Vellore Institute of Technology (VIT) is a private university located in&nbsp;Tamil Nadu, India. Founded in 1984, as Vellore Engineering College, the institution offers 20 undergraduate, 34 postgraduate, four integrated and four research programs. It has campuses in Vellore, Amravati, Bhopal and Chennai.

VIT is one of the top ranked private universities in India according to NIRF, THE and QS Rankings.&nbsp;Govt. of India has recognized&nbsp;VIT, Vellore as an&nbsp;Institution of Eminence. This has allowed VIT to take independent quality initiatives and move up in world ranking.

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N-Acyldopamines (NADAs), which are present in mammalian nervous tissues, exhibit interesting biological and pharmacological properties. In the present study, a homologous series of NADAs with varying acyl chains (n = 12-20) have been synthesized and characterized. Differential scanning calorimetric studies show that in the dry state the transition temperatures, enthalpies, and entropies of NADAs exhibit odd-even alternation with the values corresponding to the even chain length series being slightly higher. Both even and odd chain length NADAs display a linear dependence of the transition enthalpies and entropies on the chain length. However, odd-even alternation was not observed in the calorimetric properties upon hydration, although the transition enthalpies and entropies exhibit linear dependence. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-Lauroyldopamine (NLDA) crystallized in the monoclinic space group C2/c with eight symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that NLDA molecules are organized in the bilayer form, with a head-to-head (and tail-to-tail) arrangement of the molecules. Water-mediated hydrogen bonds between the hydroxyl groups of the dopamine moieties of opposing layers and N-H···O hydrogen bonds between the amide groups of adjacent molecules in the same layer stabilize the crystal packing. These results provide a thermodynamic and structural basis for investigating the interaction of NADAs with other membrane lipids, which are expected to provide clues to understand how they function in vivo, e.g., as signaling molecules in the modulation of pain. © 2013 American Chemical Society.

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Department of Chemistry

School of Advanced Sciences

Vellore Campus

Journal of Physical Chemistry B

Structure and thermotropic phase behavior of a homologous series of bioactive N-acyldopamines

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP fold. Currently, insights into the folding stability and conformational states that regulate GLTP fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP fold, we investigated the effect of a change in pH on the fungal HET-C2 GLTP fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and 10 tyrosines in human GLTP). pH-induced conformational alterations were determined by changes in (i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, and anisotropy), (ii) circular dichroism over the near-UV and far-UV ranges, including thermal stability profiles of the derivatized molar ellipticity at 222 nm, (iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid, and (iv) glycolipid intermembrane transfer activity monitored by Förster resonance energy transfer. Analyses of our recently determined crystallographic structure of HET-C2 (1.9 Å) allowed identification of side chain electrostatic interactions that contribute to HET-C2 GLTP fold stability and can be altered by a change in pH. Side chain interactions include numerous salt bridges and interchain cation interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low-pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP fold. © 2011 American Chemical Society.

Fulltext

Biochemistry

Conformational folding and stability of the HET-C2 glycolipid transfer protein fold: Does a molten globule-like state regulate activity?

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of &gt;90%, ̃85%, and ̃45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp 96-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold. © 2010 by the Biophysical Society.

Biophysical Journal

Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold

HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 Å) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp66, Asn70, Lys73, Trp109, and His147) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by α-helices and a cooperative thermal unfolding transition of 49°C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UVCDwas unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at ∼355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (∼6 nm) permitting determination of binding affinities. The unique positioning of Trp208 at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Journal of Biological Chemistry

Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP fold

Recent studies show that N-, O -diacylethanolamines (DAEs) can be derived by the O -acylation of N-acylethanolamines (NAEs) under physiological conditions. Because the content of NAEs in a variety of organisms increases in response to stress, it is likely that DAEs may also be present in biomembranes. In view of this, a homologous series of DAEs with matched acyl chains (n = 10-20) have been synthesized and characterized. Transition enthalpies and entropies obtained from differential scanning calorimetry show that dry DAEs with even and odd acyl chains independently exhibit linear dependence on the chainlength. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-, O-Didecanoylethanolamine (DDEA), N-, O-dilauroylethanolamine (DLEA), and N-, O-dimyristoylethanolamine (DMEA) crystallized in the orthorhombic space group Pbc21 with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that DDEA, DLEA, and DMEA are isostructural and adopt an L-shaped structure with the N-acyl chain and the central ethanolamine moiety being essentially identical to the structure of N-acylethanolamines, whereas the O-acyl chain is linear with all-trans conformation. In all three DAEs, the lipid molecules are organized in a bilayer fashion wherein the N-acyl and O-acyl chains from adjacent layers oppose each other. Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.

Journal of Lipid Research

Synthesis, calorimetric studies, and crystal structures of N, O-diacylethanolamines with matched chains

N-Acylethanolamines (NAEs) and N-acylphosphatidylethanolamines (NAPEs) are naturally occurring membrane lipids, whose content increases dramatically in a variety of organisms when subjected to stress, suggesting that they may play a role in the stress-combating mechanisms of organisms. In the light of this, it is of great interest to characterize the structure, physical properties, phase transitions and membrane interactions of these two classes of lipids. This review will present the current status of our understanding of the structure and phase behaviour of NAEs and NAPEs and their interaction with major membrane lipids, namely phosphatidylcholine, phosphatidylethanolamine and cholesterol. The relevance of such interactions to the putative stress-combating and membrane stabilizing properties of these lipids will also be discussed. © 2010 Elsevier Ireland Ltd. All rights reserved.

Chemistry and Physics of Lipids

Structure, phase behaviour and membrane interactions of N-acylethanolamines and N-acylphosphatidylethanolamines

The dramatic increase in the content of N-acylethanolamines (NAEs) having different acyl chains in various tissues when subjected to stress has resulted in significant interest in investigations on these molecules. Previous studies suggested that N-myristoylethanolamine (NMEA) and cholesterol interact to form a 1:1 (mol/mol) complex. In studies reported here, pressure-area isotherms of Langmuir films at the air-water interface have shown that at low fractions of cholesterol, the average area per molecule is lower than that predicted for ideal mixing, whereas at high cholesterol content the observed molecular area is higher, with a cross-over point at the equimolar composition. A plausible model that can explain these observations is the following: addition of small amounts of cholesterol to NMEA results in a reorientation of the NMEA molecules from the tilted disposition in the crystalline state to the vertical and stabilization of the intermolecular interactions, leading to the formation of a compact monolayer film, whereas at the other end of the composition diagram, addition of small amounts of NMEA to cholesterol leads to a tilting of the cholesterol molecules resulting in an increase in the average area per molecule. In Brewster angle microscopy experiments, a stable and bright homogeneous condensed phase was observed at a relatively low applied pressure of 2 mN.m- 1 for the NMEA:Chol. (1:1, mol/mol) mixture, whereas all other samples required significantly higher pressures (&gt; 10 mN.m- 1) to form a homogeneous condensed phase. These observations are consistent with the formation of a 1:1 stoichiometric complex between NMEA and cholesterol and suggest that increase in the content of NAEs under stress may modulate the composition and dynamics of lipid rafts in biological membranes, resulting in alterations in signaling events involving them, which may be relevant to the putative cytoprotective and stress-combating ability of NAEs. © 2008 Elsevier B.V. All rights reserved.

Biophysical Chemistry

Interaction of N-myristoylethanolamine with cholesterol investigated in a Langmuir film at the air-water interface

The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs), N-myristoylethanolamine (NMEA), and N-palmitoylethanolamine (NPEA), with the corresponding diacyl phosphatidylethanolamines (PEs), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE), respectively, have been investigated by differential scanning calorimetry (DSC), spin-label electron spin resonance (ESR), and 31P-NMR spectroscopy. Temperature- composition phase diagrams for both NMEA/DMPE and NPEA/DPPE binary systems were established from high sensitivity DSC. The structures of the phases involved were determined by 31P-NMR spectroscopy. For both systems, complete miscibility in the fluid and gel phases is indicated by DSC and ESR, up to 35 mol % of NMEA in DMPE and 40 mol % of NPEA in DPPE. At higher contents of the NAEs, extensive solid-fluid phase separation and solid-solid immiscibility occur depending on the temperature. Characterization of the structures of the mixtures formed with 31P-NMR spectroscopy shows that up to 75 mol % of NAE, both DMPE and DPPE form lamellar structures in the gel phase as well as up to at least 65°C in the fluid phase. ESR spectra of phosphatidylcholine spin labeled at the C-5 position in the sn-2 acyl chain present at a probe concentration of 1 mol % exhibit strong spin-spin broadening in the low-temperature region for both systems, suggesting that the acyl chains pack very tightly and exclude the spin label. However, spectra recorded in the fluid phase do not exhibit any spin-spin broadening and indicate complete miscibility of the two components. The miscibility of NAE and diacyl PE of matched chainlengths is significantly less than that found earlier for NPEA and dipalmitoylphosphatidylcholine, an observation that is consistent with the notion that the NAEs are most likely stored as their precursor lipids (N-acyl PEs) and are generated only when the system is subjected to membrane stress. © 2007 by the Biophysical Society.

Miscibility and phase behavior of N-acylethanolamine/ diacylphosphatidylethanolamine binary mixtures of matched acyl chainlengths (n = 14, 16)

Earlier studies have suggested the formation of a 1:1 (mol/mol) complex between N-myristoylethanolamine (NMEA) and cholesterol in aqueous dispersion. In this study, this interaction has been investigated further by differential scanning calorimetry (DSC) on dry mixtures of NMEA, N-palmitoylethanolamine (NPEA) and N-stearoylethanolamine (NSEA) with cholesterol. The results obtained indicate that addition of cholesterol to NMEA leads to a new phase transition at 86.5°C, besides the solid-liquid phase transition of NMEA at 95°C. The intensity of the peak corresponding to the new transition increases with cholesterol content up to 50 mol%, but decreases thereafter, whereas the intensity of the peak corresponding to the melting of NMEA decreases with increasing cholesterol content, with concomitant and gradual shift to lower temperatures and vanishes at 50 mol% cholesterol. These results are consistent with the formation of a 1:1 molar complex between NMEA and cholesterol proposed earlier and indicate that these two amphiphiles are associated in the solid state as well. DSC studies on hydrated mixtures of NPEA and NSEA with cholesterol yielded results that parallel those obtained with the NMEA/cholesterol system, indicating that these two long-chain NAEs also form 1:1 (mol/mol) complexes with cholesterol.

Current Science

Differential scanning calorimetric studies on the interaction of N-acylethanolamines with cholesterol

The molecular structure, packing properties, and intermolecular interactions of two structural polymorphs of N-palmitoylethanolamine (NPEA) have been determined by single-crystal X-ray diffraction. Polymorphs α and β crystallized in monoclinic space group P21/c and orthorhombic space group P bca, respectively. In both polymorphs, NPEA molecules are organized in a tail-to-tail manner, resembling a bilayer membrane. Although the molecular packing in polymorph α is similar to that in N-myristoylethanolamine and N-stearoylethanolamine, polymorph β is a new form. The acyl chains in both polymorphs are tilted by ∼35° with respect to the bilayer normal, with their hydrocarbon moieties packed in an orthorhombic subcell. In both structures, the hydroxy group of NPEA forms two hydrogen bonds with the hydroxy groups of molecules in the opposite leaflet, resulting in extended, zig-zag type H-bonded networks along the b-axis in polymorph α and along the a-axis in polymorph β. Additionally, the amide N-H and carbonyl groups of adjacent molecules are involved in N-H⋯O hydrogen bonds that connect adjacent molecules along the b-axis and a-axis, respectively, in α and β. Whereas in polymorph α the L-shaped NPEA molecules in opposite layers are arranged to yield a Z-like organization, in polymorph β one of the two NPEA molecules is rotated 180°, leading to a W-like arrangement. Lattice energy calculations indicate that polymorph α is more stable than polymorph β by ∼2.65 kcal/mol. Copyright © 2006 by the American Society for Biochemistry and Molecular Biology, Inc.

Molecular packing and intermolecular interactions in two structural polymorphs of N-palmitoylethanolamine, a type 2 cannabinoid receptor agonist

The critical micellar concentration (CMC) of stearoylcarnitine was determined at different pH values at room temperature by fluorescence spectroscopy, monitoring the spectral changes of 8-anilinonaphthalene-1- sulfonate (ANS). The CMC was found to vary with pH, increasing from about 10 μM at pH 3.0 to ca. 25 μM at pH 7.0, but decreasing slightly with further increase in pH to approximately 19 μM at pH 10.0. Differential scanning calorimetry (DSC) shows that stearoylcarnitine dispersed in water at low concentration undergoes a broad thermotropic phase transition at 44.5°C, with a transition enthalpy of 15.0 kcal/mol. The transition temperature (T t) shifts to ca. 50.5°C in the presence of 1 mM EDTA or when the concentration is increased significantly. The turbidity of aqueous dispersions of stearoylcarnitine was found to be considerably high at low temperatures, which decreases quite abruptly over a short temperature range, indicating that a transition occurs from a phase of large aggregates to one of much smaller aggregates, most likely micelles. The phase transition temperature was determined as 29.1°C at pH 3.0, which increased with increasing pH up to a value of 55.3°C at pH 8.6 and remains nearly constant thereafter up to pH 11.2. The pH dependence of CMC and Tt suggest that the pKa of the carboxyl group of long chain acylcarnitines shifts to higher temperatures upon aggregation (micelles or bilayer membranes). © 2006 Springer Science+Business Media, LLC.

Bioscience Reports

Studies on the critical micellar concentration and phase transitions of stearoylcarnitine

The interaction of N-myristoylethanolamine (NMEA) with cholesterol is investigated by differential scanning calorimetry (DSC), fast-atom-bombardment mass spectrometry (FAB-MS) and computational modelling. Addition of cholesterol to NMEA leads to a new phase transition at 55°C besides the chain-melting transition of NMEA at 72.5°C. The enthalpy of the new transition increases with cholesterol content up to 50 mol%, but decreases thereafter, vanishing at 80 mol%. The enthalpy of the chain-melting transition of NMEA decreases with an increase in cholesterol; the transition disappears at 50 mol%. FAB-MS spectra of mixtures of NMEA and cholesterol provide clear signatures of the formation of {[NMEA+cholesterol]+} {[NMEA+cholesterol+Na]+}. These results are consistent with the formation of a 1:1 complex between NMEA and cholesterol. Molecular modelling studies support this experimental finding and provide a plausible structural model for the complex, which highlights multiple H-bond interactions between the hydroxy group of cholesterol and the hydroxy and carbonyl groups of NMEA besides appreciable dispersion interaction between the hydrocarbon domains of the two molecules. © 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

FEBS Letters

N-myristoylethanolamine-cholesterol (1:1) complex: First evidence from differential scanning calorimetry, fast-atom-bombardment mass spectrometry and computational modelling