Herein we report the effect of different nucleobase pair compositions on the association-induced fluorescence enhancement property of Thioflavin T (ThT), upon binding with 20 base pair long double-stranded DNA (dsDNA). Analysis of binding and decay constants along with the association (K ass ) and dissociation (K diss ) rate constants obtained from the fluctuation in the fluorescence intensity of ThT after binding with different DNA revealed selective affinity of ThT toward AT-rich dsDNA. Molecular docking also substantiates the experimental results. We also observed that addition of orange-emitting ethidium bromide (EtBr) to cyan-emitting ThT−DNA complexes leads to bright white light emission (WLE) through Forster resonance energy transfer. Additionally, the emission of white light is far greater in the case of intra-DNA strands. Besides endorsing the binding insights of ThT to AT-rich dsDNA, the present investigations open a new perspective for realizing promising WLE from two biomarkers without labeling the DNA.
Structure, activity, and dynamics of a plasma protein, human serum albumin (HSA), inside a crowded environment of F127 gel are studied by circular dichroism (CD), fluorescence correlation spectroscopy (FCS), and picosecond time-resolved fluorescence spectroscopy. For this purpose, the protein is covalently labeled by a maleimide dye, 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methyl-coumarin (CPM). The circular dichroism (CD) spectra suggest that the protein is more structured in the gel reflecting about the biological activities of the protein. FCS results demonstrate that compared to that in bulk water (buffer solution), translational diffusion is about 59 times slower inside the F127 gel. This indicates higher translational friction (viscosity) sensed by the probe (CPM). On the contrary, rotational relaxation (and hence, rotational friction) is more or less similar in F127 gel and in bulk water. FCS results further indicate that the time scales of conformational relaxation of the protein are substantially slow inside the crowded environment of F127 gel. The fast component of conformational relaxation is retarded by ∼55 times, and the slow component by ∼20 times. Fluorescence maximum of CPM bound to HSA show a ∼5 nm red shift, implying that the microenvironment of the probe, CPM, is more polar inside the gel. Solvation dynamics of CPM-labeled HSA inside the gel (⟨τ s ⟩ ∼ 300 ps) is faster compared to that for the protein in bulk water (⟨τ s ⟩ ∼ 600 ps).
Sub-nanomolar selective detection of Hg(ii) ions by protein (Human Serum Albumin, HSA) templated gold nanoclusters (AuNCs), both in in vitro as well as in vivo environments and specific endocytose behaviour towards breast cancer (BC) cell lines.
Herein
we report the interaction of 3-hydroxyflavone (3HF) with
various isomeric forms of Human Serum Albumin (HSA), namely, the N-isoform
(or native HSA at pH 7.4) and the B-isoform (at pH 9.2). Spectroscopic
signatures of 3HF reveal that the interaction of 3HF with the N-isoform
of HSA results in significant lowering of absorbance of the neutral
species (λabs ∼ 345 nm) with concomitant increase
of the anionic species (λabs ∼ 416 nm) whereas
interaction with the B-isoform of HSA leads to selective enhancement
of absorbance of the anionic species. The fluorescence profile of
3HF displays marked increase of intensity of the proton transferred
tautomer (λem ∼ 538 nm) as well as the anionic
species (λem ∼ 501 nm) for both the forms
of the protein. However, analyses of the associated thermodynamics
through temperature-dependent isothermal titration calorimetric (ITC)
indicate that the interaction of 3HF with the N-isoform of HSA is
more enthalpic in the lower temperature limit while the entropy contribution
predominates in the higher temperature limit. Consequently, the 3HF–HSA
(N-isoform at pH 7.4) interaction reveals an unusual thermodynamic
signature of a positive heat capacity change (ΔC
p = 3.84 kJ mol–1K–1) suggesting the instrumental role of hydrophobic hydration. On the
contrary, the 3HF–HSA (B-isoform at pH 9.2) interaction shows
qualitatively reverse effect. Consequently, the interaction is found
to be characterized by an enthalpy-dominated hydrophobic effect (negative
heat capacity change, ΔC
p = −1.15
kJ mol–1K–1) which is rationalized
on the basis of the nonclassical hydrophobic effect.
This work delineates an integrative
approach combining spectroscopic
and computational studies to decipher the association-induced fluorescence
properties of a fluorescent molecular rotor, viz., auramine O (AuO), after interacting with 20-mer duplex DNA having
diverse well-matched base pairs. While exploring the scarcely explored
sequence-dependent interaction mechanism of AuO and DNA, we observed
that DNA could act as a conducive scaffold to the formation of AuO
dimer through noncovalent interactions at lower molecular density.
The photophysical properties of AuO depend on the nucleotide compositions
as described from sequence-dependent shifting in the emission and
absorption maxima. Furthermore, we explored such DNA base pair-dependent
fluorescence spectral characteristics of AuO toward discriminating
the thermodynamically most stable single nucleotide mismatch in a
20-mer sequence. Our results are interesting and could be useful in
developing analogues with further enhanced emission properties toward
mismatched DNA sequences.
Nature has beautifully assembled its light harvesting
pigments
within protein scaffolds, which ensures a very high energy transfer.
Designing a highly efficient artificial bioinspired light harvesting
system (LHS) thus requires the nanoscale spatial orientation and electronic
control of the associated chromophores. Although DNA has been used
as a scaffold to organize chromophores, proteins or polypeptides,
however, are very rarely explored. Here, we have developed a highly
efficient, artificial, bioinspired LHS using polypeptide (poly-d-lysine, PDL) nanostructures making use of their β-sheet
structure in an aqueous alkaline medium. The chromophores used herein
are compatible for an energy transfer process and are nonfluorescent
in an aqueous medium but exhibit high fluorescence intensity when
bound to the nanostructure of PDL. The close proximity of the chromophores
results in an energy transfer efficiency of ∼92% besides generating
white light emission at a particular molar ratio between the chromophores.
Trypsin, the most abundant pancreatic protein, aids in protein digestion by hydrolysis and exhibits aggregation propensity in presence of alcohol, which can further lead to pancreatitis and eventually pancreatic cancer. Herein, by several experimental and theoretical approaches, we unearth the inhibition of alcohol-induced aggregation of Trypsin by macrocyclic cavitand, β-cyclodextrin (β-CD). β-CD interacts with the native protein and shows inhibitory effect in a dose dependent manner. Moreover, the secondary structures and morphologies of Trypsin in presence of β-CD also clearly emphasize the inhibition of fibril formation. From Fluorescence Correlation Spectroscopy, we observed an enhancement in diffusion time of Nile Red with ~2.5 times increase in hydrodynamic radius, substantiating the presence of fibrillar structure. Trypsin also shows reduction in its functional activity due to alcohol-induced aggregation. Our simulation data reports the probable residues responsible for fibril formation, which was validated by molecular docking studies.
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