Red Edge Excitation Shift Research Articles

Use of spectroscopic and zeta potential techniques to study the interaction between lysozyme and curcumin in the presence of silver nanoparticles at different sizes

This article describes, for the first time, the effect of three different sizes of silver nanoparticles on the binding of curcumin to lysozyme as examined by spectroscopic and zeta potential techniques at physiological conditions. The binding constants of curcumin to lysozyme in the presence of silver nanoparticles were measured. Based on the results of synchronous fluorescence and three-dimensional fluorescence spectroscopy, the presence of the different sizes of silver nanoparticles caused conformational changes in lysozyme during the binding of curcumin. Such changes were also observed when increasing the curcumin concentration. The results of fluorescence resonance energy transfer theory indicated that different sizes of silver nanoparticles could change the binding distance between curcumin and lysozyme. Based on the red edge excitation shift approach, we concluded that the limited mobility around the Trp residues decreased in the presence of silver nanoparticles with bigger size. Under resonance light scattering, the aggregation of curcumin on lysozyme in the presence of silver nanoparticles can play a major role in functional proteins.Communicated by Ramaswamy H. Sarma

Wavelength-Selective Fluorescence of a Model Transmembrane Peptide: Constrained Dynamics of Interfacial Tryptophan Anchors.

WALPs are prototypical, α-helical transmembrane peptides that represent a consensus sequence for transmembrane segments of integral membrane proteins and serve as excellent models for exploring peptide-lipid interactions and hydrophobic mismatch in membranes. Importantly, the WALP peptides are in direct contact with the lipids. They consist of a central stretch of alternating hydrophobic alanine and leucine residues capped at both ends by tryptophans. In this work, we employ wavelength-selective fluorescence approaches to explore the intrinsic fluorescence of tryptophan residues in WALP23 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. Our results show that the four tryptophan residues in WALP23 exhibit an average red edge excitation shift (REES) of 6nm, implying their localization at the membrane interface, characterized by a restricted microenvironment. This result is supported by fluorescence anisotropy and lifetime measurements as a function of wavelength displayed by WALP23 tryptophans in POPC membranes. These results provide a new approach based on intrinsic fluorescence of interfacial tryptophans to address protein-lipid interaction and hydrophobic mismatch.

Aggregation Behavior of pHLIP in Aqueous Solution at Low Concentrations: A Fluorescence Study.

pH (low) insertion peptide (pHLIP) is a 36-residue peptide derived from the third transmembrane helix of the membrane protein bacteriorhodopsin. The hydrophobicity of this peptide makes it prone to aggregation even at low concentrations, but this has not been studied in detail. In this work, we characterized monomeric and aggregated forms of pHLIP in aqueous solution (pH8) at low concentrations (~μM) using fluorescence-based approaches, complemented by circular dichroism (CD) spectroscopy. We show here that monomeric and aggregated pHLIP display differential red edge excitation shift (REES) and CD spectra. These spectroscopic features allowed us to show that pHLIP aggregates even at low concentrations. A detailed knowledge of the aggregation behavior of pHLIP under these conditions will be useful for monitoring and quantifying its interaction with membranes.

Constrained dynamics of the sole tryptophan in the third intracellular loop of the serotonin1A receptor

G protein-coupled receptors (GPCRs) are major signaling proteins in eukaryotic cells and are important drug targets. In spite of their role in GPCR function, the extramembranous regions of GPCRs are relatively less appreciated. The third intracellular loop (ICL3), which connects transmembrane helices V and VI, is important in this context since its crucial role in signaling has been documented for a number of GPCRs. Unfortunately, the structure of this loop is generally not visualized in x-ray crystallographic studies since this flexible loop is either stabilized using a monoclonal antibody or replaced with lysozyme. In this work, we expressed and purified the ICL3 region of the serotonin1A receptor and monitored its motional restriction and organization utilizing red edge excitation shift (REES) of its sole tryptophan and circular dichroism (CD) spectroscopy. Our results show that the tryptophan in ICL3 exhibits REES of 4 nm, implying that it is localized in a restricted microenvironment. These results are further supported by wavelength-selective changes in fluorescence anisotropy and lifetime. This constrained dynamics was relaxed upon denaturation of the peptide, thereby suggesting the involvement of the peptide secondary structure in the observed motional restriction, as evident from CD spectroscopy and apparent rotational correlation time. To the best of our knowledge, these results constitute one of the first measurements of motional constraint in the ICL3 region of GPCRs. Our results are relevant in the context of the reported intrinsically disordered nature of ICL3 and its role in providing functional diversity to GPCRs due to conformational plasticity.

Embedding a Metal-Binding Motif for Copper Transporter into a Lipid Bilayer by Cu(I) Binding.

Peptide-lipid interactions are widely involved with biologically significant phenomena, including the pathogenic mechanisms of protein misfolding diseases and transmembrane protein folding. In this paper, the interaction of the cysteine/tryptophan (Cys/Trp) motif, which is a metal-binding motif of copper transporter (Ctr) proteins, with a lipid bilayer was studied using fluorescence and circular dichroism (CD) spectroscopy. The binding of Cu(I) to the Cys/Trp motif induced a large red-edge excitation shift in the Trp fluorescence, indicating that the Trp residue is located inside the lipid bilayer following complexation of Cu(I) with the Cys/Trp motif. The Stern-Volmer quenching of the Trp fluorescence also supported the Cu(I) binding peptide embedding in the lipid bilayer. The measurement of the CD spectra indicated the increase in β-sheet content of the Cys/Trp motif peptide as a result of Cu(I) binding. These results lead to the conclusion that complexation with Cu(I) induces the change in the secondary structure of the Cys/Trp motif, which results in the peptide embedding in the lipid bilayer. Cu(I)-induced enhancement of the lipid affinity is discussed in terms of the mechanism for copper transport by Ctr.

Conformational Plasticity in Tyrosine Kinase Inhibitor-Kinase Interactions Revealed with Fluorescence Spectroscopy and Theoretical Calculations.

To understand drug-protein dynamics, it is necessary to account for drug molecular flexibility and binding site plasticity. Herein, we exploit fluorescence from a tyrosine kinase inhibitor, AG1478, as a reporter of its conformation and binding site environment when complexed with its cognate kinase. Water-soluble kinases, aminoglycoside phosphotransferase APH(3')-Ia and mitogen-activated protein kinase 14 (MAPK14), were chosen for this study. On the basis of our prior work, the AG1478 conformation (planar or twisted) was inferred from the fluorescence excitation spectrum and the polarity of the AG1478-binding site was deduced from the fluorescence emission spectrum, while red-edge excitation shift (REES) probed the heterogeneity of the binding site (protein conformation and hydration) distributions in the protein conformational ensemble. In the AG1478-APH(3')-Ia complex, both twisted (or partially twisted) and planar AG1478 conformations were evidenced from emission wavelength-dependent excitation spectra. The binding site environment provided by APH(3')-Ia was moderately polar (λmax = 480 nm) with evidence for considerable heterogeneity (REES = 34 nm). In contrast, in the AG1478-MAPK14 complex, AG1478 was in a predominantly planar conformation with a lower degree of conformational heterogeneity. The binding site environment provided by the MAPK14 protein was of relatively low polarity (λmax = 430 nm) with a smaller degree of heterogeneity (REES = 11 nm). The results are compared with the available X-ray data and discussed in the context of our current understanding of tyrosine kinase inhibitor conformation and protein conformational ensembles.

Structural Characterization of Biocompatible Reverse Micelles Using Small-Angle X-ray Scattering, 31P Nuclear Magnetic Resonance, and Fluorescence Spectroscopy

The most critical problem regarding the use of reverse micelles (RMs) in several fields is the toxicity of their partial components. In this sense, many efforts have been made to characterize nontoxic RM formulations on the basis of biological amphiphiles and/or different oils. In this contribution, the microstructure of biocompatible mixed RMs formulated by sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT) and tri- n-octylphosphine oxide (TOPO) surfactants dispersed in the friendly solvent methyl laurate was studied by using SAXS and 31P NMR and by following the solvatochromic behavior of the molecular probe 4-aminophthalimide (4-AP). The results indicated the presence of RM aggregates upon TOPO incorporation with a droplet size reduction and an increase in the interfacial fluidity in comparison with pure AOT RMs. When confined inside the mixed systems, 4-AP showed a red-edge excitation shift and confirmed the increment of interfacial fluidity upon TOPO addition. Also, the partition between the external nonpolar solvent and the RM interface and an increase in both the local micropolarity and the capability to form a hydrogen bond interaction between 4-AP and a mixed interface were observed. The findings have been explained in terms of the nonionic surfactant structure and its complexing nature expressed at the interfacial level. Notably, we show how two different approaches, i.e., SAXS and the solvatochromism of the probe 4-AP, can be used in a complementary way to enhance our understanding of the interfacial fluidity of RMs, a parameter that is difficult to measure directly.

Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide

L1A (IDGLKAIWKKVADLLKNT-NH2) is a peptide that displays a selective antibacterial activity to Gram-negative bacteria without being hemolytic. Its lytic activity in anionic lipid vesicles was strongly enhanced when its N-terminus was acetylated (ac-L1A). This modification seems to favor the perturbation of the lipid core of the bilayer by the peptide, resulting in higher membrane lysis. In the present study, we used lipid monolayers and bilayers as membrane model systems to explore the impact of acetylation on the L1A lytic activity and its correlation with lipid-packing perturbation. The lytic activity investigated in giant unilamellar vesicles (GUVs) revealed that the acetylated peptide permeated the membrane at higher rates compared with L1A, and modified the membrane's mechanical properties, promoting shape changes. The peptide secondary structure and the changes in the environment of the tryptophan upon adsorption to large unilamellar vesicles (LUVs) were monitored by circular dichroism (CD) and red-edge excitation shift experiments (REES), respectively. These experiments showed that the N-terminus acetylation has an important effect on both, peptide secondary structure and peptide insertion into the bilayer. This was also confirmed by experiments of insertion into lipid monolayers. Compression isotherms for peptide/lipid mixed films revealed that ac-L1A dragged lipid molecules to the more disordered phase, generating a more favorable environment and preventing the lipid molecules from forming stiff films. Enthalpy changes in the main phase transition of the lipid membrane upon peptide insertion suggested that the acetylated peptide induced higher impact than the non-acetylated one on the thermotropic behavior of anionic vesicles.

Lipid chain saturation and the cholesterol in the phospholipid membrane affect the spectroscopic properties of lipophilic dye nile red

We have studied the effect of composition and the phase state of phospholipid membranes on the emission spectrum, anisotropy and lifetime of a lipophilic fluorescence probe nile red. Fluorescence spectrum of nile red in membranes containing cholesterol has also been investigated in order to get insights into the influence of cholesterol on the phospholipid membranes. Maximum emission wavelength (λem) of nile red in the fluid phase of saturated and unsaturated phospholipids was found to differ by ~10nm. The λem was also found to be independent of chain length and charge of the membrane. However, the λem is strongly dependent on the temperature in the gel phase. The λem and rotational diffusion rate decrease, whereas the anisotropy and lifetime increase markedly with increasing cholesterol concentration for saturated phosoholipids, such as, dimyristoyl phosphatidylcholine (DMPC) in the liquid ordered phase. However, these spectroscopic properties do not alter significantly in case of unsaturated phospholipids, such as, dioleoyl phosphatidylcholine (DOPC) in liquid disordered phase. Interestingly, red edge excitation shift (REES) in the presence of lipid-cholesterol membranes is the direct consequences of change in rotational diffusion due to motional restriction of lipids in the presence of cholesterol. This study provides correlations between the membrane compositions and fluorescence spectral features which can be utilized in a wide range of biophysical fields as well the cell biology.

Excitation-dependent fluorescence from atomic/molecular layer deposited sodium-uracil thin films

Atomic/molecular layer deposition (ALD/MLD) offers unique possibilities in the fabrication of inorganic-organic thin films with novel functionalities. Especially, incorporating nucleobases in the thin-film structures could open new avenues in the development of bio-electronic and photonic devices. Here we report an intense blue and widely excitation-dependent fluorescence in the visible region for ALD/MLD fabricated sodium-uracil thin films, where the crystalline network is formed from hydrogen-bonded uracil molecules linked via Na atoms. The excitation-dependent fluorescence is caused by the red-edge excitation shift (REES) effect taking place in the red-edge of the absorption spectrum, where the spectral relaxation occurs in continuous manner as demonstrated by the time-resolved measurements.

A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis.

Our understanding of how enzymes work is coloured by static structure depictions where the enzyme scaffold is presented as either immobile, or in equilibrium between well-defined static conformations. Proteins, however, exhibit a large degree of motion over a broad range of timescales and magnitudes and this is defined thermodynamically by the enzyme free energy landscape (FEL). The role and importance of enzyme motion is extremely contentious. Much of the challenge is in the experimental detection of so called 'conformational sampling' involved in enzyme turnover. Herein we apply combined pressure and temperature kinetics studies to elucidate the full suite of thermodynamic parameters defining an enzyme FEL as it relates to enzyme turnover. We find that the key thermodynamic parameters governing vibrational modes related to enzyme turnover are the isobaric expansivity term and the change in heat capacity for enzyme catalysis. Variation in the enzyme FEL affects these terms. Our analysis is supported by a range of biophysical and computational approaches that specifically capture information on protein vibrational modes and the FEL (all atom flexibility calculations, red edge excitation shift spectroscopy and viscosity studies) that provide independent evidence for our findings. Our data suggest that restricting the enzyme FEL may be a powerful strategy when attempting to rationally engineer enzymes, particularly to alter thermal activity. Moreover, we demonstrate how rational predictions can be made with a rapid computational approach.

Sensing Tryptophan Microenvironment of Amyloid Protein Utilizing Wavelength-Selective Fluorescence Approach.

Structural transition among various forms of proteins involves subtle interplay between structure and dynamics and is crucial in human diseases. Red edge excitation shift (REES) represents a suitable approach to explore the environmental organization and dynamics surrounding tryptophan residues in proteins. Although REES from tryptophan residues has been reported for native, molten globule and denatured states of proteins, such data on the amyloid form of proteins is lacking. κ-casein is one of the most important constituents of casein micelles in milk and has a tendency to form amyloid fibril. We report here REES of the sole tryptophan residue for native, acid-denatured and urea-denatured forms of κ-casein. More importantly, we show that the amyloid form of κ-casein displays REES of 4nm. We analyze these results in terms of tryptophan microenvironment in various forms of κ-casein, particularly the amyloid form. We conclude that REES is a sensitive tool to monitor structural plasticity in proteins.

Influence of metal cations and cholesterol on lipid-amphotericin membrane

Amphotericin B(AmB) has been widely used in antifungal therapy. AmB molecules combine with cholesterol to form pores that can be toxic to human cells, thus greatly limiting its clinical application. The interaction between AmB and the cell membrane may be influenced by potassium, sodium and calcium ions. In this study, the bilayer in large unilamellar lipid-drug liposomes with or without cholesterol was employed as a model membrane. N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine(NBD-PE) and 1-palmitoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminodoclecanoyl]-sn-glysero-3-phosphocholine(6-NBD-PC) are two kinds of fluorescent lipid probes, and the NBD group is attached to the polar lipid headgroup in the former, but to the sn-2 fatty acyl chain in the latter. The effect of these metal cations on the lipid-drug membrane was monitored by red edge excitation shift(REES), fluorescence polarization, and the fluorescence lifetime of lipid probes in hydrophilic and hydrophobic areas of the membrane. These ions have different effects on the lipid-AmB membrane. Cholesterol can strengthen the packing ability of the membrane, which is influenced differently by potassium, sodium and calcium ions. Moreover, the influence of these ions on the membrane may be relative to the method of ion transportation through the membrane. This study is significant to understand the reduction of AmB’s cellular toxicity.

Organization and dynamics of Trp14 of hemagglutinin fusion peptide in membrane mimetic environment

The fusion peptide of influenza hemagglutinin (HA) is important for cellular entry of influenza virus. Previous studies showed that HA fusion peptide assumes a bent structure in membrane environment, which is extremely important for its fusion-promoting activity. In this work, we have measured the organization and dynamics of the tryptophan (Trp14) residue in presence of sodium dodecyl sulfate (SDS) to investigate the conformational flexibility of tryptophan in membrane-mimetic environment. The steady state and time-resolved fluorescence measurements were employed to investigate the location and rotational flexibility of the tryptophan residue in the SDS micelles. We have calculated the apparent rotational correlation time of Trp14 from the fluorescence anisotropy and fluorescence lifetime data, and apparent rotational correlation time shows a substantial increase in presence of SDS. We have further measured the red edge excitation shift (REES) of Trp14 fluorescence in absence and in presence of two different concentrations of SDS. A large magnitude of REES at post-micellar concentration of SDS indicates the polar confined environment of Trp14 residue in the micellar milieu. Taken together, our results confirm that the tryptophan is located in a motionally restricted interfacial region of micelles. The interfacial location and slow dynamics of Trp14 might be important to adopt the appropriate structure of HA fusion peptide in membrane environment.

Label-free imaging of amyloids using their intrinsic linear and nonlinear optical properties.

The optical properties of amyloid fibers are often distinct from those of the source protein in its non-fibrillar form. These differences can be utilized for label-free imaging or characterization of such structures, which is particularly important for understanding amyloid fiber related diseases such as Alzheimer's and Parkinson's disease. We demonstrate that two amyloid forming proteins, insulin and β-lactoglobulin (β-LG), show intrinsic fluorescence with emission spectra that are dependent on the excitation wavelength. Additionally, a new fluorescence peak at about 430 nm emerges for β-LG in its amyloid state. The shift in emission wavelength is related to the red edge excitation shift (REES), whereas the additional fluorescence peak is likely associated with charge delocalization along the fiber backbone. Furthermore, the spherulitic amyloid plaque-like superstructures formed from the respective proteins were imaged label-free with confocal fluorescence, multiphoton excitation fluorescence (MPEF), and second-harmonic generation (SHG) microscopy. The latter two techniques in particular yield images with a high contrast between the amyloid fiber regions and the core of amorphously structured protein. Strong multiphoton absorption (MPA) for the amyloid fibers is a likely contributor to the observed contrast in the MPEF images. The crystalline fibrillar region provides even higher contrast in the SHG images, due to the inherently ordered non-centrosymmetric structure of the fibers together with their non-isotropic arrangement. Finally, we show that MPEF from the insulin spherulites exhibits a spectral dependence on the excitation wavelength. This behavior is consistent with the REES phenomenon, which we hypothesize is the origin of this observation. The presented results suggest that amyloid deposits can be identified and structurally characterized based on their intrinsic optical properties, which is important for probe-less and label-free identification and characterization of amyloid fibers in vitro and in complex biological samples.

A dry molten globule-like intermediate during the base-induced unfolding of a multidomain protein.

The nature of the initial structural events during the base-induced unfolding of the native (N) state of proteins is poorly understood. Combining site-specific fluorescence resonance energy transfer, size exclusion chromatography, dynamic fluorescence quenching, red-edge excitation shift and circular dichroism spectroscopy, we show here that an early intermediate during the base-induced unfolding of a multidomain protein, i.e., the B form, has features of a dry molten globule. We show that the N ⇌ B transition involves protein expansion and loosening of packing of inter-domain helices near domains I and II without the disruption of intra-domain packing or any change in hydration of the inter-domain region which resembles a molten hydrocarbon. Surprisingly, the disruption of inter-domain packing accounts for 40-45% of the total change in free energy of complete unfolding. Our results show that the disruption of van der Waals packing can be decoupled in different regions of a protein and could occur prior to hydrophobic solvation during base-induced unfolding, challenging the existing notion.

Spectroscopic and in silico study of binding mechanism of cynidine-3-O-glucoside with human serum albumin and glycated human serum albumin.

The drug-serum albumin interaction plays a dominant role in drug efficacy and disposition. The glycation of serum albumin that occurs during diabetes may affect its drug-binding properties in vivo. In order to evaluate the interactivity characteristics of cyanidin-3-O-glucoside (C3G) with human serum albumin (HSA) and glycated human serum albumin (gHSA), this study was undertaken using multiple spectroscopic techniques and molecular modeling analysis. Time-resolved fluorescence and the thermodynamic parameters indicated that the quenching mechanism was static quenching, and hydrogen bonding and Van der Waals force were the main forces. The protein fluorescence could be quenched by C3G, whereas the polarity of the fluorophore was not obviously changed. C3G significantly altered the secondary structure of the proteins. Furthermore, the interaction force that existed in the HSA-C3G system was greater than that in the gHSA-C3G system. Fluorescence excitation emission matrix spectra, red edge excitation shift, Fourier transform infrared spectroscopy and circular dichroism spectra provided further evidence that glycation could inhibit the binding between C3G and proteins. In addition, molecular modeling analysis supported the experimental results. The results provided more details for the application of C3G in the treatment of diabetes.

Simultaneous High-Throughput Conformational and Colloidal Stability Screening Using a Fluorescent Molecular Rotor Dye, 4-(4-(Dimethylamino)styryl)-N-Methylpyridinium Iodide (DASPMI)

Technologies to improve the throughput for screening protein formulations are continuously evolving. The purpose of this article is to highlight novel applications of a molecular rotor dye, 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (DASPMI) in screening for the conformational stability, colloidal stability, and subtle pretransition dynamics of protein structures during early formulation development. The measurement of the apparent unfolding temperature (Tm) for a monoclonal antibody in the presence of Tween 80 was conducted and data were compared to the results of differential scanning calorimetry (DSC) measurements. Additionally, measuring the fluorescence intensity of DASPMI as a function of protein concentration shows consistent correlation to the diffusion interaction parameter (kD) for two distinct monoclonal antibody formulations measured by DLS. Lastly, due to the sensitivity of the molecular rotor dye to changes in microviscosity (ηmicro), subtle pretransition dynamics were discernable for two monoclonal antibody formulations that correlate with findings by red-edge excitation shift (REES) experiments. This novel application of molecular rotor dyes offers a valuable and promising approach for streamlining the early formulation development process due to low material consumption and rapid analysis time in a 96-well plate format.

Novel benzanthrone probes for membrane and protein studies

The applicability of a series of novel benzanthrone dyes to monitoring the changes in physicochemical properties of lipid bilayer and to differentiating between the native and aggregated protein states has been evaluated. Based on the quantitative parameters of the dye-membrane and dye-protein binding derived from the fluorimetric titration data, the most prospective membrane probes and amyloid tracers have been selected from the group of examined compounds. Analysis of the red edge excitation shifts of the membrane- and amyloid-bound dyes provided information on the properties of benzanthrone binding sites within the lipid and protein matrixes. To understand how amyloid specificity of benzanthrones correlates with their structure, quantitative structure activity relationship (QSAR) analysis was performed involving a range of quantum chemical molecular descriptors. A statistically significant model was obtained for predicting the sensitivity of novel benzanthrone dyes to amyloid fibrils.

Effect of 1,4-bis(3-dodecylimidazolium-1-yl) butane bromide on channel form of gramicidin vesicles

The present work is a brief overview of technical applications of surfactant that involves membrane interactions, and surfactant-based protocols to study biological systems. Herein, we investigate the effect of ionic liquid type gemini surfactant [C12-4-C12im]Br2, (1,4-bis(3-dodecylimidazolium-1-yl) butane bromide) on the channel form of gramicidin vesicle. Gramicidin is a linear pentadecapeptide antibiotic that adopts a wide range of environment dependent conformations. Gemini surfactants are a novel class of surfactants possessing better physicochemical properties than conventional surfactants. Several spectroscopic approaches viz. fluorescence, time resolved fluorescence, circular dichroism (CD) and red edge excitation shift (REES) were employed to elucidate the interaction between gramicidin vesicle and [C12-4-C12im]Br2. Fluorescence and time-resolved fluorescence studies confirmed the quenching and accessibility of gramicidin fluorescence by [C12-4-C12im]Br2 through static quenching mechanism. CD results show the change in orientation of tryptophan within gramicidin vesicle on the addition of [C12-4-C12im]Br2. REES results suggested the possible interaction between gramicidin and [C12-4-C12im]Br2. In order to further validate our results, we exploited techniques like dynamic light scattering (DLS), dynamic surface tension (DST) and scanning electron microcopy (SEM). DLS results reveal increase in the size of gramicidin vesicles upon interaction with [C12-4-C12im]Br2. To our surprise, the DST shows the surface activity of gramicidin vesicles decreases with increase in concentration of [C12-4-C12im]Br2, suggesting possible interaction and complex formation between gramicidin and [C12-4-C12im]Br2. SEM images of gramicidin vesicle with different concentrations of [C12-4-C12im]Br2 differ markedly showing aggregation on the surface of vesicles, again supporting the above results. Docking results provide deep molecular insight into the probable interaction between tryptophan and D-leucine residues of gramicidin with [C12-4-C12im]Br2 through hydrophobic interactions. This work has prime importance in future to design ionic liquids based biomaterials for drug/gene delivery.