Indian Institute of Technology Palakkad&nbsp;(IIT Palakkad or IIT PKD) is a public autonomous&nbsp;engineering&nbsp;and&nbsp;research&nbsp;institute located in&nbsp;Palakkad,&nbsp;Kerala. It was&nbsp;one of the five new&nbsp;IITs&nbsp;proposed in the&nbsp;2014 Union budget of India. The campus was inaugurated on 3 August 2015. The institute has 94 faculty, 978 students, and 63 non-teaching staffs.

IT Palakkad currently offers four-year Bachelor of Technology (B.Tech) and two years MS programmes in 8 different engineering disciplines including&nbsp;Computer Science, Electrical, Mechanical, Civil among others.

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IIT Palakkad

Prion-like misfolding of superoxide dismutase 1 (SOD1) is associated with the disease ALS, but the mechanism of misfolding remains unclear, partly because misfolding is difficult to observe directly. Here we study the most misfolding-prone form of SOD1, reduced un-metallated monomers, using optical tweezers to measure unfolding and refolding of single molecules. We find that the folding is more complex than suspected, resolving numerous previously undetected intermediate states consistent with the formation of individual β-strands in the native structure. We identify a stable core of the protein that unfolds last and refolds first, and directly observe several distinct misfolded states that branch off from the native folding pathways at specific points after the formation of the stable core. Partially folded intermediates thus play a crucial role mediating between native and non-native folding. These results suggest an explanation for SOD1's propensity for prion-like misfolding and point to possible targets for therapeutic intervention. © 2017 The Author(s).

Department of Chemistry

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Nature Communications

Partially native intermediates mediate misfolding of SOD1 in single-molecule folding trajectories

The role of a room temperature ionic liquid (RTIL, [pmim][Br]) on the size and conformational dynamics of a protein, horse heart cytochrome c (Cyt C) in its native, molten globule (MG-I and II), and unfolded states is studied using fluorescence correlation spectroscopy (FCS). For this purpose, the protein was covalently labeled by a fluorescent dye, Alexa Fluor 488. It is observed that the addition of the RTIL leads to an increase in the hydrodynamic radius (rH) of the protein, Cyt C in the native or MG-I state. In contrast, the addition of RTIL causes a decrease in the size (hydrodynamic radius, rH) of Cyt C unfolded by GdnHCl or MG-II state. The decrease in size indicates the formation of a relatively compact structure. We detected two types of conformational relaxation of the protein. The shorter relaxation time component (∼3-5.5 μs) corresponds to the protein folding or intrachain contact formation, while the relatively longer time component (∼63-122 μs) may be assigned to the motion of the protein side chains or concerted chain dynamics. The burst integrated fluorescence lifetime histograms indicate that the increase in size of the protein is accompanied by an increase in the contribution of the shorter component (∼0.3-0.4 ns) with a concomitant decrease of the contribution of the longer component (∼2.8-3.6 ns). An opposite trend is observed during the decrease in size of the protein. © 2012 American Chemical Society.

Journal of Physical Chemistry B

Role of ionic liquid on the conformational dynamics in the native, molten globule, and unfolded states of cytochrome C: A fluorescence correlation spectroscopy study

The effect of two room-temperature ionic liquids (RTILs) on the diffusion of three fluorescent dyes in the gel phase of a triblock copolymer, (PEO) 20-(PPO)70-(PEO)20 [Pluronic P123; poly ethylene oxide (PEO), poly propylene oxide (PPO)], was studied by using fluorescence correlation spectroscopy (FCS). We used three dyes, 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM), coumarin 480 (C480), and coumarin 343 (C343). By field-emission scanning electron microscopy (FESEM), it was observed that the macroscopic structure of the P123 gel remained unaffected upon addition of RTIL. In the absence of RTIL, the diffusion coefficient (Dt) of the hydrophobic dye DCM (1 μm 2s-1 at the core) is smaller than that of the other two hydrophilic dyes (7 μm2s-1 for C480 and C343). On addition of RTIL, the Dt values of all of the dyes increase, indicating a decrease in local viscosity (ηeff). The ηeff of the core of the RTIL-P123 gel estimated from the D t of DCM is lower than that of both the P123 gel (at the core η=90 cP) and RTIL (η=110 cP). It is shown that the RTIL affects the structure of the gel by modifying the size of the micellar aggregates and by penetrating the core. © 2012 Wiley-VCH Verlag GmbH&amp; Co. KGaA, Weinheim.

ChemPhysChem

Effect of ionic liquid on diffusion in P123 Gel: Fluorescence correlation spectroscopy

Picosecond spectroscopy under a confocal microscope is employed to study solvation dynamics of coumarin 153 (C153) inside a single giant lipid vesicle (1,2-dilauroyl-sn-glycero-3-phosphocholine, DLPC) of diameter 20 μm. Fluorescence correlation spectroscopy (FCS) indicates that the diffusion coefficient (D t) of the probe (coumarin153, C153) in the immobilized vesicle displays a wide distribution from ∼3 to 21 μm 2 s -1. The distribution of D t suggests that the microenvironment of the probe (C153) is highly heterogeneous and the local friction is different for probe molecules in different regions. The values of D t is significantly smaller than that for the same dye in bulk water (550 μm 2 s -1). This suggests that the probe is located in the interface or membrane region rather than in the water pool of the vesicle. The solvation time of C153 in different regions of the lipid vesicle varies between 750 to 1200 ps. This result clearly shows that a confocal microscope is able to resolve the spatial heterogeneity in local friction (i.e., D t) and solvation dynamics within a lipid vesicle. © 2012 American Chemical Society.

Langmuir

Solvation dynamics under a microscope: Single giant lipid vesicle

Ultrafast photoinduced electron transfer (PET) from N,N-dimethylaniline (DMA) to coumarin dyes in a room-temperature ionic liquid (RTIL, [pmim][BF 4]) and in a mixed micelle containing the RTIL and a triblock copolymer, (PEO)20-(PPO)70-(PEO)20, (Pluronic P123) is studied using femtosecond upconversion. A Marcus-like inversion in the rate of PET is observed in neat RTIL. This is attributed to high viscosity and nanostructuring of the RTIL. Diffusion and the rate of PET in the neat RTIL are slower than those in the RTIL-P123 mixed micelle. The coumarin dyes exhibit faster electron transfer and translational diffusion (anisotropy decay) in the RTIL-P123 mixed micelle compared to that in the P123 micelle. © 2011 American Chemical Society.

Marcus-like inversion in electron transfer in neat ionic liquid and ionic liquid-mixed micelles

Fluorescence correlation spectroscopy (FCS) has been used to study the formation of large nanotube aggregates involving γ-cyclodextrin (γ-CD) and coumarin 153 (C153). It is observed that the length of a γ-CD:C153 nanotube aggregate is ∼770 nm. This is ∼480 times larger than the length of a 1:1 γ-CD:C480 complex (∼1.6 nm) and ∼950 times that of a γ-CD. This implies that 950 γ-CD units are noncovalently attached in the γ-CD:C153 aggregate. Binding constants (Kb) of both the dyes to γ-CD were obtained from the fluctuation in fluorescence intensity. The rate of association and dissociation are obtained from the inverse of τoff and τon, respectively. The binding constant for the 1:1 γ-CD:C480 complex is ∼1000 M-1. The burst integrated fluorescence lifetime (BIFL) histogram reveals presence of three distinct lifetime 1.8 ns (18%), 2.8 ns (69%), 3.2 ns (13%). These three lifetimes correspond to C153 present in bulk water and at the end and middle of the γ-CD:C153 nanotube aggregate, respectively. The lifetime of C480 in the 1:1 γ-CD:C480 complex is found to be 3.7 ns. © 2011 American Chemical Society.

Study of γ-cyclodextrin host-guest complex and nanotube aggregate by fluorescence correlation spectroscopy

The effect of a room temperature ionic liquid (RTIL) on the conformational dynamics of a protein, human serum albumin (HSA), is studied by fluorescence correlation spectroscopy (FCS). For this, the protein was covalently labeled by a fluorophore, 7-dimethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). On addition of a RTIL ([pmim][Br]) to the native protein, the diffusion coefficient (Dt) decreases and the hydrodynamic radius (Rh) increases. This suggests that the RTIL ([pmim][Br]) acts as a denaturant when the protein is in the native state. However, addition of [pmim][Br] to a protein denatured by GdnHCl causes an increases in Dt and decrease in R h. This suggests that in the presence of GdnHCl addition of RTIL helps the protein to refold. In the native state, the conformational dynamics of protein is described by three distinct time constants: ∼3.6 ± 0.7, ∼29 ± 4.5, and 133 ± 23 μs. The faster components (∼3.6 ± 0.7 and ∼29 ± 4.5 μs) are ascribed to chain dynamics of the protein, while the slowest component (133 μs) is responsible for interchain interaction or concerted motion. On addition of [pmim][Br], the conformational dynamics of HSA becomes slower (∼5.1 ± 1, ∼43.5 ± 2.8, and ∼311 ± 2.3 μs in the presence of 1.5 M [pmim][Br]). The time constants for the protein denatured by 6 M GdnHCl are 3.2 ± 0.4, 34 ± 6, and 207 ± 38 μs. When 1.5 M [pmim][Br] is added to the denatured protein (in 6 M GdnHCl), the time constants become ∼5 ± 1, ∼41 ± 10, and ∼230 ± 45 μs. The lifetime histogram shows that, on addition of GdnHCl to HSA, the contribution of the shorter lifetime component decreases and vanishes at 6 M GdnHCl. The shorter lifetime component immediately reappears after addition of RTIL to unfolded HSA. This suggests recoiling of the unfolded protein by RTIL. © 2011 American Chemical Society.

An FCS study of unfolding and refolding of CPM-labeled human serum albumin: Role of ionic liquid

Ultrafast forster resonance energy transfer (FRET) in a RTIL-P123 gel is studied by femtosecond up-conversion technique. The mixed gel consists of a tri-block copolymer, (PEO)20-(PPO)70-(PEO)20 and a room temperature ionic liquid (RTIL) (1-pentyl-3-methyl-imidazolium bromide, [pmim][Br]). The acceptor rhodamine 6G (R6G) being ionic, stays in the peripheral corona region of the micellar aggregates of the gel. We use two donor dyes of different hydrophobicity. The hydrophobic neutral donor coumarin 153 (C153) resides in the core while the hydrophilic ionic donor coumarin 343 (C343) is located in the corona. As a result donor-acceptor distances are different for the two donors. Thus by varying the donor one can study region dependent FRET processes in the RTIL-P123 gel.

Journal of the Indian Chemical Society

Region dependent ultrafast FRET in an ionic liquid-P123 gel: A femtosecond study

The mobility of the organic dye DCM (4-dicyanomethylene-2-methyl-6-p- dimethyl aminostyryl-4H-pyran) in the gel and fluid phases of a lipid vesicle is studied by fluorescence correlation spectroscopy (FCS). Using FCS, translational diffusion of DCM is determined in the gel phase and fluid phase of a single lipid vesicle adhered to a glass surface. The size of a lipid vesicle (average diameter ∼100 nm) is smaller than the diffraction limited spot size (∼250 nm) of the microscope. Thus, the vesicle is confined within the laser focus. Three lipid vesicles (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC)) having different gel transition temperatures (-1, 23, and 41 °C, respectively) were studied. The diffusion coefficient of the dye DCM in bulk water is ∼300 μm2/s. In the lipid vesicle, the average Dt decreases markedly to ∼5 μm 2/s (∼60 times) in the gel phase (for DPPC at 20 °C) and 40 μm2/s (∼8 times) in the fluid phase (for DLPC at 20 °C). This clearly demonstrates higher mobility in the fluid phase compared with the gel phase of a lipid. It is observed that the Dt values vary from lipid to lipid and there is a distribution of Dt values. The diffusion of the hydrophobic dye DCM (Dt ∼ 5 μm2/s) in the DPPC vesicle is found to be 8 times smaller than that of a hydrophilic anioinic dye C343 (Dt ∼ 40 μm2/s). This is attributed to different locations of the hydrophobic (DCM) and hydrophilic (C343) dyes. © 2010 American Chemical Society.

A fluorescence correlation spectroscopy study of the diffusion of an organic dye in the gel phase and fluid phase of a single lipid vesicle

Effect of a room temperature ionic liquid (RTIL) and water on the ultrafast excited state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6- trisulfonate, HPTS) inside a microemulsion is studied by femtosecond up-conversion. The microemulsion consists of the surfactant, triton X-100 (TX-100) in benzene (bz) and contains the RTIL, 1-pentyl-3-methyl-imidazolium tetrafluoroborate ([pmim] [BF4]) as the polar phase. In the absence of water, HPTS undergoes ultrafast ESPT inside the RTIL microemulsion (RTIL/TX-100/bz) and the deprotonated form (RO-) exhibits three rise components of 0.3, 14, and 375 ps. It is proposed that in the RTIL microemulsion, HPTS binds to the TX-100 at the interface region and participates in ultrafast ESPT to the oxygen atoms of TX-100. On addition of water an additional slow rise of 2150 ps is observed. Similar long rise component is also observed in water/TX-100/benzene reverse micelle (in the absence of [pmim] [BF4]). It is suggested that the added water molecules preferentially concentrate (trapped) around the palisade layer of the RTIL microemulsion. The trapped water molecules remain far from the HPTS both in the presence and absence of ionic liquid and gives rise to the slow component (2150 ps) of ESPT. Replacement of H2 O by D2 O causes an increase in the time constant of the ultraslow rise to 2350 ps. © 2010 American Institute of Physics.

Journal of Chemical Physics

Ultrafast and ultraslow proton transfer of pyranine in an ionic liquid microemulsion

Fluorescence correlation spectroscopy (FCS) has been used to study the motion of fluorescent dyes in a giant (diameter 20 000 nm = 20 μm) catanionic vesicle comprised of the surfactant sodium dodecyl sulfate (SDS) and dodecyltrimethyl ammonium bromide (DTAB). The diffusion in the anion (SDS) rich catanionic vesicle was studied both in bulk water and in an immobilized vesicle attached to a positively charged glass surface. In the case of the immobilized vesicle, the diffusion coefficients (Dt) of R6G (rhodamine 6G), DCM (4-dicyanomethylene-2-methyl-6-p-dimethyl aminostyryl-4H-pyran), and C343 (coumarin 343) are found to be 1.5, 2.5, and 10 μm2/s, respectively, which are 280, 120, and 55 times slower compared to those for the same dyes in bulk water. The magnitude of Dt is found to vary for different vesicles. This was attributed to the difference in size and shape of the immobilized vesicles. In bulk, R6G binds completely to the vesicle and exhibits extremely slow diffusion with Dt = 0.5 ± 0.1 μm2/s (∼850 and 3 times slower compared to that of R6G in bulk water and within the immobilized vesicle). This is attributed to very slow overall diffusion of the very large size vesicles (20 μm = 20 000 nm). Both of the dye molecules (DCM and C343) show two different diffusion coefficients for the vesicles in bulk. In this case, the small Dt (0.5 ± 0.1 μm2/s) corresponds to the diffusion of the vesicle as a whole and the large Dt value (300 and 550 μm2/s for DCM and C343, respectively) corresponds to the free dye molecules in bulk water. © 2010 American Chemical Society.