Ganglioside GM1 Drives Hemin and Protoporphyrin Adsorption in Phospholipid Membranes: A Structural Study.

Monosialoganglioside (GM1), a ubiquitous component of lipid rafts, and hemin, an integral part of heme proteins such as hemoglobin, are essential to the cell membranes of brain neurons and erythrocyte red blood cells for regulating cellular communication and oxygen transport. Protoporphyrin IX (PPIX) and its derivative hemin, on the contrary, show significant cytotoxic effects when in excess causing hematological diseases, such as thalassemia, anemia, malaria, and neurodegeneration. However, the in-depth molecular etiology of their interactions with the cell membrane has so far been poorly understood. Herein, the structure of the polymer cushion-supported lipid bilayer (SLB) of the binary mixture of phospholipid and GM1 in the presence of PPIX and its derivative hemin has been investigated to predict the molecular interactions in model phospholipid membranes. A high-resolution synchrotron-based X-ray scattering technique has been employed to explore the out-of-plane structure of the assembly at different compositions and concentrations. The structural changes have been complemented with the isobaric changes in the mean molecular area obtained from the Langmuir monolayer isotherm to predict the additive-induced membrane condensation and fluidization. PPIX-induced fluidization of phospholipid SLB without GM1 was witnessed, which was reversed to condensation with 2-fold higher structural changes in the presence of GM1. A hemin concentration-dependent linear condensing effect was observed in the pristine SLB. The effect was significantly reduced, and the linearity was observed to be lost in the mixed SLB containing GM1. Our study shows that GM1 alters the interaction of hemin and PPIX with the membrane, which could be explained with the aid of hydrophobic and electrostatic interactions. Our study indicates favorable and unfavorable interactions of GM1 with PPIX and hemin, respectively, in the membrane. The observed structural changes in both SLB and the underlying polymer cushion layer lead to the proposal of a molecule-specific interaction model that can benefit the pharmaceutical industries specialized for drug designing. Our study potentially enriches our fundamental biophysical understanding of neurodegenerative diseases and drug-membrane interactions.

Spectrin: an alternate target for cytoskeletal drugs.

Cytoskeletal drugs having enormous therapeutic potential act on the cytoskeletal components like actin, tubulin either by promoting polymerization or destabilizing the same. Here we present the interaction of the popular cytoskeletal drugs such as taxol, latrunculin and cytochalasin with spectrin, a huge protein with multi domains that forms the cytoskeletal network. Particularly, the actin binding domain of spectrin regulates the dynamics of the actin cytoskeleton. We followed the binding of these drugs to its actin binding domain and intact spectrin as well. These drugs bind with moderate affinity (Kb ∼ 104 M-1) and the interaction with actin binding domain is entropy driven and hydrophobic in nature as determined by Van't Hoff plot. The docking studies and molecular dynamics simulations further corroborate the experimental findings. Particularly the higher binding constants in the case of latrunculin and cytochalasin to the actin binding domain of spectrin suggest the binding sites are presumably located in its actin binding domain.Communicated by Ramaswamy H. Sarma.

Structural Flexibility of Proteins Dramatically Alters Membrane Stability─A Novel Aspect of Lipid-Protein Interaction.

Protein isoforms are structural variants with changes in the overall flexibility predominantly at the tertiary level. For membrane associated proteins, such structural flexibility or rigidity affects membrane stability by playing modulatory roles in lipid-protein interaction. Herein, we investigate the protein chain flexibility mediated changes in the mechanistic behavior of phospholipid model membranes in the presence of two well-known isoforms, erythroid (ER) and nonerythroid (NER) spectrin. We show dramatic alterations of membrane elasticity and stability induced by spectrin in the Langmuir monolayers of phosphatidylocholine (PC) and phosphatidylethanolamine (PE) by a combination of isobaric relaxation, surface pressure-area isotherm, X-ray scattering, and microscopy measurements. The NER spectrin drives all monolayers to possess an approximately equal stability, and that required 25-fold increase and 5-fold decrease of stability in PC and PE monolayers, respectively. The untilting transition of the PC membrane in the presence of NER spectrin observed in X-ray measurements can explain better membrane packing and stability.

Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins.

Hemoglobin oxidation due to oxidative stress and disease conditions leads to the generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the red blood cell (RBC) membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2', 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-ß-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.

Comparative Analysis of Tryptophan Dynamics in Spectrin and Its Constituent Domains: Insights from Fluorescence.

Spectrin is a cytoskeletal protein ubiquitous in metazoan cells that acts as a liaison between the plasma membrane and the cellular interior and imparts mechanical stability to the plasma membrane. Spectrin is known to be highly dynamic, with an appreciable degree of torsional and segmental mobility. In this context, we have earlier utilized the red edge excitation shift (REES) approach to report the retention of restricted solvation dynamics and local structure in the vicinity of spectrin tryptophans on urea denaturation and loss of spectrin secondary structure. As a natural progression of our earlier work, in this work, we carried out a biophysical dissection of tryptophan solvation and rotational dynamics in spectrin and its constituent domains, in order to trace the origin of local structure retention observed in denatured spectrin. Our results show that the ankyrin binding domain (and, to a lesser extent, the ß-tetramerization domain) is capable of retention of local structure, similar to that observed for intact spectrin. However, all α-chain domains studied exhibit negligible retention of local structure on urea denaturation. Such a stark chain-specific retention of local structure could originate from the fact that the ß-chain domains possess specialized functions, where conservation of local (structural) integrity may be a prerequisite for optimum cellular function. To the best of our knowledge, these observations represent one of the first systematic biophysical dissections of spectrin dynamics in terms of its constituent domains and add to emerging literature on comprehensive domain-based analysis of spectrin organization, dynamics, and function.

Thermodynamics of adsorption of alcohol dehydrogenase on the gold nanoparticle surface: a model based analysis versus direct measurement.

Characterization of the nanoparticle protein corona has gained tremendous importance lately. The parameters which quantitatively establish a specific nanoparticle-protein interaction need to be measured accurately since good quality data are necessary for the elucidation of the underlying mechanism and accurate molecular dynamics simulation. Here, we have employed surface sensitive second harmonic light scattering (SHLS) for investigating the adsorption of a tetrameric protein, alcohol dehydrogenase (ADH, Saccharomyces cerevisiae 147 kDa), on 16 nm, 27 nm, 41 nm, and 69 nm citrate capped gold nanoparticles (GNPs) in aqueous phosphate buffer at pH 7. We have extracted the binding constant, number of ADH bound per GNP, Gibbs free energy (ΔG°) from the decay of the second harmonic scattered signal as a function of protein concentration using a modified version of the Langmuir adsorption isotherm. The data obtained were checked with another technique, dynamic light scattering, using the same modified Langmuir model (MLM). While the binding constants measured by the two methods are in agreement, the number of ADH bound to each GNP obtained by the two methods varies a lot. In order to further probe this binding independent of a model fitting, we used an orthogonal fluorescence assay which measures the number of ADH bound to a GNP directly, and no model-fitting is necessary. We then used temperature dependent SHLS to measure the heat of adsorption (ΔH°) and entropy (ΔS°) for ADH-GNP corona formation. We found that the equilibrium binding constant (Kb) obtained from SHLS is of the order of 109 M-1 and the formation of the GNP-ADH corona is spontaneous with ΔG° ∼ -55 kJ mol-1. However, the adsorption is modestly endothermic, accompanied by a large increase in entropy. Stated differently, GNP-ADH corona formation is entropically driven. This is perhaps due to the tremendous disruption of the water structure at the negatively charged interface upon the arrival of the protein within the bonding distance to it. We believe that the SHLS technique is highly sensitive and reliable, at very low concentrations of both nanoparticles and proteins, for the quantitative estimation of the thermodynamic parameters of nanoparticle-protein corona formation, where many other techniques may fall short.

Phospholipid Asymmetry in Biological Membranes: Is the Role of Phosphatidylethanolamine Underappreciated?

The asymmetric distribution of phospholipids in cell membranes has been the focus of a lot of important research keeping its biological importance in mind. Most of this research is focused on phosphatidylserine (PS) since it is an apoptotic marker, and there is a robust and easy method available its selective quantification. The aim of this commentary is to argue in favour of another highly abundant membrane lipid, phosphatidylethanolamine (PE) almost always associated with PS. PE has one of the smallest headgroups and shows distinctly asymmetric transbilayer distribution. It is a neutral aminophospholipid and capable of a vastly wider range of interactions as seen in its unique ability to act as a molecular chaperone, implicated role in disease biology and its possible role as an anti-cancer target. There are ample evidences to the fact that PE may also bind to Annexin V (ANV), the PS-specific probe, at higher than 10 mol% PE concentrations and absence of Ca2+ ions. An update of the major takeaways from the literature regarding PE asymmetry is also provided.

Effects of free soluble iron on thermal aggregation of hemoglobin.

Aggregation of hemoglobin is implicated in the presentation of diseases like sickle cell disease and thalassemia. Hallmark of the disease being imbalance in the production of globin chains leading to aggregation of excess globin chains and aberrant hemoglobins associated with the disease, broadly categorized as hemoglobinopathy. We have studied thermal aggregation of hemoglobin at 70 °C and pH 6.5 using light scattering, flow cytometry and optical microscopy and tried to investigate effects of few abundant soluble metal ions on such aggregation. Our study indicate that only iron, both in Fe2+ and Fe3+ forms, could inhibit hemoglobin aggregation and the extent of inhibition was 60% in presence of 100 mgL-1 FeCl3. Similar effect was not seen in lysozyme aggregation. Metal ions such as, Cu2+, Zn2+ and Ni2+ also did not have any significant effects on hemoglobin aggregation. Results show this important chaperone like behavior of free iron affecting the kinetics and yield of the aggregation process which could have important consequence in the extent of severity of such hematological diseases.

Multiple Functions of Spectrin: Convergent Effects.

Spectrin is a multifunctional, multi-domain protein most well known in the membrane skeleton of mature human erythrocytes. Here we review the literature on the crosstalk of the chaperone activity of spectrin with its other functionalities. We hypothesize that the chaperone activity is derived from the surface exposed hydrophobic patches present in individual "spectrin-repeat" domains and show a competition between the membrane phospholipid binding functionality and chaperone activity of spectrin. Moreover, we show that post-translational modifications such as glycation which shield these surface exposed hydrophobic patches, reduce the chaperone function. On the other hand, oligomerization which is linked to increase of hydrophobicity is seen to increase it. We note that spectrin seems to prefer haemoglobin as its chaperone client, binding with it preferentially over other denatured proteins. Spectrin is also known to interact with unstable haemoglobin variants with a higher affinity than in the case of normal haemoglobin. We propose that chaperone activity of spectrin could be important in the cellular biochemistry of haemoglobin, particularly in the context of diseases.

Chaperone potential of erythroid spectrin: Effects of hemoglobin interaction, macromolecular crowders, phosphorylation and glycation.

Spectrin, the major protein component of the erythrocyte membrane skeleton has chaperone like activity and is known to bind membrane phospholipids and hemoglobin. We have probed the chaperone activity of spectrin in presence of hemoglobin and phospholipid SUVs of different compositions to elucidate the effect of phospholipid/hemoglobin binding on chaperone function. It is seen that spectrin displays a preference for hemoglobin over other substrates leading to a decrease in chaperone activity in presence of hemoglobin. A competition is seen to exist between phospholipid binding and chaperone function of spectrin, in a dose dependent manner with the greatest extent of decrease being seen in case of phospholipid vesicles containing aminophospholipids e.g. PS and PE which may have implications in diseases like hereditary spherocytosis where mutation in spectrin is implicated in its detachment from cell membrane. To gain a clearer understanding of the chaperone like activity of spectrin under in-vivo like conditions we have investigated the effect of macromolecular crowders as well as phosphorylation and glycation states on chaperone activity. It is seen that the presence of non-specific, protein and non-protein macromolecular crowders do not appreciably affect chaperone function. Phosphorylation also does not affect the chaperone function unlike glycation which progressively diminishes chaperone activity. We propose a model where chaperone clients adsorb onto spectrin's surface and processes that bind to and occlude these surfaces decrease chaperone activity.

Localizing the chaperone activity of erythroid spectrin.

Spectrin, the major protein of the erythrocyte membrane skeleton has canonically been thought to only serve a structural function. We have previously described a novel chaperone-like property of spectrin and also hypothesized that the chaperone activity and binding of a hydrophobic ligand, Prodan are localized in the self-association domain. Here we probe the location and molecular origin of the chaperone activity of multi-domain spectrin using a selection of individual recombinant spectrin domains, which we have characterized using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Aggregation assays using insulin, ADH, α- and ß-globin as well as enzyme refolding assays using alkaline phosphatase and α-glucosidase show that the chaperone activity is not only localized in the self-association domain but is a generalized property of spectrin domains. This is to our understanding, a unique feature in the case of modular multi-repeat proteins, possibly implicating that the large family of "spectrin-repeat" domain containing proteins may also have chaperone like property. Substrate selectivity of chaperone activity as evidenced by the preferential protection of α- over ß-globin chains is seen; which has implications in hemoglobin diseases. Moreover, enzyme-refolding assays also indicate alternate modes of chaperone action. We propose that the molecular origin of chaperone activity resides in the surface exposed hydrophobic patches of the spectrin domains as shown by ANS (1-anilinonaphthalene-8-sulfonic acid) and Prodan (6-propionyl-2[dimethylamino]-naphthalene) binding. We also show that Prodan does indeed have a unique binding site on spectrin located at the self-association domain.

Effects of GM1 on brain spectrin-aminophospholipid interactions.

Spectrin, a major component of the membrane skeletal meshwork of metazoan cells, is implicated to associate with membrane domains and is known to act as a scaffold for stabilization and activation of different signalling modules. We have studied the effect of GM1 (monosialotetrahexosyl ganglioside), a well-known model ganglioside and a signalling moiety, on the interaction of non-erythroid brain spectrin with both saturated and unsaturated aminophospholipids by spectroscopic methods. We observe that GM1 modulates brain spectrin-aminophospholipid interaction to the greatest degree whereas its effect on erythroid spectrin is not as pronounced. Fluorescence quenching studies show that brain spectrin interacts with DMPC/DMPE-based vesicles with a 10-fold increased affinity in presence of very low amounts of 2% and 5% GM1, and the extent of quenching decreases progressively in presence of increasing amounts of GM1. Interaction of brain spectrin with unsaturated membrane systems of DOPC/DOPE weakens in presence GM1. Increase in the mean lifetime of the Trp residues of brain spectrin in presence of GM1 indicates change in the microenvironment of spectrin, without affecting the secondary structure of the protein significantly. Studies on pressure - area isotherm of Langmuir-Blodgett monolayer and Brewster's angle microscopy show that GM1 has an expanding effect on the aminophospholipid monolayers, and ordered regions in DMPC/DMPE mixed monolayers are formed and are stabilized at higher pressure. GM1-induced fluidization of the phospholipid membranes and probable physical contact between bulky sugar head group of GM1 and spectrin, may explain the modulatory role of GM1 on aminophospholipid interactions with nonerythroid brain spectrin.

Structure-activity relationship of heme and its analogues in membrane damage and inhibition of fusion.

Under pathological conditions, such as sickle cell disease and malaria, heme concentration increases considerably, and it induces membrane damage. As sickled and normal erythrocytes contain high cholesterol: phospholipid ratio, we investigated the role of lipid composition, chain length, and unsaturation on the partitioning and leakage of hemin in phospholipid vesicles. To establish structure-activity relationship in membrane damage, experiments with two other analogues, protoporphyrin-IX and hematoporphyrin (HP) were also carried out. Hemin and its analogues localize differently in membranes and exhibit distinct roles in partitioning, leakage and fusion. Hemin and HP trigger more leakage in the presence of aminophospholipids, whereas cholesterol buffers the destabilizing effect remarkably. Inhibition of fusion by hemin further suggests its unexplored and important role in membrane trafficking, particularly under diseased conditions.

Status of Membrane Asymmetry in Erythrocytes: Role of Spectrin.

Spectrin-based proteinaceous membrane skeletal network has been found to be implicated in membrane disorders like hereditary spherocytosis (HS). HS greatly affects eryptosis via loss of membrane asymmetry which is seen to be the case in haemoglobin disorders like thalassemia and sickle cell disease as well. The biological implications of the status of membrane asymmetry are strongly correlated to spectrin interactions with aminophospholipids, e.g. PE and PS. Fluorescence and X-ray reflectivity (XRR) measurements of spectrin interactions with small unilamellar vesicles (SUVs) and cushioned bilayers of phospholipids, respectively, were studied. Both the XRR and fluorescence measurements led to the characterization of spectrin orientation on the surface of lipid bilayer of phosphatidylcholine (PC) and PC/aminophospholipid mixed membrane systems showing formation of a uniform layer of spectrin on top of the mixed phospholipid bilayer. Fluorescence studies show that spectrin interacts with PC and phosphatidylethanolamine (PE)/phosphatidylserine (PS) membranes with binding dissociation constants (Kd) in the nanomolar range indicating the role of spectrin in the maintenance of the overall membrane asymmetry of erythrocytes.

Flow Cytometric Analysis of Protein Aggregates.

BACKGROUND: Misfolding of proteins often leads to aggregation. Accumulation of diverse protein aggregates in various cells, tissue and organs is the hallmark of many diseases, such as Alzheimer's disease and Parkinson's disease. OBJECTIVES: The main objective of this study was to present a novel method of characterization of protein aggregates, associated with differential toxicity with different size and composition in vitro using flow cytometry. METHODS: A Beckman Coulter Epics XL flow cytometer with argon ion laser operating at 488 nm was used for flow cytometry analysis. The voltage and the gain settings for individual channels were set at high voltage and gain for the detections of autofluorescence, fluorescence of adsorbed Congo red, forward scattering (FSC) and side scattering (SSC) intensities from the aggregates of proteins and nanoparticles. Each sample was analyzed to characterize and quantify the number of aggregates with a limit of maximum 20,000 events. The flow cytometry data were analyzed using Flowing software version 2.5.1 and Origin 8.0. RESULTS: Autofluorescence and scattering intensities could distinguish between amyloid and nonamyloid aggregates. Dot plots of both side scattering (SSC) and forward scattering (FSC) intensities also showed characteristic fingerprint of both the types of aggregates when compared with those of well known nanoparticles of oxides of Fe and Cu. CONCLUSION: This work reports a novel, simple and robust flow cytometric method of characterization of protein aggregates of different size and composition which would find wider application in characterization of biomolecular aggregates, in general.

Protein-Protein Interaction Probed by Label-free Second Harmonic Light Scattering: Hemoglobin Adsorption on Spectrin Surface as a Case Study.

In this article, we have studied the binding of different naturally occurring hemoglobin (Hb) variants on erythrocyte skeletal protein, spectrin surface using the label free nondestructive second harmonic light scattering (SHLS) technique in aqueous buffer. Hemoglobin variants like sickle hemoglobin (HbS) and hemoglobin E (HbE) were chosen as they associate with sickle cell disease and HbEß-thalassemia, respectively, and their interaction with spectrin is compared with normal adult hemoglobin (HbA). The concentration dependent change in the second harmonic light intensity from nanomolar spectrin solution has been measured after addition of small aliquots of hemoglobins. From the second harmonic titration data, the binding constant is calculated using a modified Langmuir adsorption model of hemoglobin binding to the spectrin surface. Interestingly, it is found that the binding constant for HbE (13.8 × 108 M-1) is 1 order of magnitude higher than that of HbS (1.6 × 108 M-1) or HbA (2.1 × 108 M-1) which indicates higher affinity of HbE for spectrin compared to HbA and HbS. The number of the Hb molecules bound to the spectrin surface was estimated to be of the order of hundred's which is determined for the first time.

Substrate specificity in the context of molecular chaperones.

Molecular chaperones are one of the key players in protein biology and as such their structure and mechanism of action have been extensively studied. However the substrate specificity of molecular chaperones has not been well investigated. This review aims to summarize what is known about the substrate specificity and substrate recognition motifs of chaperones so as to better understand what substrate specificity means in the context of molecular chaperones. Available literature shows that the majority of chaperones have broad substrate range and recognize non-native conformations of proteins depending on recognition of hydrophobic and/or charged patches. Based on these recognition motifs chaperones can select for early, mid or late folding intermediates. Another major contributor to chaperone specificity are the co-chaperones they interact with as well as the sub-cellular location they are expressed in and the inducability of their expression. Some chaperones which have only one or a few known substrates are reported. In their case the mode of recognition seems to be specific structural complementarity between chaperone and substrate. It can be concluded that the vast majority of chaperones do not show a high degree of specificity but recognize elements that signal non-native protein conformation and their substrate range is modulated by the context they function in. However a few chaperones are known that display exquisite specificity of their substrate e.g. mammalian heat shock protein 47 collagen interaction. © 2017 IUBMB Life, 69(9):647-659, 2017.

Effect of pH on stability, conformation, and chaperone activity of erythroid & non-erythroid spectrin.

Spectrin, a major component of the eukaryotic membrane skeleton, has been shown to have chaperone like activity. Here we investigate the pH induced changes in the structure and stability of erythroid and brain spectrin by spectroscopic methods. We also correlate these changes with modulations of chaperone potential at different pH. We have followed the pH induced structural changes by circular dichroism spectroscopy and intrinsic tryptophan fluorescence. It is seen that lowering the pH from 9 has little effect on structure of the proteins till about pH6. At pH4, there is significant change of the secondary structure of the proteins, along with a 5nm hypsochromic shift of the emission maxima. Below pH4 the proteins undergo acid denaturation. Probing exposed hydrophobic patches on the proteins using protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates that there is higher solvent accessibility of hydrophobic surfaces in both forms of spectrin at around pH4. Dynamic light scattering and 90° light scattering studies show that the both forms of spectrin forms oligomers at pH~4. Chemical unfolding data shows that these oligomers are less stable than the tetrameric form. Aggregation studies with BSA show that at pH4, both spectrins exhibit better chaperone activity. This enhancement of chaperone like activity appears to result from an increase in regions of solvent-exposed hydrophobicity and oligomeric state of the spectrins which in turn are induced by moderately acid pH. This may have in-vivo implications in cells facing stress conditions where cytoplasmic pH is lowered.

Cholesterol-Induced Structural Changes in Saturated Phospholipid Model Membranes Revealed through X-ray Scattering Technique.

Lateral and out-of-plane organization of cholesterol and its effect on regulating the physicochemical properties of zwitterionic phospholipid model membranes have been investigated by a pressure-area isotherm study from the Langmuir monolayer, atomic force microscopy (AFM), and X-ray reflectivity (XRR) measurements from supported binary monolayer films. The systematic isotherm studies on the Langmuir monolayer of phospholipids and the subsequent extraction of excess Gibbs free energy (ΔGexc) revealed the mechanism of cholesterol interaction and the molecular cooperativeness for different arrangements in the phospholipid model membranes. We have found a critical cholesterol molar concentration (χc) up to which the lipid-cholesterol miscibility gradually increases and then further increase in the concentration leads to an inhomogeneous structure formation similar to raft structures. The thickening in the lipid acyl chain and the subsequent lowering of the lipid head group thickness up to χc are also evident from the XRR study. Beyond χc, large-sized domains are observed in the AFM images from the deposited monolayer. χc has also been observed to depend on the phase of the monolayer, in particular, ∼25 molar % in the gel phase and ∼40 molar % in the fluid phase, wherein a regular distribution has been found with the highest separation between the cholesterol molecules. The extracted isothermal compressibility coefficient (CS) and ΔGexc from the monolayer isotherms indicate that the molecular arrangement at χc are the most stable configurations of the monolayer. Our study provides direct evidence into cholesterol-induced evolution in phase behavior and the consequent model on the structure at different phases in the phospholipid Langmuir monolayers.

Enzyme Adsorption on Nanoparticle Surface Probed by Highly Sensitive Second Harmonic Light Scattering.

Recent developments in second harmonic light scattering technique and the associated theoretical models have provided a deeper insight of molecular interactions on micro- and nanoparticle surfaces. This technique is extended to probe the thermodynamics of protein adsorption on nanoparticle surface which is crucial for understanding the fate of nanoparticle-based formulations in biomedical applications. A modified Langmuir adsorption model has been applied to extract the thermodynamic parameters from the experimental data. The general applicability of the technique is established by extracting free energy change, association constant, and binding stoichiometry of adsorption of a moderate size protein, alcohol dehydrogenase, and a small size protein, insulin, on gold nanoparticles. The free energy change for the adsorption is found to be of the order of -55kJ/mol, which indicates that the interaction of proteins with the nanoparticle surface involves weak forces. On the other hand, the low value of the free energy change makes the detachment of the protein from the particle surface easier and guarantees reversibility of the binding process. In addition, one gets the binding stoichiometry of the proteins with the nanoparticle surface which opens up the possibility of controlling the payload of the protein- or peptide-based therapeutics in future biomedical applications.