thus leading to intense optical intramolecular charge-transfer (ICT) absorbances over a wide spectral range. The chemical and electrochemical oxidations of 1 result in an unprecedented TTF(⋅+) radical cation dimerization, thereby leading to the formation of [1(⋅+)](2) at room temperature in solution due to the stabilizing effect, which arises from strong π-π interactions. Moreover, ICT fluorescence is observed with large solvent-dependent Stokes shifts and quantum efficiencies of 0.05 for 1 and 0.035 for 8 in dichloromethane.

DEACTIVATION PROCESSES AND HYDROGEN BONDING OF EXCITED N-SUBSTITUTED ACRIDONES

Abstract The photophysical properties of 10-decyl- and 10-propenyl-9-acridones, important emitters in electro- and chemiluminescence, are investigated in dependence of the solvent polarity and its hydrogen bonding ability at room temperature as well as in frozen matrix at 77 K. Fluorescence is inefficient in non-polar solvents at room temperature due to fast intersystem crossing but increases with solvent polarity due to the inversion of the S 1 (ππ ∗ ) and the T (nπ ∗ ) states. In the case of 9-[10-(1-propenyl)]-acridone the dependencies of the fluorescence spectrum and quantum yield on the ET(30) solvent parameters indicate that intermolecular hydrogen bonded solute-solvent complexes are formed in the excited state, inducing efficient non-radiative deactivation.

Photophysical properties and quantum chemical calculations of differently substituted 2(2-phenylethenyl)-benzoxazoles and benzothiazoles

The low fluorescence quantum yield of 2(2-phenylethenyl)-benzoxazoles and benzothiazoles in solution at room temperature is attributed to intramolecular librations with great amplitude of the two heavy fragments at the both ends of the allyl chain. Freezing the solutions at 77 K leads to remarkable increase of fluorescence quantum yield up to three orders of magnitude. No phosphorescence at 77 K is observed. The analysis of the results from quantum-chemical calculations (AM1 approximation) supports the hypothesis for the quenching processes in excited sate.

Fluorescence Spectroscopy of the Tryptophan Microenvironment in Carcinus aestuarii Hemocyanin

The steady-state and time-resolved fluorescence properties of the multitryptophan minimal subunit CaeSS2 from Carcinus aestuarii hemocyanin have been studied with the aim of probing the environment of the fluorophores within the protein matrix. Subunit a of Panulirus interruptus hemocyanin, whose X-ray structure is known, has been also studied. The results are compared with those collected with other two monomeric fractions (CaeSS1, CaeSS3) produced by dissociation of the native, oligomeric protein as well as with those of the hexameric aggregate. Three classes of tryptophan residues can be singled out by a combination of fluorescence quenching and lifetime measurements on the holo-Hc (the copper containing, oxygen binding form) and the apo-Hc (the copper-free derivative). One class of tryptophans is exposed to the protein surface. Some of these residues are proposed to be involved in the intersubunit interactions in CaeSS1 and CaeSS3 fractions whereas in CaeSS2 the protein matrix masks them. This suggests the occurrence of conformational rearrangements after detachment of the subunit from the native aggregate, which could explain the inability of CaeSS2 to reassociate. A second class of tryptophan has been correlatively assigned, by comparison with the results obtained with Panulirus interruptus hemocyanin, to residues in close proximity to the active site. The third class includes buried, active site-distant, residues.

Steady State and Dynamic Photophysical Properties of 2-Benzoimidazoleacetonitrile- α-Phenylmethylenes and 1-Aryl-2,2-Dicyanoethenes

The steady state and dynamic photophysical characteristics of newly synthesized 2-benzoimidazoleacetonitrile- α-phenylmethylenes (BIA’s) and 1-aryl-2,2-dicyanoethenes (DCE’s) have been investigated in solution at 300 K and in a frozen matrix at 77 K. The compounds have a very low or no emission in solution at 300 K both in non-polar and polar solvents. While in BIA’s a significant fluorescence ability is registered in a frozen ethanol matrix at 77 K, freezing solutions of DCE’s at 77 K does not lead to the appearance of either fluorescence or phosphorescence at 77 K even in the presence of C2H5J. The comparison of the fluorescence ability in aprotic and protic solvents does not show any evidence for the specific influence of the protondonating ability of the solvents on the weak fluorescence caused by the formation of intramolecular hydrogen bonds in excited states. The high fluorescence quantum yield of BIA’s in a frozen matrix at 77 K indicates that intramolecular libration of two heavy groups in the allyl chain is the reason for the weak fluorescence at 300 K. Quantum-chemical calculations support the hypothesis of quenching processes in the excited state of BIA’s at 300 K.

Design, synthesis and photophysical study of fluorophore modified noble metal nanoparticles

Metal / dielectric nanostructures, such as metal nanoparticles of different shape (spheres, rods, stars) capped with organic adsorbates, have a strong potential for the development of new materials for nanotechnology. Among these structures, those including monolayer protected clusters of noble metal nanoparticles modified with organic fluorophores are of great interest for the fabrication of new fluorescent bio- and chemical sensors, and for optical devices as well. The nature of the interactions between metals and organic fluorophores is still not totally understood. These interactions, which depend on various factors, including the geometry of the systems, the physicochemical properties of the interacting materials and their environment, often result in an enhancement or a quenching of the emission. The performed investigations comprise design, preparation, characterization and photophysical study of monolayer protected clusters of noble metal nanoparticles modified with newly synthesized organic fluorophore.

Media Effect upon the Fluorescence Ability of Differently Substituted α-Cyanostilbenes

Abstract The steady state and dynamic photophysical characteristics of α-cyanostilbene derivatives have been investigated in solution and in Polyvinylchloride (PVC) film at 300 K as well as in a frozen matrix at 77 K. While no emission is observed in solution, a significant fluorescence ability is registered both in PVC and frozen media. This fact is attributed to the matrix effect, which lessens the possible intramolecular motions around the central double bond. No phosphorescence at 77 K is observed. The fluorescence lifetimes at 77 K are compared with literature data for similar chemically fixed structures.

Electronic Spectral Investigations upon the Photochemical Transformations of Some Substituted 1-(N,N-bisacyl)amino-4,5-diphenyl-l,2,3-triazoles

Abstract The objective of this report is to investigate the steady state and dynamic photophysical and photochemical properties of 1-(N,N-bisacyl)amino-4,5-diphenyl-1,2,3-triazoles in solvents of different polarity at room temperature and in frozen matrix at 77 K. On the basis of the comparison of their UV absorption and luminescence spectra with those of 4,5-diphenyl-1,2,3-triazole and diben-zamide (model compounds), cleavage of the N-N bond in the title compounds after irradiation with polychrome UV light is proved.