Conformational transitions of cytochrome c in sub-micron-sized capsules at air/buffer interface.

This work presents the design of sub-micron-sized capsules of Cytochrome c (cyt c) in the range 300-350 nm and the conformational transitions of the protein that occur when the films of these capsules spread at the air/buffer interface are subjected to repeated compression-expansion cycles. Steady state fluorescence, time-resolved fluorescence, and circular dichroic (CD) spectra have been used to study the highly compact native conformation (70% helicity) of the protein in the capsules and its stability has been analyzed using cyclic voltammetry. The capsules have been characterized using zeta sizer and high resolution transmission electron microscopy (HRTEM). Surface concentration-surface pressure (Γ-π) isotherms of the films of the capsules spread at air/buffer interface following compression-expansion show destabilizing effect on cyt c. FTIR and CD spectra of these films skimmed from the surface show that the protein transitions gradually from its native helical to an anomalous beta sheet aggregated state. This results from a competition between stabilizing hydrated polar segments of the protein in the capsule and destabilizing nonspecific hydrophobic interactions arising at the air/buffer interface. This 2D model could further our understanding of the spatial and temporal roles of proteins in confined spaces and also in the design of new drug delivery vehicles using proteins.

Interaction of chromium(III) complexes with model lipid bilayers: implications on cellular uptake.

To understand molecular cytotoxicity of chromium(III) and how it affects the stability of biological membranes, studies on the interaction of chromium(III) complexes aquapentaminechromium complex (complex I) and trans- [Cr(5-methoxysalcyclohex) (H(2)O) (2)] ClO(4) (complex II) with model biomembranes have been carried out. Langmuir films of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidic acid (DPPA), dioctadecyldimethylammoniumbromide (DOMA) at air/water interface interacting with the chromium(III) complexes have been characterized using the surface pressure-molecular area (π-A) isotherms. Initial surface pressures changes for the two complexes show that the chromium(III) complexes inserted in the Langmuir films and complex I interacted strongly compared to complex II. Supported bilayers (SB) of the lipids on solid substrates formed by hydrating their Langmuir-Blodgett films (LB films) have been characterized using linear dichroic spectra, low angle X-ray diffraction and steady state fluorescence anisotropy. Depending on the geometry of the ligands and concentration, the complexes either insert in the alkyl or in the head group region of the SB and sometimes in both regions. The Supported lipid bilayers are well-layered and at low concentration, the metal complexes are incorporated near the head group region. Order and increase in lamellar spacing show stronger interaction of complex I with the lipids compared with complex II. This study provides some insights into the mechanism of chromium(III) toxicity and uptake of chromium(III) by the cells.

Physico-chemical studies of elastic compliance and adsorption of DOPC vesicles and its mixture with charged lipids at fluid/solid interface.

Lipid bilayer mechanics is crucial to membrane dynamics and in design of liposomes for delivery applications. In this work, vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (size from 50 nm to 1 µm) and its mixtures with anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DOPG) and cationic dimethyldioctadecylammonium bromide (DODAB), have been studied under shear stress at fluid/solid interface and their elastic compliance evaluated. Results show that the rate of spreading of the smaller vesicles (∼70 nm) is about 1.4 times slower than those of larger ones (∼1 µ) and that DOPC has the highest elastic compliance compared with DOPC + DOPG and DOPC + DODAB vesicles. A direct correlation between the elastic compliance and the size of the vesicles shows larger vesicles are more structurally labile during adsorption and subsequent adhesion to solid surfaces than the smaller ones. Specific role of bound water in DODAB is reflected in the lowest elastic compliance of DODAB compared to other lipids. Results show that during the process of adhesion at the fluid/air interface, the vesicles undergo contraction, thereby transmitting mechanical stresses to their microenvironment, which matches the SAXS electron density profiles that indicates larger vesicles have thicker bilayer membranes with larger volume of water compared to the smaller sized ones.

Hydrodynamically coupled water in surface adsorbed amino acids as a tool to study hydrated peptides.

The influence of different amino acid residues on properties of a protein surface is of great interest and importance. Hydrodynamically coupled water in the amino acids has the potential to be used as a tool to study surface properties of proteins. The contribution of this coupled water fraction in design of a hydropathy scale in surface adsorbed amino acid films on solid using quartz crystal microbalance is presented in this work. This scale compares well with the hydropathy scale of Guy reported in the literature and can be correlated with the solid/liquid interfacial tension and work of adhesion of the adsorbed amino acid films. Using Graphical Representation and Analysis of Surface Properties (GRASP) the free energy of transfer from Octanol to water for the amino acids has been estimated and shows approximately an inverse relationship with the coupled water fraction. This scale has been applied in a benchmark test for a native Laminin peptide YIGSR and its mutated sequences (with mutations carried out at 'Y and 'R' positions). The experimentally measured coupled water fractions seem to compare well with that obtained from the present scale assuming the total solvent fraction to be a linear function of the amino acids in the sequence. A survey of the protein data bank showed that sets of sequences based on this scale occur in membrane insertion domain or in trans-membrane proteins suggesting that the scale is suitable to study structure-function correlation in proteins.

Elastic compliance of fibrillar assemblies in type I collagen.

Fibrillary assemblies of Type I collagen find important applications in tissue engineering and as matrices for biophysical studies. The mechanical and structural properties of these structures are governed by factors such as protein concentration, temperature, pH and ionic strength. This study reports on an impedance based analysis of the elastic compliance of fibrillary assemblies of Type I collagen using quartz crystal microbalance with dissipation (QCM-D) at a fundamental frequency of 5 MHz and overtones (n = 3,5,7,9,11). Here, In situ partial fibrillation of the adsorbing collagen followed by its fibrillary assemblies on hydrophilic gold coated quartz surface have been crosslinked using Gallic acid (GA), Chromium (III) gallate (Cr-GA), Catechin (Cat), Tetrakis(hydroxymethyl)phosphonium sulfate (THPS) and Oxazolidine (Ox). This approach allows direct comparison of how viscoelastic properties track the structural evolution of the fiber and network length scales. The collagen crosslinking shows significant positive impact on the protein's mechanical behaviour and on the type of crosslinking agents used. The elastic modulus increases as collagen 

Do properties of bovine serum albumin at fluid/electrolyte interface follow the Hofmeister series?--An analysis using Langmuir and Langmuir-Blodgett films.

Folding and solubility of proteins are dependent on their state of hydration. How does a protein-bovine serum albumin (BSA) behave in the presence of Hofmeister electrolytes, especially at interfaces? Langmuir films of bovine serum albumin (BSA) in the presence of different Hofmeister electrolytes at air/solution interface and as Langmuir-Blodgett films (LB films) at solid/solution interface have been studied using the surface pressure-molecular area (pi-A) isotherms and surface energy parameters. Changes in secondary structure have been analyzed using circular dichroism (CD) and fluorescence spectroscopy. Hydrodynamically coupled water fraction of BSA in different environments has been estimated using quartz crystal microbalance (QCM) and related to the secondary structural changes. Molecular modeling of BSA in different environments showed that the protein has a compact structure at the interface compared to vacuum. The contact areas estimated using molecular modeling agreed with the experimental results. The results show that the properties of BSA at the interface follow the Hofmeister series with NaF leading to maximum compaction in the protein. Further, in addition to ion specific solvation and different ion size, water structure alteration and the bound water fractions contribute importantly to the Hofmeister effect.

Towards understanding structure-stability and surface properties of laminin peptide YIGSR and mutants.

Properties of laminin peptide YIGSR and its mutated sequences YIGSD, YIGSS, YIGSN and YIGSQ have been investigated using molecular dynamics simulations (MDS) and Langmuir films at air/water interface. Simulation studies on laminin peptide YIGSR were performed in the isothermal-isobaric (N, P, T) ensemble, with run up to 5 ns in water as well as lipid environment at 298 K. From different initial configurations, shape transformations of the peptides on the timescale of nanoseconds were observed. The results showed YIGSR to be the most stable peptide with the order of minimized energy being YIGSR

Langmuir and Langmuir-Blodgett films of proline-rich N-terminal domain peptide of gamma-zein.

The proline-rich N-Terminal domain peptides of gamma-zein (VHLPPP)(n) with n=1 and 3 (peptides I and II) are shown to form stable Langmuir films at air/water interface and the films have been characterized using surface pressure-molecular area (pi-A), surface potential-molecular area (DeltaV-A) isotherms, respectively. The longer peptide sequence does not show dramatic increase in surface or interfacial properties suggesting that the minimum length of n=1 is sufficient to achieve the necessary surface properties. Brewster angle micrographs also agreed with these results. The high surface-active nature of the peptide suggests a fairly non-polar character at air/water interface and at solid/air interface when coated expresses a high surface energy. Additives such as isopropyl alcohol (IPA) and polyvinyl alcohol (PVA) with the peptides showed more homogenous films at the air/water interface and also improved mechanical and tensile properties. The organized assembly of peptide I at the air/water and solid/air interface suggests that even thin layer of the peptide could play an important role in coating the inner surface of protein body membrane in storage proteins. Composite films of such short peptides with biocompatible polymers may find applications as surface coatings and in biomaterials.

Influence of crosslinking agents on the pore structure of skin.

Analysis of pore structure of skin is important to understand process of diffusion and adsorption involved during any application of the skin matrix. In this study, the effect of thermal shrinkage on the pore structure of chromium and vegetable treated skin has been analyzed as these tanning agents are known to bring about thermal stability to the matrix. The changes brought about in the pore structure have been studied using mercury intrusion porosimetry and scanning electron microscopy. Response of the chromium treated and vegetable tanning treated skin structure to heat has been found to be quite different from each other. About 41% decrease in porosity is observed for chromium treated skin as against 97% decrease for the skin treated with vegetable tannins. This is primarily attributed to the basic nature of these materials and the nature of interaction of them towards skin.

Langmuir and Langmuir-Blodgett films of N-(4-octadecyloxy-2-hydroxybenzylidene) derivatives of amino acids.

Langmuir and Langmuir-Blodgett monolayers of N-(4-octadecyloxy-2-hydroxybenzylidene) derivatives of glycine, tyrosine, and phenylalanine were studied using pi-A isotherms and photoelastic modulated FTIR (PEM-FTIR). Based on compression modulus and interaction parameters, mixed monolayers of these compounds with stearylamine (SAM) showed well-organized monolayers compared to mixed systems with stearic acid (SA) and stearyl alcohol (SAL). The pure amphiphiles exhibited fairly well-ordered packing in the films, and in the mixtures, the ordering increased and showed a triclinic packing arrangement. For the phenylalanine amphiphile the packing showed slight disorder compared to the other two compounds. Surface properties of the LB films of these compounds on solid substrates were analyzed using static and dynamic contact angles of a series of liquids. The surface tension of coated substrates reflected clearly the highly acidic character. Fluidlike monolayers having a molecularly rough surface indicated high wettability for n-alkanes. In contrast, the monolayer containing well-ordered, well-packed alkyl chains indicated low wettability and small hysteresis.

Initiating fibro-proliferation through interfacial interactions of myoglobin colloids with collagen in solution.

This work examines fibro-proliferation through interaction of myoglobin (Mb), a globular protein with collagen, an extracellular matrix fibrous protein. Designed colloids of Mb at pH 4.5 and 7.5 have been mixed with collagen solution at pH 7.5 and 4.5 in different concentrations altering their surface charges. For the Mb colloids, 100-200nm sizes have been measured from Transmission electron micrographs and zeta sizer. CD spectra shows a shift to beta sheet like structure for the protein in the colloids. Interaction at Mb/Collagen interface studied using Dilational rheology, Quartz crystal microbalance with dissipation and Differential Scanning calorimetry show that the perturbation is not only by the charge compensation arising from the difference in pH of the colloids and collagen, but also by the organized assembly of collagen at that particular pH. Results demonstrate that positive Mb colloids at pH 4.5, having more% of entrained water stabilize the collagen fibrils (pH 7.5) around them. Ensuing dehydration leads to effective cross-linking and inherently anisotropic growth of fibrils/fibres of collagen. In the case of Mb colloids at pH 7.5, the fibril formation seems to supersede the clustering of Mb suggesting that the fibro-proliferation is both pH and hydrophilic-hydrophobic balance dependent at the interface.

Interaction of Myoglobin colloids with BSA in solution: Insights into complex formation and elastic compliance.

This work focusses on the supramolecular complex formed between Myoglobin (Mb) and Bovine Serum Albumin (BSA) at colloids/solution interface at pH 4.0 and pH 7.5. Electrostatic interactions between Mb as colloids and BSA solution (pH=7.5 and 4.0) have been confirmed by Zeta potential that suggest that while Mb has a narrow interaction range, BSA has a wider interaction space. The organization of Mb colloids in BSA characterized using dilational rheological parameters show that the Mb colloids are elastic and the strong adsorbed water layers on the surface restrict the deformation, regulated by the viscoelastic surface layer. Stability of the complexes analyzed using UV-vis, Fluorescence and Circular dichroic spectroscopy indicate that there is a 1:1 interaction between Mb and BSA with a binding constant of about 105M-1. Quartz Crystal microbalance with dissipation has been used to evaluate the elastic compliance of the complexes of Mb colloids dispersed in very dilute BSA solution. The higher elastic compliance at pH=4.0 (than at pH=7.5) and the complex sizes correlate with changes in zeta potential suggesting that the mechanical properties of the protein in colloids are dependent on both the electrostatic interaction as well as the degree of hydration of the colloids.

Simple coacervates of zein to encapsulate Gitoxin.

This work reports the use of simple coacervates of the hydrophobic protein zein to encapsulate Gitoxin, a cardiotonic glycoside. The microspheres obtained using ethanol, methanol, iso-propyl alcohol were characterized using viscosity index, scanning electron microscopy (SEM) and laser light scattering particle analyzer. Scanning electron micrographs indicated that the zein film was made of microspheres with diameter in the 1-1.5 microm range, which could be controlled. Sizes of Gitoxin-loaded zein microspheres changed little before and after release of the drug because of conglutination among zein microspheres. Release of Gitoxin from zein microspheres, were performed in vitro to investigate the mechanism of model drug release. The results show that the zein microspheres obtained using ethanol are best suited for use as a sustained-release form of Gitoxin. The microspheres may also be useful in drug targeting system since the diameter of the microspheres is appropriate for phagocytosis by macrophages. Both zein film and Gitoxin-loaded zein microsphere film were effective in suppressing platelet adhesion.

Amphiphilic laminin peptides at air/water interface--effect of single amino acid mutations on surface properties.

Amphiphilic derivative of the laminin peptide YIGSR and three other mutated peptides with mutation at Y with V (valine), I (isoleucine), and L (leucine) have been synthesized. The monolayer formation and the stability of these peptide analogues at air/water interface and the interaction with phospholipid monolayers have been studied using surface pressure-molecular area (pi-A) and surface potential-molecular area (DeltaV-A) isotherms. The single amino acid mutation in the native sequence leads to appreciable changes in surface activity, orientation and insertion into lipid monolayers with LIGSR showing most hydrophobic character while YIGSR showed most polar nature. The morphology of spread monolayers in the most close packed state was carried out using Brewster angle microscopy (BAM). LB films of these amphiphilic peptide derivatives transferred to hydrophilic quartz surfaces and hydrophobically modified surfaces showed significant changes in the work of adhesion as well as spreading behavior of water with the L substituted sequence showing maximum work of adhesion and the native sequence YIGSR, the least work of adhesion. From theoretical estimates, the long-range effects of the different amino acid residues in position 1 on the alkyl chains have been studied from charge on the carbon and hydrogen atoms of the alkyl tails. The present study demonstrates that amphiphilic derivatives of the laminin peptide YIGSR show enhanced activity compared to the original sequence. This work shows that the amino acid substituents on the head group clearly influence the distal methylene groups of the tail. Thus, any mutation of even single amino acid in a peptide sequence influences and plays an important role in determining macroscopic properties such as surface energy and adhesion both at air/solution and solid/solution interfaces.

Mucin at solution/air and solid/solution interfaces.

In this paper the surface activity of protein mucin at solution/air interface has been studied. The experiments of the adsorbed protein at solution/air interface have been carried out with a range of protein concentrations at a defined pH. The adsorption of the protein to solid surfaces and the degree of hydrophobicity at solid/solution interface of mucin have been evaluated at different pH and in the presence of Hofmeister electrolyte. The results from these studies have been further substantiated by surface potential measurements of mucin covered surface on stainless steel. Quartz crystal microbalance (QCM) has been used to follow the protein adsorption kinetics from solution to solid surface. The results from these measurements show that the adsorption behavior has a remarkable dependence on the degree of maximum coverage and is almost independent of the ionic strength. Other characteristic features such as maximum adsorption values at the protein isoelectric point (IEP4.7) and low-affinity isotherms that showed surface saturation even under unfavorable electrostatic conditions have been observed. The amount of mucin adsorbed in the presence of electrolytes has been estimated using electron spectroscopy for chemical analysis (ESCA). The study clearly shows that there exists an inverse relationship between the hydrophobicity and surface tension of the protein and also on the hydrated radius of Hofmeister electrolyte used.

Relevance of interfacial viscoelasticity in stability and conformation of biomolecular organizates at air/fluid interface.

Soft materials are complex macromolecular systems often exhibiting perplexing non-Newtonian viscoelastic properties, especially when the macromolecules are entangled, crowded or cross-linked. These materials are ubiquitous in the biology, food and pharma industry and have several applications in biotechnology and in the field of biosensors. Based on the length scales, topologies, flexibility and concentration, the systems behave both as liquids (viscous) and solids (elastic). Particularly, for proteins and protein-lipid systems, viscoelasticity is an important parameter because it often relates directly to stability and thermodynamic interactions of the pure biological components as well as their mixtures. Despite the large body of work that is available in solution macro-rheometry, there are still a number of issues that need to be addressed in dealing with proteins at air/fluid interfaces and with protein-polymer or protein-lipid interfaces that often exhibit very low interfacial viscosity values. Considering the important applications that they have in biopharmaceutical, biotechnological and nutraceutical industries, there is a need for developing methods that meet the following three specific issues: small volume, large dynamic range of shear rates and interfacial properties of different biomolecules. Further, the techniques that are developed should include Newtonian, shear thinning and yielding properties, which are representative of the different solution behaviors typically encountered. The review presented here is a comprehensive account of the rheological properties of different biomolecules at air/fluid and solid/fluid interfaces. It addresses the usefulness of 'viscoelasticity' of the systems at the interfaces analyzed at the molecular level that can be correlated with the microscopic material properties and touches upon some recent techniques in microrheology that are being used to measure the unusually low viscosity values sensitively.

Balancing soft elasticity and low surface polarity in films of charged BSA capsules at air/fluid interface.

Interaction between charged BSA colloids and the buffer at air/fluid interface has been studied using spread films of the capsules of the protein prepared at pH 4.5 and 7.5 (below and above the pI of BSA). Surface pressure-surface concentration plots, interfacial dilational rheology and Quartz crystal microbalance with dissipation have been used to characterize the films. The study shows that below the pI of the protein, the positively charged colloids entrain more water on the surface which leads to partial neutralization of the charges. Results suggest that the charged capsules are elastic due to the strongly adsorbed protein layers that restrict deformation and any small shape fluctuations is likely due to the distortion of the viscoelastic surface layer at pH=4.5. Capsules of BSA behave as 'soft elastic membrane' with interfacial properties lying between that of an elastic membrane and a slightly soluble diffuse capsule with low interfacial tension. Such elastic capsules would find applications in drug delivery and food colloids.

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.

Fusion of vesicles in manganese complex of a single-chain schiff base amphiphile--3-cyano-N-benzylidene hexadecylamine.

A single-chain amphiphile containing a rigid Schiff base segment, 3-cyano-N-benzylidene hexadecylamine (CNBHB) in the polar head group was synthesized and studied for its vesicle-forming properties. The dependence of the aggregation behavior of the vesicles as such and in the presence of manganese ions were studied as a function of temperature using differential scanning calorimetry and turbidity measurements. Transmission electron microscopy (TEM) was used to analyze the morphology of the vesicles, showed interesting features with fusion of regular structures, and were quite stable. In the presence of manganese ions, fusion of vesicles takes place. This could be due to the metal ions that are bound to the surface of the vesicles that cause a partial destruction of the hydration shell on the surface of the vesicles. The reduction in the hydration force could thus be responsible for the fusion.

Investigation of surface properties of amino acids: polarity scale for amino acids as a means to predict surface exposed residues in films of proteins.

It is of great interest and importance to study how different amino acid residues contribute to and affect the properties of proteins coated as films on solid surface. This work shows that the solid/liquid interfacial energy of surface localized amino acid films and their Gibbs energies of transfer at the air/solution interface have the potential to be used as a rapid and simple method for studying the surface properties of proteins. Based on these results, a new polarity scale for amino acids has been proposed. This scale is compared with existing hydropathy scales in a benchmark test using some proteins with solved 3D structure. The proteins were characterized in terms of surface-exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP). It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface.