Ratiometric Zinc Biosensor Based on Bioluminescence Resonance Energy Transfer: Trace Metal Ion Determination with Tunable Response.

Determination of metal ions such as zinc in solution remains an important task in analytical and biological chemistry. We describe a novel zinc ion biosensing approach using a carbonic anhydrase-Oplophorus luciferase fusion protein that employs bioluminescence resonance energy transfer (BRET) to transduce the level of free zinc as a ratio of emission intensities in the blue and orange portions of the spectrum. In addition to high sensitivity (below nanomolar levels) and selectivity, this approach allows both quantitative determination of "free" zinc ion (also termed "mobile" or "labile") using bioluminescence ratios and determination of the presence of the ion above a threshold simply by the change in color of bioluminescence, without an instrument. The carbonic anhydrase metal ion sensing platform offers well-established flexibility in sensitivity, selectivity, and response kinetics. Finally, bioluminescence labeling has proven an effective approach for molecular imaging in vivo since no exciting light is required; the expressible nature of this sensor offers the prospect of imaging zinc fluxes in vivo.

Genetic counseling student demographics: an empirical comparison of two cohorts.

Genetic counseling student characteristics may be evolving with the expansion and diversification of the genetic counseling field. We compared characteristics and previously accrued experiences of genetic counseling students enrolled in the 2018-2019 academic year with genetic counseling students surveyed by Lega et al. (Journal of Genetic Counseling, 14, 395; 2005). Four-hundred thirty students completed a survey (60% response rate) assessing demographics, select application experiences, encouragement and discouragement to apply to genetic counseling programs, and career certainty and motivations. Data analyses comprised descriptive statistics, content analysis of open-ended responses, and t tests and chi-square tests to compare responses to variables also assessed by Lega et al. Similarities between the two cohorts included most students being female, White/Caucasian, and biology majors; they reported a similar amount and type of support and discouragement; and they had strong career certainty. Salient group differences included the current cohort having a larger proportion of males (8% versus 3%; p=.007), greater percentage of parent(s) with a high socioeconomic status (SES; 31% versus 17%; p=.005), a lower first application cycle acceptance rate (71% versus 80%; p<.001), and they were more strongly influenced to pursue genetic counseling by future income (p<.001), desire to help others (p=.002), the profession's prestige (p<.001), and programs' 2-year duration (p<.001). Students applied to an average of six programs during their first application cycle and paid, on average, $1,648 for all application and interview expenses in their acceptance year. A vast majority (99%) had advocacy experiences (most commonly crisis intervention) and shadowing opportunities (94%), and 26% worked as genetic counseling assistants prior to their acceptance. Most students were interested primarily in cancer genetics at the time of survey completion. The genetic counseling field should continue efforts to improve racial and gender diversity and identify ways to increase program accessibility/affordability for individuals at all SES levels.

High-Throughput Screening at the Membrane Interface Reveals Inhibitors of Amyloid-ß.

Aggregation and the formation of oligomeric intermediates of amyloid-ß (Aß) at the membrane interface of neuronal cells are implicated in the cellular toxicity and pathology of Alzheimer's disease. Small molecule compounds have been shown to suppress amyloid aggregation and cellular toxicity, but often the presence of a lipid membrane negates their activity. A high-throughput screen of 1800 small molecules was performed to search for membrane active inhibitors, and 21 primary hits were discovered. Through the use of fluorescence-based assays, transmission electron microscopy, and dot blot assays, the initial 21 primary hits were narrowed down to five lead compounds. Nuclear magnetic resonance and circular dichroism experiments were used for further confirmation of amyloid inhibition at the membrane interface and to obtain insights into the secondary structure of amyloid-ß, while size exclusion chromatography was used to characterize the size of Aß species. Lastly, dye-leakage assays allowed us to understand how the addition of the five lead compounds affected amyloid-ß's ability to permeate the lipid bilayer. These results provide insights into small molecules that stabilize small amyloid species in the presence of membranes for the development of tool compounds for deeper investigations of these transient species.

Structural Interaction of Apolipoprotein A-I Mimetic Peptide with Amyloid-ß Generates Toxic Hetero-oligomers.

Apolipoproteins are involved in pathological conditions of Alzheimer's disease (AD), and it has been reported that truncated apolipoprotein fragments and ß-amyloid (Aß) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with Aß(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of Aß(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-Aß(M1-42) heteromers comprised of unstructured Aß(M1-42) and helical 4F. A uniform two-fold reduction in 15N/1H NMR signal intensities of Aß(M1-42) with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond-scale atomistic molecular dynamics simulations showed that 4F interaction with Aß(M1-42) is electrostatically driven and induces unfolding of Aß(M1-42). Neurotoxicity profiling of Aß(M1-42) complexed with 4F confirms a significant reduction in cell viability and neurite growth. Thus, the molecular architecture of heteromerization between 4F and Aß(M1-42) discovered in this study provides evidence toward our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.

A cationic polymethacrylate-copolymer acts as an agonist for ß-amyloid and an antagonist for amylin fibrillation.

In humans, ß-amyloid and islet amyloid polypeptide (IAPP, also known as amylin) aggregations are linked to Alzheimer's disease and type-2 diabetes, respectively. There is significant interest in better understanding the aggregation process by using chemical tools. Here, we show the ability of a cationic polymethacrylate-copolymer (PMAQA) to quickly induce a ß-hairpin structure and accelerate the formation of amorphous aggregates of ß-amyloid-1-40, whereas it constrains the conformational plasticity of amylin for several days and slows down its aggregation at substoichiometric polymer concentrations. NMR experiments and microsecond scale atomistic molecular dynamics simulations reveal that PMAQA interacts with ß-amyloid-1-40 residues spanning regions K16-V24 and A30-V40 followed by ß-sheet induction. For amylin, it binds strongly close to the amyloid core domain (NFGAIL) and restrains its structural rearrangement. High-speed atomic force microscopy and transmission electron microscopy experiments show that PMAQA blocks the nucleation and fibrillation of amylin, whereas it induces the formation of amorphous aggregates of ß-amyloid-1-40. Thus, the reported study provides a valuable approach to develop polymer-based amyloid inhibitors to suppress the formation of toxic intermediates of ß-amyloid-1-40 and amylin.

Alzheimer's amyloid-beta intermediates generated using polymer-nanodiscs.

Polymethacrylate-copolymer (PMA) encased lipid-nanodiscs (∼10 nm) and macro-nanodiscs (>15 nm) are used to study Aß1-40 aggregation. We demonstrate that PMA-nanodiscs form a ternary association with Aß and regulate its aggregation kinetics by trapping intermediates. Results demonstrating the reduced neurotoxicity of nanodisc-bound Aß oligomers are also reported.

Nanodisc-Forming Scaffold Protein Promoted Retardation of Amyloid-Beta Aggregation.

Peptidic nanodiscs are useful membrane mimetic tools for structural and functional studies of membrane proteins, and membrane interacting peptides including amyloids. Here, we demonstrate anti-amyloidogenic activities of a nanodisc-forming 18-residue peptide (denoted as 4F), both in lipid-bound and lipid-free states by using Alzheimer's amyloid-beta (Aß40) peptide as an example. Fluorescence-based amyloid fibrillation kinetic assays showed a significant delay in Aß40 amyloid aggregation by the 4F peptide. In addition, 4F-encased lipid nanodiscs, at an optimal concentration of 4F (>20 µM) and nanodisc size (<10 nm), significantly affect amyloid fibrillation. A comparison of experimental results obtained from nanodiscs with that obtained from liposomes revealed a substantial inhibitory efficacy of 4F-lipid nanodiscs against Aß40 aggregation and were also found to be suitable to trap Aß40 intermediates. A combination of atomistic molecular dynamics simulations with NMR and circular dichroism experimental results exhibited a substantial change in Aß40 conformation upon 4F binding through electrostatic and π-π interactions. Specifically, the 4F peptide was found to interfere with the central ß-sheet-forming residues of Aß40 through substantial hydrogen, π-π, and π-alkyl interactions. Fluorescence experiments and coarse-grained molecular dynamics simulations showed the formation of a ternary complex, where Aß40 binds to the proximity of peptidic belt and membrane surface that deaccelerate amyloid fibrillation. Electron microscopy images revealed short and thick amyloid fibers of Aß40 formed in the presence of 4F or 4F-lipid nanodsics. These findings could aid in the development of amyloid inhibitors as well as in stabilizing Aß40 intermediates for high-resolution structural and neurobiological studies.

Fluorescence lifetime imaging of physiological free Cu(II) levels in live cells with a Cu(II)-selective carbonic anhydrase-based biosensor.

Copper is a required trace element that plays key roles in a number of human enzymes, such that copper deficiency or genetic defects in copper transport lead to serious or fatal disease. Rae, et al., had famously predicted that free copper ion levels in the cell cytoplasm were extremely low, typically too low to be observable. We recently developed a variant of human apocarbonic anhydrase II for sensing metal ions that exhibits 25-fold better selectivity for Cu(II) over Zn(II) than the wild type protein, enabling us to accurately measure Cu(II) in the presence of ordinary cellular (picomolar) concentrations of free zinc. We inserted a fluorescent labeled Cu(II)-specific variant of human apocarbonic anhydrase into PC-12 cells and found that the levels are indeed extremely low (in the femtomolar range). We imaged the free Cu(II) levels in living cells by means of frequency-domain fluorescence lifetime microscopy. Implications of this finding are discussed.

Long wavelength fluorescence ratiometric zinc biosensor.

A protein-based emission ratiometric fluorescence biosensor is described that exhibits sensitivity to free zinc ion in solution down to picomolar concentrations. Ratiometric measurements are widely used to assure accurate quantitation, and emission ratios are preferred for laser scanning microscopes such as confocal fluorescence microscopes. The relatively long emission wavelengths used are well suited to studies in tissues and other matrices which exhibit significant fluorescence background, and the apo-carbonic anhydrase moiety recognizes zinc ion with high and controllable specificity.

Determination of zinc using carbonic anhydrase-based fluorescence biosensors.

This chapter summarizes the use of carbonic anhydrase (CA)-based fluorescent indicators to determine free zinc in solution, in cells, and in subcellular organelles. Expression (both in situ and in vitro) and preparation of CA-based indicators are described, together with techniques of their use, and procedures to minimize contamination. Recipes for zinc buffers are supplied.

Measuring picomolar intracellular exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor.

Zinc plays both physiological and pathological roles in biology, making it of increasing interest. To date, intracellular free zinc has been measured in cell types supplemented with or enriched in zinc, such as hippocampal neurons. Here we quantitatively image intracellular exchangeable zinc in an ordinary resting cell culture line (PC-12), using an excitation ratiometric fluorescent biosensor based on carbonic anhydrase (CA). Human CA II has a K d of 4 pM for zinc and suffers no interference from millimolar calcium or magnesium ions. The CA-based biosensor was readily introduced into the cell by a novel approach: fusing a transactivator of transcription (TAT)-derived cell penetrating peptide to the CA molecule and adding it to the cells. Our results indicate that the resting concentration is approximately 5-10 pM in cytoplasm and nucleus. Interestingly, the tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)-Zn complex and TPEN are both apoptogenic for this cell line.

DsRed as a highly sensitive, selective, and reversible fluorescence-based biosensor for both Cu(+) and Cu(2+) ions.

The wild type form of Red fluorescent protein (DsRed), an intrinsically fluorescent protein found in tropical corals, is found to be highly selective, reversible and sensitive for both Cu(+) and Cu(2+), with a nanomolar detection limit. The selectivity towards these ions is retained even in the presence of other heavy metal ions. The K(d) values for monovalent and divalent copper, based on single binding isotherms, are 450 and 540 nM, respectively. The wild type DsRed sensitivity to Cu(2+) (below 1 ppb) is seven orders of magnitude better than that of the related wild type Green Fluorescent protein (GFP), and it is even 40 times more sensitive than engineered mutants of GFP. Potential binding sites have been proposed, based on amino acid sequences for copper binding and the distance from the chromophore, with the aid of computer modeling.

A continuous fluorescent assay for protein prenyltransferases measuring diphosphate release.

Protein farnesyltransferase and protein geranylgeranyltransferase type I catalyze the transfer of a 15- and a 20-carbon prenyl group, respectively, from a prenyl diphosphate to a cysteine residue at the carboxyl terminus of target proteins, with the concomitant release of diphosphate. Common substrates include oncogenic Ras proteins, which are implicated in up to 30% of all human cancers, making prenyltransferases a viable target for chemotherapeutic drugs. A coupled assay has been developed to measure the rate constant of diphosphate (PPi) dissociation during the prenyltransferase reaction under both single and multiple turnover conditions. In this assay, the PPi group produced in the prenyltransferase reaction is rapidly cleaved by inorganic pyrophosphatase to form phosphate (Pi), which is then bound by a coumarin-labeled phosphate binding protein from Escherichia coli, resulting in a fluorescence increase. The observed rate constant for PPi release is equal to the rate constant of prenylation of the peptide, as measured by other assays, so that this nonradioactive assay can be used to measure prenyltransferase activity under either single or multiple turnover conditions. This assay can be adapted for high-throughput screening for potential prenyltransferase substrates and inhibitors.