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The nature of binding of specially designed charge transfer (CT) fluorophore at the hydrophobic protein interior of human serum albumin (HSA) has been explored by massive blue-shift (82 nm) of the polarity sensitive probe emission accompanying increase in emission intensity, fluorescence anisotropy, red edge excitation shift, and average fluorescence lifetimes. Thermal unfolding of the intramolecular CT probe bound HSA produces almost opposite spectral changes. The spectral responses of the molecule reveal that it can be used as an extrinsic fluorescent reporter for similar biological systems. Circular dichrosim spectra, molecular docking, and molecular dynamics simulation studies scrutinize this binding process and stability of the protein probe complex more closely., (Copyright © 2012 Wiley Periodicals, Inc.)

We report extrinsic fluorescent probe 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid (DMAPPDA) as a molecular reporter for studying microheterogeneous environment of protein Human Serum Albumin (HSA) via spectral modification of the probe under physiological condition. Steady state emission, fluorescence anisotropy, Red Edge Excitation Shift (REES), far-UV Circular Dichroism (CD), Atomic Force Microscopy (AFM) imaging, time resolved spectral measurements, Molecular Docking and Molecular Dynamics (MD) Simulation techniques have been used to fulfill this achievement. Interaction of the probe with HSA is signaled by the blue shift of the fluorophore emission maxima with enhancement of fluorescence intensity. The increase in steady state anisotropy, REES and fluorescence lifetime values with increasing protein concentrations indicates interaction and movement of the probe from free aqueous media to the more restricted less polar hydrophobic interior of protein. Experimental results obtained from Benesi-Hildebrand plot support the formation of 1:1 HSA-DMAPPDA complex with high binding constant and negative free energy change. Thermal denaturation of the probe bound protein has also been tracked using the spectral response of DMAPPDA. Molecular Docking studies revealed binding of the probe with in the hydrophobic cavity of subdomain IIA of HSA. MD Simulation supports greater stability of HSA-DMAPPDA complex compared to free protein., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Abstract: Binding interaction of plasma protein bovine serum albumin (BSA) with external flexible charge transfer fluorophore 5-(4-dimethylamino-phenyl)-penta-2,4-dienenitrile (DMAPPDN) has been explored at physiological pH (7.4) by steady state absorption, emission, fluorescence anisotropy, Red Edge Excitation Shift (REES), far-UV circular dichroism (CD), time resolved spectral measurements in combination with molecular docking and molecular dynamics (MD) simulation studies. Chemical denaturation of the protein bound probe by guanidine hydrochloride (GdnHCl) has also been tracked using the spectral response of DMAPPDN. Interaction of the probe with BSA is reflected by the massive blue shift of the fluorophore emission maxima (78nm) with the enhancement of fluorescence intensity due to change of hydrophobic micro-environment of the probe compared to a little change in protein secondary structure. Benesi–Hildebrand plot reveals spontaneous formation of 1:1 BSA–DMAPPDN complex with binding constant 8.821±0.01×103 M−1 and binding free energy change −5.359kcalmol−1. Molecular docking study supports the binding of probe in the hydrophobic cavity of sub domain IIA of BSA. The distance for energy transfer from tryptophan of BSA to DMAPPDN measured from fluorescence resonance energy transfer (FRET) results is in good agreement with results of molecular docking study. MD simulation predicts greater stability of BSA–DMAPPDN complex compared to the free protein. [Copyright &y& Elsevier]

Structural modification through binding interaction of plasma protein bovine serum albumin (BSA) with an extrinsic charge transfer fluorophore 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid (DMAPPDA) and its response to external perturbation due to interactions with surfactant sodium dodecyl sulphate (SDS) have been explored at physiological pH by steady state absorption, emission, fluorescence anisotropy, red edge excitation shift, far-UV circular dichroism and time resolved spectral measurements in combination with Molecular Docking and Molecular Dynamics (MD) simulation. Interaction of the probe with BSA is reflected by a small change in protein secondary structure with fluorescence enhancement and blue shift of probe emission. Molecular docking studies revealed that the probe binds to the hydrophobic cavity of sub-domain IIA of BSA. The distance for energy transfer from the tryptophan of BSA to the bound DMAPPDA measured by Fluorescence Resonance Energy Transfer is in good agreement with the molecular docking results. MD simulation predicts stabilization of the complex with respect to the bare molecule. Interaction of BSA and SDS with DMAPPDA supports the movement of the probe from hydrophilic free water region to a more restricted hydrophobic zone inside the protein. [ABSTRACT FROM AUTHOR]