How the biotin–streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer

The interaction between SA (streptavidin) and biotin is one of the strongest non-covalent interactions in Nature. SA is a widely used tool and a paradigm for protein–ligand interactions. We previously developed a SA mutant, termed Tr (traptavidin), possessing a 10-fold lower off-rate for biotin, with increased mechanical and thermal stability. In the present study, we determined the crystal structures of apo-Tr and biotin–Tr at 1.5 Å resolution. In apo-SA the loop (L3/4), near biotin's valeryl tail, is typically disordered and open, but closes upon biotin binding. In contrast, L3/4 was shut in both apo-Tr and biotin–Tr. The reduced flexibility of L3/4 and decreased conformational change on biotin binding provide an explanation for Tr's reduced biotin off- and on-rates. L3/4 includes Ser45, which forms a hydrogen bond to biotin consistently in Tr, but erratically in SA. Reduced breakage of the biotin–Ser45 hydrogen bond in Tr is likely to inhibit the initiating event in biotin's dissociation pathway. We generated a Tr with a single biotin-binding site rather than four, which showed a simi-larly low off-rate, demonstrating that Tr's low off-rate was governed by intrasubunit effects. Understanding the structural features of this tenacious interaction may assist the design of even stronger affinity tags and inhibitors

Encapsulation and modulation of protolytic equilibrium of β-carboline-based norharmane drug by cucurbit[7]uril and micellar environments for enhanced cellular uptake

The effect of supramolecular nanocavity on photophysical and acid-dissociation properties of Norharmane (NHM), a physiologically important, anxiety control and memory-enhancing β-carboline-based drug, has been investigated using steady-state absorption and fluorescence spectroscopy. Self-assembled organization derived from surfactants and rigid water-soluble macrocyclic host Cucurbit[7]uril (CB7) have been selected for this investigation. The confined-space offered by the supramolecular assemblies modulates the pKa value of NHM (up to 3 units) as it can exist in two protolytic forms at near neutral pH. Therefore, the pH-dependent binding properties, modulation of pKa value and its consequences on the photophysical, chemical and solubility properties are investigated in detail. This investigation shows a large shift in the protolytic equilibrium which in turn causes ca. 15 times solubility-enhancement at near neutral pH. Moreover, the effect of enhanced solubility has been further investigated by the augmentation in the cellular uptake of NHM entrapped inside CB7. Thus, the modulation of the acid-base properties and solubility of β-carboline-based drugs will have immense potential for their formulation, cellular uptake and bioavailability

Interaction of Lucifer yellow with cetyltrimethyl ammonium bromide micelles and the consequent suppression of its non-radiative processes

The interaction of the fluorophore Lucifer yellow with micelles has been monitored using steady state and time resolved fluorescence techniques. Contrary to the popular belief that the fluorophore is too polar to associate with the stern layer or hydrophobic core of micelles, we have observed that it binds with the micelles of the positively charged surfactant cetyl trimethyl ammonium bromide and that such interaction causes an decrease in the rates of its non-radiative processes. This phenomenon cannot be explained solely in the light of a reduced polarity, as we have demonstrated that the photophysics of Lucifer yellow is complex and intramolecular charge transfer does not seem to be the only excited state process that is operative.© Elsevie

Selective sensing of lysosomal iron(III) via three-component fluorescence-based strategy in living cells

Iron-rich lysosomal compartment causes a significant amount of cellular damage. A lysosome targetable, pH sensitive fluorescent chemosensor based on naphthalimide dye containing a catechol and morpholine moiety has been synthesized, which selectively senses lysosomal Fe(III) with the detection limit of 0.5 ppm. The probe has also been employed for real-time monitoring of lysosomal Fe(III) via live-cell fluorescence imaging

Iridium Complexes as a Roadblock for DNA Polymerase during Amplification

Iridium-based metal complexes containing polypyridyl-pyrazine ligands show properties of DNA intercalation. They serve as roadblocks to DNA polymerase activity, thereby inhibiting the polymerization process. Upon the addition of increasing concentrations of these iridium complexes, a rapid polymerase chain reaction (PCR)-based assay reveals the selective inhibition of the DNA polymerization process. This label-free approach to study the inhibition of fundamental cellular processes via physical roadblock can offer an alternative route toward cancer therapy

Substituent electronic effects on the persistence and absorption spectra of (Z)-o-xylylenols. A nanosecond laser flash photolysis study

A systematic investigation on a broad set of aldehydes reveals that the lifetimes of (Z)-photoenols can be modulated by variation of the substituents. We have found that the lifetimes of (Z)-enols (in benzene) can be varied by more than 1 order of magnitude with a judicious choice of the substituents that exert mesomeric and inductive effects as, for example, in the case of pentamethylbenzaldehyde (τ=35 ns) and dicyanomesitaldehyde (τ=760 ns). This study thus points to the fact that the electronic factors in conjunction with hydrogen bonding stabilization can considerably broaden the uni-as well as bimolecular chemistry based on photoenolization. Further, we have shown that the photoenols exhibit dramatic shifts in their absorption properties with variation of the substituents; although the photoenols have long been considered to be colored, their absorption properties have not been heretofore comprehensively examined

Intramolecular O-H···O hydrogen-bond-mediated reversal in the partitioning of conformationally restricted triplet 1,4-biradicals and amplification of diastereodifferentiation in their lifetimes

The photoreactivity and nanosecond transient phenomena have been investigated for a rationally designed set of ketones 4-9 in order to gain comprehensive insights concerning the influence of intramolecular hydrogen bonding on (i) the lifetimes of triplet 1,4-biradicals and (ii) the partitioning of the latter between cyclization and elimination. Comparisons of the photochemical results and lifetime data for the biradicals of ketones 6 versus 8 and 7 versus 9 revealed a remarkable influence of hydrogen bonding when superimposed upon steric factors: while 6 and 7 yielded cyclobutanols in poor yields, cyclization was found to be overwhelmingly predominant for 8-anti and moderately so for 9-anti, with a high stereoselectivity in the formation of cyclobutanols (&gt;95% for 8-anti). The diastereochemistry in the case of 8 permitted the occurrence of fragmentation or cyclization almost exclusively (&gt;90% cyclization for 8-anti and &gt;75% elimination for 8-syn). Significantly, the intramolecular hydrogen bonding in the biradicals of 8 and 9 was found to reverse their partitioning between cyclization and elimination compared with the behavior of the biradicals of ketones 3; the ketones 8-anti and 9-anti underwent cyclization in benzene, predominantly leading to cyclobutanols with syn stereochemistry between the C2 and C3 substituents. In accordance with photoproduct profiles, an unprecedented ~2-fold difference in the lifetimes of the intermediate diastereomeric triplet biradicals of ketones 8 in nonpolar solvents (e.g., t<SUB>syn</SUB>=123 ns and t<SUB>anti</SUB>=235 ns in cyclohexane) was observed via nanosecond laser flash photolysis, while no such difference in lifetimes was found for the triplet biradicals of acetoxy ketones 9. The intriguing diastereodifferentiation in the lifetimes of the diastereomeric triplet 1,4-biradicals of 8 and the product profiles of ketones 6, 7, and 9 are best reconciled via a unified mechanistic picture in which superposition of steric factors over varying magnitudes of O-H···O hydrogen bonding selectively facilitates a particular pathway. In particular, the diastereodifferentiation in the photochemical outcomes for the diastereomers of ketone 8 and in the lifetimes of their triplet biradicals can be understood on the basis of rapid deactivation of the 8-syn triplet biradical via fragmentation and slow cyclization of the 8-anti triplet biradical from chair-and twist-boat-like hydrogen-bonded conformations, respectively. The photolysis in polar aprotic solvents such as DMSO and pyridine was found to reverse the chemoselectivity, yielding reactivity paralleling that of ketones 3, for which the steric factors between the C2 and C3 substituents control the photochemical outcome

Diastereomer-differentiating photochemistry of β - arylbutyrophenones: Yang cyclization versus type II elimination

The diastereomers of ketones 2 and 3 are shown to exhibit distinct photochemical reactivities due to conformational preferences; while the anti isomers of 2 and 3 undergo efficient Yang cyclization in 75-90% yields with a remarkable diastereoselectivity (> 90%), the syn isomers predominantly undergo Norrish Type II elimination. The differences in the product profiles of the diastereomers are consistent with a mechanistic picture involving the formation of precursor diastereomeric triplet 1,4-biradicals in which the substituents at a and ß-positions stabilize the cisoid (cyclization) or transoid (elimination) geometry. The fact that such a diastereomeric relationship does indeed ensue at the triplet-excited-state itself is demonstrated via the nanosecond laser-flash photolysis of model ketones 1. The diastereomeric discrimination in the product profiles observed for ketones 2 and 3 as well as in the triplet lifetimes observed for ketones 1 can both be mechanistically traced back to different conformational preferences of the ground-state diastereomeric ketones and the intermediary 1,4-biradicals. Additionally, it emerges from the present study that the syn and anti diastereomers of ketones 2 and 3 represent two extremes of a broad range of widely examined butyrophenones, which lead to varying degrees of Yang photocyclization depending on the alkyl substitution pattern

Hydroxy-Terminated Conjugated Polymer Nanoparticles Have Near-Unity Bright Fraction and Reveal Cholesterol-Dependence of IGF1R Nanodomains

Fluorescent nanoparticles have enabled many discoveries regarding how molecular machines function. Quantum dots have been the dominant class of fluorescent nanoparticles but suffer from blinking and from a substantial dark fractionparticles where the fluorescence is never seencomplicating any analysis of biological function. Nanoparticles composed of conjugated fluorescent polymers (Pdots) have recently been shown to have high brightness and no blinking. Here we develop a robust and efficient means to measure the dark fraction of Pdots, conjugating Atto dyes to the nanoparticles and testing fluorescence colocalization of dye and Pdot puncta. This established that the Pdots we generated had minimal dark fraction: ∼3%. The application of nanoparticles in biological environments is highly sensitive to surface functionalization. For Pdots we found that passivation with uncharged hydroxy-terminated polyethylene glycol caused a dramatic reduction in nonspecific cell binding and aggregation compared to a charged coating. Using carbonyl di-imidazole the hydroxy-Pdots were functionalized efficiently with streptavidin for high stability targeting, allowing specific labeling of mammalian cells. Type I insulin-like growth factor receptor (IGF1R) regulates cell survival and development, with roles in aging, heart disease, and cancer. We used hydroxy-Pdots to track the dynamics of IGF1R on a breast cancer cell-line, determining the diffusion characteristics and showing cholesterol-containing membrane nanodomains were important for receptor mobility at the plasma membrane. The near-unity bright fraction and low nonspecific binding of hydroxy-Pdots, combined with Pdot photostability and lack of blinking, provides many advantages for investigations at the single molecule level