Increase In Steady-state Anisotropy Research Articles

Study of microheterogeneous environment of protein Human Serum Albumin by an extrinsic fluorescent reporter: A spectroscopic study in combination with Molecular Docking and Molecular Dynamics Simulation

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.

Quest for Mode of Binding of 2-(4-(Dimethylamino)styryl)-1-methylpyridinium Iodide with Calf Thymus DNA

The mode of binding of 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) with calf thymus DNA as revealed from different steady state and time-resolved emission spectroscopic measurements has been reported in this paper. Fluorescence enhancement of DASPMI and its quenching by potassium iodide (KI) points to groove binding of dye with ct-DNA, rather than intercalation in the ct-DNA helix. An increase in steady state anisotropy and fluorescence lifetime hints at binding with ct-DNA. The value of binding constant from emission and association constant from circular dichroic spectrum also indicates weak binding. The strong dependence on ionic strength or salt in controlling the binding of DASPMI with ct-DNA by electrostatic interaction confirms groove binding. The high semicone angle of DASPMI in ct-DNA certainly rules out the possibility of intercalated bonding. A theoretical modeling shows that the probe is bound to ct-DNA as a crescent with a curvature of 11.35 A, which is the previously known curvature of probe in the minor groove.

Illuminating the Dynamic Changes Associated with Heterotropic Allosteric Communication in Phosphofructokinase from Bacillus stearothermophilus

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) undergoes dynamic changes in its otherwise rigid structure upon binding of ligands, fructose 6-phosphate (F6P) in the active sites, and allosteric inhibitor phosphoenolpyruvate (PEP) in the allosteric sites. PFK can exist in four distinct ligation states: Apo BsPFK, BsPFK-F6P, PEP-BsPFK, and PEP-BsPFK-F6P, however, the biophysical properties of each ligation state are poorly understood particularly with respect to their dynamic character. BsPFK is a homotetramer with four active sites and four allosteric sites, creating an intricate network of both homotropic and heterotropic interactions. The intrinsic tryptophan fluorescence emanating from hybrid enzymes in which a single tryptophan was used to report local side-chain dynamics upon ligand binding to sites involved in a single heterotropic interaction. Tryptophan was positioned by constructing conservative tryptophan-shift mutant enzymes in which one of the sixteen native phenylalanine and tyrosine residues in each subunit was substituted with tryptophan and the native tryptophan was changed to tyrosine. The first site of interest was a phenylalanine at position 230. Fluorescence intensity and steady-state anisotropy measurements reveal no change in response to F6P binding, but a 10% increase in intensity and a slight increase in steady-state anisotropy for PEP alone. Time-resolved experiments indicated that rotational correlation time significantly increased (by greater than 1 ns) for the PEP-BsPFK and PEP-BsPFK-F6P ligation states, but only slightly increased in the presence of F6P alone when compared to the unligated state of BsPFK. These trends are opposite comparable measurements made with PFK from E. coli (EcPFK). These results may suggest a structural basis for the observation that allosteric communication in BsPFK is entropically dominated whereas it is enthalpically dominated in EcPFK. Funding is provided by NIH grant GM33216 and Welch Foundation grant A1543.

C-phycocyanin incorporated into reverse micelles: a fluorescence study

The solubilization of C-PC into aerosol-OT(AOT, sodium bis(2-ethylhexyl)sulphosuccinate)/water/ i-octane reverse micelles has been investigated by fluorescence spectroscopy by following its behaviour as a function of the water-to-surfactant molar ratio, w 0. The maximum wavelength emission decreased steadily with an increase in water content, together with a concomitant increase in steady-state anisotropy. These effects saturated at a w 0 of around 30 and remained practically constant at w 0>30. These results are explained in terms of an inclusion into the inner core of the reverse AOT micelles. Strong interactions also took place between amino-acid residues neighbouring bilin chromophores and the AOT sulfonate headgroups, which resulted in the chromophores being located in the structured interfacial micellar water layer. The stabilization rate of the spectral parameters from micelles of protein solutions at pH 5.0 followed pseudo-first-order kinetics and afterwards remained stable for some days. The fluorescence intensity decay of C-PC in reverse micelles is described by a triple exponential function, showing a similar complex pattern to that in an aqueous solution. The fluorescence lifetime values of micelled C-PC indicate that the chromophores are shielded against solvent quenching by the structured interfacial water layer.

Effects of lung surfactant proteolipid SP-C on the organization of model membrane lipids: A fluorescence study

Lipid-protein interactions of pulmonary surfactant-associated protein SP-C in model DPPC/DPPG and DPPC/DPPG/eggPC vesicles were studied using steady-state and time-resolved fluorescence measurements of two fluorescent phospholipid probes, NBD-PC and NBD-PG. These fluorescent probes were utilized to determine SP-C-induced lipid perturbations near the bilayer surface, and to investigate possible lipid headgroup-specific interactions of SP-C. The presence of SP-C in DPPC/DPPG membrane vesicles resulted in (1) a dramatic increase in steady-state anisotropy of NBD-PC and NBD-PG at gel phase temperatures, (2) a broadening of the gel-fluid phase transition, (3) a decrease in self-quenching of NBD-PC and NBD-PG probes, and (4) a slight increase in steady-state anisotropy of NBD-PG at fluid phase temperatures. Time-resolved measurements, as well as steady-state intensity measurements indicate that incorporation of SP-C into DPPC/DPPG or DPPC/DPPG/eggPC vesicles results in a increase in the fraction of the long-lifetime species of NBD-PC. The results presented here indicate that SP-C orders the membrane bilayer surface, disrupts acyl chain packing and may increase the lateral pressure within the bilayer.