Priyankar Sen

Ligand binding strategies of human serum albumin: how can the cargo be utilized?

Human serum albumin (HSA), being the most abundant carrier protein in blood and a modern day clinical tool for drug delivery, attracts high attention among biologists. Hence, its unfolding/refolding strategies and exogenous/endogenous ligand binding preference are of immense use in therapeutics and clinical biochemistry. Among its fellow proteins albumin is known to carry almost every small molecule. Thus, it is a potential contender for being a molecular cargo/or nanovehicle for clinical, biophysical and industrial purposes. Nonetheless, its structure and function are largely regulated by various chemical and physical factors to accommodate HSA to its functional purpose. This multifunctional protein also possesses enzymatic properties which may be used to convert prodrugs to active therapeutics. This review aims to highlight current overview on the binding strategies of protein to various ligands that may be expected to lead to significant clinical applications.

Interaction of Bovine (BSA), Rabbit (RSA), and Porcine (PSA) Serum Albumins with Cationic Single-Chain/Gemini Surfactants: A Comparative Study

The interactions among bovine, rabbit, and porcine serum albumins and single-chain cationic surfactant cetyltrimethylammonium bromide (CTAB) versus its gemini counterpart (designated as G4) have been studied. The studies were carried out in an aqueous medium at pH 7.0 using UV, intrinsic and extrinsic fluorescence spectroscopy, and far-UV circular dichroism techniques. The results indicate that compared to CTAB, G4 interacts strongly with the serum albumins, resulting in a significantly larger unfolding or decrease in alpha-helical content as reflected by the significantly larger decrease in ellipticity in the far-UV range. Unlike CTAB, a remarkable increase in the alpha-helical content of BSA at 625 microM G4 and at 250 microM G4 for RSA and PSA is observed. The appearance of conformational changes and saturation points in the proteins occurs at considerably lower [G4] compared to [CTAB]. The results obtained from the multi-technique approach are ascribed to the stronger forces in G4 owing to the presence of two charged headgroups and two hydrocarbon tails. Keeping the results in view, it is suggested that the gemini surfactants may be effectively used in the renaturation of proteins produced in genetically engineered cells via the artificial chaperone protocol and may also prove useful in drug delivery as solubilizing agents to recover proteins from insoluble inclusion bodies.

Interactions of thioflavin T with serum albumins: Spectroscopic analyses

The interaction of thioflavin T (ThT) with serum albumins from four different mammalian species i.e. human, bovine, porcine and rabbit, has been investigated by circular dichroism (CD), fluorescence spectroscopy and ITC. The binding constant (K) for HSA was found to be 9.9 x 10(4)M(-1), 4.3 x 10(4)M(-1) for RSA, 1.07 x 10(4)M(-1) for PSA and 0.3 x 10(4)M(-1) for BSA and the number of binding sites (n) were 1.14, 1.06, 0.94 and 0.8, respectively, which is very significant. By using unfolding pathway of HSA in the presence of urea, domain II of HSA has been assigned to possess binding site of ThT. Its binding constant is comparable to many drugs that bind at domain II of HSA, like salicylate, warfarin, digitoxin, etc. Acting force between HSA and ThT is showing that both hydrophobic and electrostatic forces have contributed for the interaction. DeltaG(binding), DeltaH and DeltaS were calculated to be -28.46 kJ mol(-1), -3.50 kJ mol(-1) and 81.04 JK(-1)mol(-1), respectively. The data described here will help to increase our understanding about the interaction of ThT with native proteins. The results also indicate that care must be taken while using ThT as a probe for detecting amyloid fibrils.

Spectroscopic studies on the comparative interaction of cationic single-chain and gemini surfactants with human serum albumin.

To gain insights into the comparative effect of single-chain/gemini surfactants on proteins and the possible implications, the interaction of human serum albumin (HSA) with cationic single-chain surfactant cetyltrimethylammonium bromide (CTAB) and its gemini counterpart bis(cetyldimethylammonium)butane dibromide with spacer -(CH(2))(4)- (designated as G4) using turbidity measurements, far-UV and near-UV circular dichroism (CD), intrinsic fluorescence and extrinsic fluorescence spectroscopy at pH 7.0 are reported in this contribution. A decrease of 33.5% alpha-helical content at 22.5 microM G4 was monitored compared to a 15% decrease at 2,250 microM CTAB. Against a 3.5% increase at 11,250 microM CTAB, a rise of 21.1% in the alpha-helical content was observed 375 microM G4. The result is related to the stronger electrostatic and hydrophobic forces in G4, owing to the presence of two charged headgroups and two hydrophobic hydrocarbon tails that make it to bind strongly to the protein compared to its single chain counterpart, CTAB, resulting in larger unfolding. The stabilization at higher concentrations is attributed to the highly hydrophobic microdomain of the G4 aggregates formed at such concentrations. The results of the multi-technique approach are consistent with the fact that the gemini surfactants are more efficient than their conventional single-chain counterparts and hence may be used more effectively in the renaturation of proteins produced in the genetically engineered cells via the artificial chaperone protocol, as solubilizing agents to recover proteins from insoluble inclusion bodies and in drug delivery.

Structural Stability as a Probe for Molecular Evolution of Homologous Albumins Studied by Spectroscopy and Bioinformatics

Equilibrium unfolding by guanidinium hydrochloride (GuHCl) and urea as well as evolutionary trends of two homologous albumins, pig serum albumin (PSA) and rabbit serum albumin (RSA), has been studied with circular dichroism, tryptophanyl fluorescence and bioinformatics. GuHCl cannot distinguish the contribution of electrostatic interactions to the proteins which were otherwise effectively monitored by urea. Higher differences in free energy changes due to urea than GuHCl show electrostatic interactions among charged amino acids are possibly responsible for higher structural stability of RSA in comparison to PSA. From the sequence of HSA and RSA, deletion of arginine at position 117 and the presence of one extra tryptophan at position 135 may possess some clue for lesser stability of PSA. Here, for comparison, chemical unfolding data of HSA and BSA had been taken into consideration. We found that thermodynamically RSA and PSA are closer to HSA and BSA, respectively, in accordance with their sequence homologies. Taxonomically, rabbit belongs to lagomorph which is closer to hominids than ungulates. Hence, on the basis of these thermodynamic data of protein denaturation of different species we can use this new approach to analyze the phylogenetic relationship among the major clades of eutherian mammals to obtain their evolutionary trends.

2,2,2-Trifluroethanol induces simultaneous increase in α-helicity and aggregation in alkaline unfolded state of bovine serum albumin

Little work has been done to understand the folding of proteins at alkaline conditions. BSA acquires a partially reversible unfolded state at pH 13.0, devoid of any native structure. Introduction of methanol, ethanol and 2-propanol with the alkaline unfolded protein resulted in beta-sheet-like structure formation, and 2,2,2-trifluroethanol found to enhance alpha-helical conformations with simultaneous increase in aggregation. The extent of secondary and tertiary structure formation is in the order of methanol < ethanol < 2-propanol < 2,2,2-trifluroethanol. Exposure of hydrophobic core of protein molecules in apolar environment of 2,2,2-trifluroethanol seems to promote intermolecular cluster formation. This is one of the very few reports that alpha-helical structures can also aggregate.

How methyl cyanide induces aggregation in all-alpha proteins: A case study in four albumins

Serum albumins are chief carrier of ligands in blood, hence important in clinical biotechnology. The effects of methyl cyanide (MeCN), a chief solvent of reverse phase chromatography, on four mammalian serum albumins (human, bovine, porcine and rabbit sources) were studied at neutral pH with the help of scattering, circular dichroism, IR and fluorescence spectroscopy. We have detected an intermediate state in the presence of 20% (v/v) MeCN, having 8-9% higher alpha-helical structure than that of their native states. In the presence of 60% (v/v) MeCN another intermediate was observed with non-native beta-sheet structure and high tendency to form aggregates.

Plasmodium vivax tryptophan-rich antigen PvTRAg33.5 contains alpha helical structure and multidomain architecture.

Tryptophan-rich proteins from several malarial parasites have been identified where they play an important role in host-parasite interaction. Structural characterization of these proteins is needed to develop them as therapeutic targets. Here, we describe a novel Plasmodium vivax tryptophan-rich protein named PvTRAg33.5. It is expressed by blood stage(s) of the parasite and its gene contains two exons. The exon 1 encodes for a 23 amino acids long putative signal peptide which is likely to be cleaved off whereas the exon 2 encodes for the mature protein of 252 amino acids. The mature protein contains B-cell epitopes which were recognized by the human immune system during P.vivax infection. The PvTRAg33.5 contains 24 (9.5%) tryptophan residues and six motifs whose patterns were similar among tryptophan-rich proteins. The modeled structure of the PvTRAg33.5 consists of a multidomain architecture which is stabilized by the presence of large number of tryptophan residues. The recombinant PvTRAg33.5 showed predominantly α helical structure and alpha helix to beta sheet transition at pH below 4.5. Protein acquires an irreversible non-native state at temperature more than 50°C at neutral pH. Its secondary and tertiary structures remain stable in the presence of 35% alcohol but these structures are destabilized at higher alcohol concentrations due to the disturbance of hydrophobic interactions between tryptophanyl residues. These structural changes in the protein might occur during its translocation to interact with other proteins at its final destination for biological function such as erythrocyte invasion.

Characterization of Fluoroalcohols-Induced Intermediates of Mucor miehei Lipase at Low pH

We have previously characterized an acid-unfolded (U(A)) state of Mucor miehei lipase at pH 2. The effect of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) resulted in characterization of molten-globule (MG) like states with beta-sheet secondary structure at 15% (v/v) TFE and 6% (v/v) HFIP. alpha-Helical states accumulate at 80% (v/v) TFE and 30% (v/v) HFIP.

Unveiling the stimulatory effects of tartrazine on human and bovine serum albumin fibrillogenesis: Spectroscopic and microscopic study

Amyloid fibrils are playing key role in the pathogenesis of various neurodegenerative diseases. Generally anionic molecules are known to induce amyloid fibril in several proteins. In this work, we have studied the effect of anionic food additive dye i.e., tartrazine (TZ) on the amyloid fibril formation of human serum albumins (HSA) and bovine serum albumin (BSA) at pHs7.4 and 3.5. We have employed various biophysical methods like, turbidity measurements, Rayleigh Light Scattering (RLS), Dynamic Light Scattering (DLS), intrinsic fluorescence, Congo red assay, far-UV CD, transmission electron microscopy (TEM) and atomic force microscopy (AFM) to decipher the mechanism of TZ-induce amyloid fibril formation in both the serum albumins at pHs7.4 and 3.5. The obtained results suggest that both the albumins forms amyloid-like aggregates in the presence of 1.0 to 15.0mM of TZ at pH3.5, but no amyloid fibril were seen at pH7.4. The possible cause of TZ-induced amyloid fibril formation is electrostatic and hydrophobic interaction because sulfate group of TZ may have interacted electrostatically with positively charged amino acids of the albumins at pH3.5 and increased protein-protein and protein-TZ interactions leading to amyloid fibril formation. The TEM, RLS and DLS results are suggesting that BSA forms bigger size amyloids compared to HSA, may be due to high surface hydrophobicity of BSA.