Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.     

Molecular basis for water-promoted supramolecular chirality inversion in helical rosette nanotubes

Helical rosette nanotubes (RNTs) are obtained through the self-assembly of the GwedgeC motif, a self-complementary DNA base analogue featuring the complementary hydrogen bonding arrays of both guanine and cytosine. The first step of this process is the formation of a 6-membered supermacrocycle (rosette) maintained by 18 hydrogen bonds, which then self-organizes into a helical stack defining a supramolecular sextuple helix whose chirality and three-dimensional organization arise from the chirality, chemical structure, and conformational organization of the GwedgeC motif. Because a chiral GwedgeC motif is predisposed to express itself asymmetrically upon self-assembly, there is a natural tendency for it to form one chiral RNT over its mirror image. Here we describe the synthesis and characterization of a chiral GwedgeC motif that self-assembles into helical RNTs in methanol, but undergoes mirror image supramolecular chirality inversion upon the addition of very small amounts of water (<1% v/v). Extensive physical and computational studies established that the mirror-image RNTs obtained, referred to as chiromers, result from thermodynamic (in water) and kinetic (in methanol) self-assembly processes involving two conformational isomers of the parent GwedgeC motif. Although derived from conformational states, the chiromers are thermodynamically stable supramolecular species, they display dominant/recessive behavior, they memorize and amplify their chirality in an achiral environment, they change their chirality in response to solvent and temperature, and they catalytically transfer their chirality. On the basis of these studies, a detailed mechanism for supramolecular chirality inversion triggered by specific molecular interactions between water molecules and the GwedgeC motif is proposed.     

Mechanism of titanocene-mediated epoxide opening through homolytic substitution

The mechanism of titanocene-mediated epoxide opening was studied by a combination of voltammetric, kinetic, computational, and synthetic methods. With the aid of electrochemical investigations the nature of a number of Ti(III) complexes in solution was established. In particular, the distribution of monomeric and dimeric Ti(III) species was found to be strongly affected by the exact steric conditions. The overall rate constants of the reductive epoxide opening were determined for the first time. These data were employed as the basis for computational studies of the structure and energies of the epoxide-titanocene complexes, the transition states of epoxide opening, and the beta-titanoxy radicals formed. The results obtained provide a structural basis for the understanding of the factors determining the regioselectivity of ring opening and match the experimentally determined values. By employing substituted titanocenes even more selective epoxide openings could be realized. Moreover, by properly adjusting the steric demands of the catalysts and the substrates the first examples of reversible epoxide openings were designed.     

Theoretical analysis on changes in thermodynamic quantities upon protein folding: essential role of hydration

The free energy change associated with the coil-to-native structural transition of protein G in aqueous solution is calculated by using the molecular theory of solvation, also known as the three-dimensional reference interaction site model theory, to uncover the molecular mechanism of protein folding. The free energy is decomposed into the protein intramolecular energy, the hydration energy, and the hydration entropy. The folding is accompanied with a large gain in the protein intramolecular energy. However, it is almost canceled by the correspondingly large loss in the hydration energy due to the dehydration, resulting in the total energy gain about an order of magnitude smaller than might occur in vacuum. The hydration entropy gain is found to be a substantial driving force in protein folding. It is comparable with or even larger than the total energy gain. The total energy gain coupled with the hydration entropy gain is capable of suppressing the conformational entropy loss in the folding. Based on careful analysis of the theoretical results, the authors present a challenging physical picture of protein folding where the overall folding process is driven by the water entropy effect.     

Type II polyketide synthases: gaining a deeper insight into enzymatic teamwork

This review covers advances in understanding of the biosynthesis of polyketides produced by type II PKS systems at the genetic, biochemical and structural levels.     

Theoretical study of the cosolvent effect on the partial molar volume change of staphylococcal nuclease associated with pressure denaturation

We explain the molecular mechanism of the effect of urea and glycerol cosolvents on the partial molar volume (PMV) change associated with the pressure denaturation of staphylococcal nuclease (SNase) protein recently observed in experiments. Native and denatured conformations of SNase are produced by using molecular dynamics simulations in water, and the PMV is obtained from the integral equation theory of molecular liquids called 3D-RISM, which is based on statistical mechanics. The PMV of the native SNase in water predicted by 3D-RISM theory is in good agreement with experiment. The PMV changes associated with pressure denaturation in water and in water-urea and water-glycerol mixtures are qualitatively reproduced. By analyzing the results obtained, we found two interesting cosolvent effects on the PMV: (1) both urea and glycerol cosolvents increase the PMVs of both native and denatured SNase compared to those in water and (2) both urea and glycerol cosolvents increase the PMV of denatured SNase more than that of native SNase. We also showed that these two observations can be explained in terms of the thermal volume, which is related to the packing effect of solvent molecules.     

Interaction of meso-tetrakis(4-sulphonatophenyl)porphine with chitosan in aqueous solutions

Interaction of meso-tetrakis(4-sulphonatophenyl)porphine (TPPS4) with chitosan (Mr approximately 400 kDa, N-acetyls approximately 20 mol.%) was studied in aqueous solutions. UV-vis absorption and circular dichroism (CD) spectroscopic titration of 10 micromol l-1 TPPS4 with chitosan demonstrated that an addition of the polysaccharide at appropriate concentrations and pH values induce and support self-aggregation of the macrocycles. The mode of aggregation was strongly dependent on pH: stacking (H-type) aggregates predominated at weak acidic conditions (pH 4.8-6.8) and tilted (J-type) aggregates at pH 2.5. At the intermediate pH value (3.6) both types of TPPS4 aggregates were detected. High amount of chitosan (>0.05 mmol l-1 of GlcN) disrupts H-aggregates forming monomeric porphyrin-chitosan complexes (pH 3.6-6.8), while J-aggregates (pH 2.5) are stable even at very high chitosan concentrations. CD titration experiments confirmed the formation of optically active species of TPPS4 in the presence of chitosan. The complex nature of CD bands assigned to both types of porphyrin aggregates indicated the occurrence of several chiral macrocyclic species dependently on pH value and chitosan concentration.     

Conformational flexibility of DNA

Pulsed Electron-Electron Double Resonance (PELDOR) on double-stranded DNA (ds-DNA) was used to investigate the conformational flexibility of helical DNA. Stretching, twisting, and bending flexibility of ds-DNA was determined by incorporation of two rigid nitroxide spin labels into a series of 20 base pair (bp) DNA duplexes. Orientation-selective PELDOR experiments performed at both X-band (9 GHz/0.3 T) and G-band (180 GHz/6.4 T) with spin label distances in the range of 2-4 nm allowed us to differentiate between different simple models of DNA dynamics existing in the literature. All of our experimental results are in full agreement with a dynamic model for ds-DNA molecules, where stretching of the molecule leads to a slightly reduced radius of the helix induced by a cooperative twist-stretch coupling.     

Layered hydrazinium titanate: advanced reductive adsorbent and chemical toolkit for design of titanium dioxide nanomaterials

LHT-9, a layered hydrazinium titanate with an interlayer spacing of ~9 ?, is a new nanohybrid compound combining the redox functionality of hydrazine, the ion-exchange properties of layered titanate, the large surface area of quasi-two-dimensional crystallites, surface Br?nsted acidity, and the occurrence of surface titanyl bonds. LHT-9, ideally formulated as (N(2)H(5))(1/2)Ti(1.87)O(4), relates to a family of lepidocrocite-type titanates. It possesses a high uptake capacity of ~50 elements of the periodic table. Irreversibility of reductive adsorption allows LHT-9 to be used for cumulative extraction of reducible moieties (noble metals, chromate, mercury, etc.) from industrial solutions and wastewaters. Unlike sodium titanates that do not tolerate an acidic environment, LHT-9 is capable of uptake of transition metals and lanthanides at pH > 3. Adsorption products loaded with the desired elements retain their layered structures and can be used as precursors for tailored titanium dioxide nanomaterials. In this respect, the uptake of metal ions by LHT-9 can be considered as a method complementary to electrostatic self-assembly deposition (ESD) and layer-by-layer self-assembly (LBL) techniques. LHT-9 is readily synthesized in one step by a mild fluoride route involving hydrazine-induced hydrolysis of hexafluorotitanic acid under near-ambient conditions.