A fluorescence-based high-throughput assay for antimicrotubule drugs

With the advent of combinatorial chemistry and the extensive libraries of potential drugs produced from it, there is a growing need for rapid sensitive, high-throughput screening for drug potency. Microtubules are important targets for anticancer agents, and new antimicrotubule compounds are of continued interest in drug development. The in vitro potency of antimicrotubule drugs may be evaluated by measuring the extent of tubulin assembly. The extent of polymerization is proportional to the turbidity of the solution, which usually has been measured as apparent absorption. The turbidity method has inherent problems that hinder its adaptation to a high-throughput format, such as a requirement for high protein concentrations and a high coefficient of variation. We present here a high-throughput assay for antimicrotubule activity in which fluorescence is used to monitor microtubule assembly. Both assembly-inhibiting and assembly-promoting compounds can be evaluated. The assay is rapid and easy to perform, and the data are reliable, with good accuracy and reproducibility.     

Reversible unfolding of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2-(iodoacetyl)amino) ethyl)naphthalene-1-sulfonic acid) probes. The unfolding reaction is described minimally as a 4-state transition from folded dimer-->partially unfolded dimer-->monomer-->unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.     

Monitoring and quantifying dynamic physiological processes in live neurons using fluorescence recovery after photobleaching

The direct visualization of subcellular dynamic processes is often hampered by limitations in the resolving power achievable with conventional microscopy techniques. Fluorescence recovery after photobleaching has emerged as a highly informative approach to address this challenge, permitting the quantitative measurement of the movement of small organelles and proteins in living functioning cells, and offering detailed insights into fundamental cellular phenomena of physiological importance. In recent years, its implementation has benefited from the increasing availability of confocal microscopy systems and of powerful labeling techniques based on genetically encoded fluorescent proteins or other chemical markers. In this review, we present fluorescence recovery after photobleaching and related techniques in the context of contemporary neurobiological research and discuss quantitative and semi‐quantitative approaches to their interpretation.     

Small photoactivatable molecules for controlled fluorescence activation in living cells

The search for chemical probes which allow a controlled fluorescence activation in living cells represent a major challenge in chemical biology. To be useful, such probes have to be specifically targeted to cellular proteins allowing thereof the analysis of dynamic aspects of this protein in its cellular environment. The present paper describes different methods which have been developed to control cellular fluorescence activation emphasizing the photochemical activation methods known to be orthogonal to most cellular components and, in addition, allowing a spatio-temporal controlled triggering of the fluorescent signal.     

Measurement of Reactive Oxygen Species in the Culture Media Using Acridan Lumigen PS-3 Assay

Reactive oxygen species (ROS) are generated continuously during aerobic metabolism. ROS are highly reactive molecules and in excessive amounts, can lead to protein and DNA oxidation, protein cross-linking, and cell death. Cell-culture models provide a valuable tool in understanding the mechanisms that lead to cell death. Accumulation of ROS within cells and/or their release into the culture media are highly cell type-specific. The ability to estimate ROS levels in the culture media is an important step in understanding the mechanisms contributing to disease processes. In this paper, we describe the optimization of a simple method to estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK). We have shown that the Acridan Lumigen PS-3 assay generates ROS-specific chemiluminescence in fresh as well as media stored at −20°C, in as little as 10–20 μl of samples. The method was able to detect the dose (of stimulants)- and time (acute and chronic)-dependent changes in ROS levels in media collected from various cell types. Our results suggest that the kit reagents, PBS buffer, and various media did not contribute significantly to the overall chemiluminescence generated in the assay; however, we suggest that the unused medium specific for each cell type should be used as blanks and final readings of test samples normalized against these readings. As this method uses commonly available laboratory equipment and commercially available reagents, we believe this assay is convenient, economical, and specific in estimating ROS released extracellularly into the culture media.     

Enhancing single-molecule fluorescence with nanophotonics

Single-molecule fluorescence spectroscopy has become an important research tool in the life sciences but a number of limitations hinder the widespread use as a standard technique. The limited dynamic concentration range is one of the major hurdles. Recent developments in the nanophotonic field promise to alleviate these restrictions to an extent that even low affinity biomolecular interactions can be studied. After motivating the need for nanophotonics we introduce the basic concepts of nanophotonic devices such as zero mode waveguides and nanoantennas. We highlight current applications and the future potential of nanophotonic approaches when combined with biological systems and single-molecule spectroscopy.     

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

The self‐assembling MexA‐MexB‐OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR‐wt as well as a selected set of MDR single mutants distant from the proposed DNA‐binding helix. Although DNA affinity and MexA‐MexB‐OprM repression were both drastically impaired in the selected MexR‐MDR mutants, MexR‐wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR‐MDR mutants, secondary structure content and oligomerization properties were very similar to MexR‐wt despite their lack of DNA binding. Despite this, the MexR‐MDR mutants showed highly varying stabilities compared with MexR‐wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA‐binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR‐wt in both free and DNA‐bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations—stability, domain interactions, and internal hydrophobic surfaces—are also critical for the regulation of MexR DNA binding.     

Alpha repeat proteins (αRep) as expression and crystallization helpers

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.     

Opioid receptor-like 1 (ORL1) receptor binding and the biological properties of Ac-Arg-Tyr-Tyr-Arg-Ile-Arg-NH2 and its analogs.

Hexapeptides such as Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) and Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH(2) have been isolated from a combinatorial peptide library as small peptide ligands for the opioid peptide-like 1 (ORL1) receptor. To investigate the detailed structural requirements of hexapeptides, 25 analogs of these hexapeptides, based on the novel analog Ac-Arg-Tyr-Tyr-Arg-Ile-Arg-NH(2) (1), were synthesized and tested for their ORL1 receptor affinity and agonist/antagonist activity on mouse vas deferens (MVD) tissues. Analog 1 and its Cit(6)-analog (10) were found to possess high affinity to the ORL1 receptor, comparable to that of nociceptin/orphanin FQ, and exhibited potent antagonist activity (pA(2) values of 7.77 for 1 and 7.51 for 10, which are higher than that of [NPhe(1)]nociceptin(1-13)-NH(2) (6.90) on MVD assay. It was also found that the amino acid residue in position 5 plays a key role in agonist/antagonist activity, i.e. an L-configuration aliphatic amino acid is required for potent antagonist activity, while a nonchiral or D-configuration residue produces potent agonist activity. These lines of evidence may provide insight into the mechanisms controlling agonist/antagonist switching in the ORL1 receptor, and may also serve to help developing more potent ORL1 agonists and antagonists.