When R > 0.8 R0: fluorescence anisotropy, non-additive intensity, and cluster size

Assembly and clustering feature in many biological processes and homo-FRET and fluorescence anisotropy can assist in estimating the aggregation state of a system. The distance dependence of resonance energy transfer is well described and tested. Similarly, assessment of cluster size using steady state anisotropy is well described for non-oriented systems when R 0.8 R0. Fused trimeric DNA clusters labelled with fluorescein were engineered to provide inter-fluorophore distances from 0.7 to 1.6 R/R0 and intensity and anisotropy were measured. These constructs cover a range where anisotropy effects depend on distance. Analytical expressions were derived for fully labelled and fractionally labelled clusters and the experimental results analysed. The experimental results showed that: 1) the system underwent distance dependent quenching; 2) when incompletely labelled both doubly and triply labelled forms could be assessed to obtain distance dependent intensity factors; 3) the anisotropy behaviour of a multiply labelled cluster of a particular size depends on the behaviour of the fluorophores and their distance in a cluster. This work establishes that when emission intensity data are available the analytically useful range for investigating clusters does not have to be restricted to R < 0.8 R0 and is applicable to cases where the anisotropy of a cluster of N fluorophores is not well approximated by r1/N

Theoretical and lab based studies of fluorescence anisotropy toward the analysis of multiple homo-FRET pairs

Many biological functions, involve assembly of proteins. Protein clustering has an important role in signal transduction through the cell membrane. Model systems that simulate receptor aggregation can be used to monitor oligomerization. Techniques based on homo-FRET can reveal the aggregation state of receptor proteins because homo-FRET causes the fluorescence anisotropy of the system to decrease when the number of identical fluorophores within energy transfer distance increases. Theories that describe these systems apply an assumption of equal fluorescence efficiency for all sites [1, 2], that means emission intensity of fluorophores do not change when in close proximity with each other in a cluster. However, most fluorophores in close proximity show either self-quenching or emission enhancement. Previous theories that were devised to study fractional labelling of proteins were only applicable to cases where the distances between the labels were less than 0.8Ro, while in practice the distances between the dyes are more in protein aggregates. Three different systems were chosen to mimic aggregation of fluorescently labelled receptor proteins and analytical expressions were presented for fully labelled and fractionally labelled clusters and the experimental results from the three systems were analyzed. The systems were: a)stochastically labelled BSA containing up to 24 FITC, b) DNA fused trimeric clusters of fluorescein (stochastically labelled), and c)DNA Holliday junctions labelled with fluorescein (in a range of sizes). The inter-probe distances on the DNA constructs were designed to be up to 1.5 Ro to cover cluster sizes of larger protein clusters. The experimental results showed that: 1) none of the clustered species followed the assumption of equal fluorescence efficiency, 2) by applying the assumption of equal fluorescence efficiency, anisotropies were under-predicted and cluster sizes were under-estimated in systems that show quenching and 3) anisotropy behaviour of a multiply labelled cluster of a particular size depends on the behaviour of the fluorophores and their distance in a cluster. As a result of the theory presented here, the size of larger clusters than currently considered possible can be determined; if they are strongly quenched and their fractional labelling is carefully selected

A biophysical study on the mechanism of interactions of DOX or PTX with α-lactalbumin as a delivery carrier

© 2018, The Author(s). Doxorubicin and paclitaxel, two hydrophobic chemotherapeutic agents, are used in cancer therapies. Presence of hydrophobic patches and a flexible fold could probably make α-Lactalbumin a suitable carrier for hydrophobic drugs. In the present study, a variety of thermodynamic, spectroscopic, computational, and cellular techniques were applied to assess α-lactalbumin potential as a carrier for doxorubicin and paclitaxel. According to isothermal titration calorimetry data, the interaction between α-lactalbumin and doxorubicin or paclitaxel is spontaneous and the K (M−1) value for the interaction of α-lactalbumin and paclitaxel is higher than that for doxorubicin. Differential scanning calorimetry and anisotropy results indicated formation of α-lactalbumin complexes with doxorubicin or paclitaxel. Furthermore, molecular docking and dynamic studies revealed that TRPs are not involved in α-Lac’s interaction with Doxorubicin while TRP 60 interacts with paclitaxel. Based on Pace analysis to determine protein thermal stability, doxorubicin and paclitaxel induced higher and lower thermal stability in α-lactalbumin, respectively. Besides, fluorescence lifetime measurements reflected that the interaction between α-lactalbumin with doxorubicin or paclitaxel was of static nature. Therefore, the authors hypothesized that α-lactalbumin could serve as a carrier for doxorubicin and paclitaxel by reducing cytotoxicity and apoptosis which was demonstrated during our in vitro cell studies

Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells