Kamatchi Sankaranarayanan

Reversible and irreversible conformational transitions in myoglobin: role of hydrated amino acid ionic liquid.

Hydrated phenylalanine ionic liquid (Phe-IL) has been used to solubilize myoglobin (Mb). Structural stability of Mb in Phe-IL analyzed using fluorescence and circular dichroism spectroscopy shows that for low levels of hydration of Phe-IL there is a large red shift in the fluorescence emission wavelength and the protein transforms to complete β sheet from its native helical conformation. Rehydration or dilution reverses the β sheet to an α helix which on aging organizes to micrometer-sized fibrils. At concentrations higher than 200 μM, the protein changes from β to a more random coiled structure. Organization of the protein in Phe-IL in a Langmuir film at the air/water interface has been investigated using the surface pressure-molecular area isotherm and shows nearly the same surface tension for both pure Mb and Mb in Phe-IL. Scanning electron microscopy of the films of Mb in Phe-IL transferred using the Langmuir-Blodgett film technique show layered morphology. This study shows that the conformation of Mb is completely reversible going from β → helix → β sheet up to 200 μM of Phe-IL. Similar surface tension values for Mb in water and in Phe-IL suggests that direct ion binding interactions with the protein coupled with the change in local viscosity from the IL seems to not only alter the secondary structure of individual proteins but also drives the self-assembly of the protein molecules leading finally to fibril formation.

Interfacial Viscoelasticity of Myoglobin at Air/Water and Air/Solution Interfaces: Role of Folding and Clustering

This study describes the folding and organization of myoglobin (Mb) at the solution/air interface at different pH values of 2.5, 3.5, 5.5, 7.5, and 8.5. Dynamic surface tension and the associated dilational and shear viscoelasticity for Mb at these pHs have been studied using a sinusoidal surface compression and expansion for frequencies ranging from 0.01 to 0.4 Hz. The changes in dilational viscosity, elasticity, and fluorescence lifetime measurements have been related to the conformational changes of the protein films at the interface. It is observed that while acid-induced denaturation of the protein does not lead to large changes in dilational properties, the shear properties on the other hand are strongly influenced by it, and the protein behaves like a shear-thickening fluid. At higher pH, particularly at the isoelectric point, Mb is pseudoplastic indicating an increase in the shear viscosity. These results are strongly suggestive of formation of hydrophobic clusters at the protein-buffer interface because of the change in the overall charge distributions.

Microviscosity-induced conformational transition in β-lactoglobulin in the presence of an ionic liquid.

This study reports on the helix-beta conformation transition of bovine β-lactoglobulin (βLG) prepared at two different pH conditions (pH 4 and 7.5) and in the presence of the ionic liquid 1-ethyl-3-methylimidazolium ethyl sulfate (IL-emes). The investigation was carried out by combining a range of techniques such as circular dichroic (CD) spectroscopy, steady-state fluorescence spectroscopy, isothermal titration calorimetry (ITC), and transmission electron microscopy. The influence of microviscosity induced by IL-emes on the secondary structure of βLG was studied using a quartz crystal microbalance and correlated with the steady-state fluorescence emission. The effect of heat on the helix-beta transition in βLG was directly measured by ITC by titrating βLG with IL-emes. The net effect of heat after subtraction of the heat of dilution was negative in both cases, suggesting that the protein moves to a stable conformation. The changes in the overall aggregated structures were confirmed by transmission electron microscopy, where a shift in the size and morphology of aggregates was found, from large clusters (size of 70 nm) at pH 4 to smaller aggregates (size of 20 nm) at pH 7.5, which reduced to 7 nm in the presence of the IL. The transformation of helical to beta structure at pH 4 show that the folding pathway in the presence of the ionic liquid is hierarchical, whereas at neutral pH, it appeared to be nonhierarchical and the final native structure was acquired by nonlocal interactions through typical forces involved in the stabilization of the tertiary structure.

Assembling fibrinogen at air/water and solid/liquid interfaces using Langmuir and Langmuir-Blodgett films.

Langmuir films of pure fibrinogen (Fg) and Fg spread at the air/buffer interface and subphase containing electrolytes, NaCl, KCl, CaCl(2), and ZnCl(2), have been analyzed to understand the role of the surface/interface in mediating the organization of the protein eventually to fibrils. These films have been characterized by the surface pressure and surface potential-molecular area ((pi-A) and (DeltaV-A)) isotherms and Brewster angle microscopy (BAM). The Langmuir-Blodgett (LB) films of the protein transferred to the solid substrates have been characterized by scanning electron microscopy (SEM) and circular dichroism (CD). Our results suggest that fibrils are formed during organization at air/solution interface and also in LB films. The rate of formation of the fibril is the maximum for Fg with ZnCl(2). Adsorption of Fg to surfaces coated with a neutral lipid, dimyristoylphosphatidylcholine (DMPC), and a cationic lipid, dioctadecyldimethylammonium bromide (DOMA), from a range of solution concentrations has been studied using a quartz crystal microbalance (QCM). The work of adhesion of the protein on the solid surface shows fibril formation and positive adhesion for Fg in the presence of electrolytes. SEM results show that the adherent protein exhibits the widely reported nodulelike structure in the presence of CaCl(2) and ZnCl(2). These results provide definite evidence that specifically designed surfaces can promote adhesion of Fg and also activate fibril formation even in the absence of thrombin.

Role of viscogens on the macromolecular assemblies of fibrinogen at liquid/air and solid/air interfaces

In this study, an attempt has been made to understand the organization and association of fibrinogen (Fg) in solvent environment induced by viscogens such as 1-ethyl 3-methyl imidazolium ethyl sulfate (IL-emes), Ficoll, and Trehalose. The author observed that Fg in IL-emes adsorbed on solid surface shows higher β-sheet conformation. Shear viscosity measured using quartz crystal microbalance, for Fg in IL-emes was highest with a corresponding higher adsorbed mass 3.26 μg/cm(2). Associated assemblies of the protein at the liquid/air interface were monitored with changes in surface tension and were used to calculate work of adhesion. Changes in work of adhesion were used as a tool to measure the adsorption of Fg to solid surfaces in presence of viscogens and highest adsorption was observed for hydrophilic surfaces. Scanning electron microscopy images show Fg in trehalose forms elongated bead like structures implying organization of the protein at the interface. Crowding in the solvent environment induced by viscogens can slow down organization of Fg, leading to macromolecular assemblies near the interface.

Fibrils of Phenylalanine Adsorbed to Langmuir-Blodgett Films: Role of Lipids

Phenylalanine (F) aggregation and its organized assemblies seem to play an important role in amyloid structures associated with physiological impairments in diseases such as Alzheimer’s, Huntington’s, and Type II Diabetes. Recently, Phenylalanine in milli-molar levels has been reported to accumulate on the plasma, cerebrospinal fluid, and brain tissue leading to phenylketonuria (PK). This work demonstrates the role of hydrophilic and hydrophobic surfaces in organized assemblies of phenylalanine using different Langmuir-Blodgett films from stearic acid (SA), anionic (dipalmitoyl phosphatidyl glycerol [DPPG]), cationic (dioctadecyldimethylammoniumbromide [DOMA]), and neutral (Dioleyol phosphatidyl choline [DOPC]) and their supported lipid bilayers. CLSM shows the typical F aggregate pattern with fiber like morphology with diameters ranging from several nanometers to tens of micrometers on LB films of cationic lipid. Needle-like structures are formed on cationic and or neutral lipids and the rate of formation is controlled by choice of the mixed lipids. The contact angle titrations show hydrophobic surface is more effective in driving this organization process. This study shows that even very low concentrations of L-Phe (60 μM) can lead to fibrils and aggregation can be tuned by varying charge and hydrophobicity of the surface.

Influence of Ficoll on urea induced denaturation of fibrinogen

Ficoll is a neutral, highly branched polymer used as a molecular crowder in the study of proteins. Ficoll is also part of Ficoll-Paque used in biology laboratories to separate blood to its components (erythrocytes, leukocytes etc.,). Role of Ficoll in the urea induced denaturation of protein Fibrinogen (Fg) has been analyzed using fluorescence, circular dichroism, molecular docking and interfacial studies. Fluorescence studies show that Ficoll prevents quenching of Fg in the presence of urea. From the circular dichroism spectra, Fg shows conformational transition to random coil with urea of 6 M concentration. Ficoll helps to shift this denaturation concentration to 8 M and thus constraints by shielding Fg during the process. Molecular docking studies indicate that Ficoll interacts favorably with the protein than urea. The surface tension and shear viscosity analysis shows clearly that the protein is shielded by Ficoll.

Does l to d-amino acid substitution trigger helix → sheet conformations in collagen like peptides adsorbed to surfaces?

The present work reports on the structural order, self assembling behaviour and the role in adsorption to hydrophilic or hydrophobic solid surfaces of modified sequence from the triple helical peptide model of the collagenase cleavage site in type I collagen (Uniprot accession number P02452 residues from 935 to 970) using (D)Ala and (D)Ile substitutions as given in the models below: Model-1: GSOGADGPAGAOGTOGPQGIAGQRGVV GLOGQRGER. Model-2: GSOGADGP(D)AGAOGTOGPQGIAGQRGVVGLOGQRGER. Model-3: GSOGADGPAGAOGTOGPQG(D)IAGQRGVVGLOGQRGER. Collagenase is an important enzyme that plays an important role in degrading collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism by which this degradation occurs is not completely understood. Our results show that adsorption of the peptides to the solid surfaces, specifically hydrophobic triggers a helix to beta transition with order increasing in peptide models 2 and 3. This restricts the collagenolytic behaviour of collagenase and may find application in design of peptides and peptidomimetics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

Evaluation of hydropathy of amino acids from a comparison of their viscosities inside vesicles and on supported lipid bilayers

The viscosity of amino acids enclosed in giant lipid vesicles (η(out)) subjected to a shear flow near a solid surface has been studied using quartz crystal microbalance (QCM). This viscosity has been compared with shear viscosity for the different amino acids adsorbed on supported bilayers (SLBs) (η(in)) of the lipids on quartz. Using a first approximation of vesicles as model rigid spheres, the measured viscosities and the extent of deformation of vesicles observed using optical microscopy, two non-dimensional parameters: the reduced volume and the ratio of (η(in))/(η(out)) have been analyzed as a function of physical parameters: vesicle substrate distance (vesicle vs. supported lipid bilayers), vesicle size and their variation as a function of the viscosity. The kinematics of the vesicles with the amino acids compared with the shear at supported lipid bilayers seems to describe a reasonable hydropathy scale for the amino acids. The results show that there is a direct correlation between the above parameters and the polarity variations in amino acids suggesting that the viscous force may be an important parameter and should be taken into account in studies on membrane proteins interacting with cells and cell adhesion in flow chambers where cell membrane and the adhesive substrate are in relative motion.