Temperature-Induced Luminescence Intensity Fluctuation of Protein-Protected Copper Nanoclusters: Role of Scaffold Conformation vs Nonradiative Transition.

Protein-scaffolded atomically precise metal nanoclusters (NCs) have emerged as a promising class of biofriendly nanoprobes at the forefront of modern research, particularly in the area of sensing. The photoluminescence (PL) intensity of several nanoclusters showed a systematic temperature-dependent fluctuation, but the mechanism remains ambiguous and is poorly understood. We tried to shed some light on this mechanistic aspect by testing a couple of hypotheses: (i) conformational fluctuation of the protein scaffold-mediated PL intensity fluctuation and (ii) PL intensity fluctuation due to the variation in the radiative and nonradiative transition rates. Herein, the PL intensity of the lysozyme-capped copper nanocluster (Lys-Cu NC) showed excellent temperature dependency; upon increasing the temperature, the PL intensity gradually decreased. However, contrasting effects can be seen when the nanocluster is exposed to a chemical denaturant (guanidine hydrochloride (GdnHCl)); the PL intensity increased with the increase in the GdnHCl concentration due to the change in the ionic strength of the medium. This discrepancy clearly suggests that the thermal PL intensity fluctuation cannot be explained by a change in the scaffold conformation. Furthermore, upon closer investigation, we observed a 2-fold increase in the nonradiative decay rate of the Lys-Cu NC at the elevated temperature, which could reasonably explain the decrease in the PL intensity of the nanocluster at the higher temperature. Additionally, from the result, it was evident that the protein scaffold-metal core interaction played a key role here in stabilizing each other; hence, the scaffold structure remained unaffected even in the presence of chemical denaturants.

Rapid Detection of Ag(I) via Size-Induced Photoluminescence Quenching of Biocompatible Green-Emitting, l-Tryptophan-Scaffolded Copper Nanoclusters.

Atomically precise metal nanoclusters capped with small molecules like amino acids are highly favored due to their specific interactions and easy incorporation into biological systems. However, they are rarely explored due to the challenge of surface functionalization of nanoclusters with small molecules. Herein, we report the synthesis of a green-emitting (λex = 380 nm, λem = 500 nm), single-amino-acid (l-tryptophan)-scaffolded copper nanocluster (Trp-Cu NC) via a one-pot route under mild reaction conditions. The synthesized nanocluster can be used for the rapid detection of a heavy metal, silver (Ag(I)), in the nanomolar concentration range in real environmental and biological samples. The strong green photoluminescence intensity of the nanocluster quenched significantly upon the addition of Ag(I) due to the formation of bigger nanoparticles, thereby losing its energy quantization. A notable color change from light yellow to reddish-brown can also be observed in the presence of Ag(I), allowing its visual colorimetric detection. Portable paper strips fabricated with the Trp-Cu NC can be reliably used for on-site visual detection of Ag(I) in the micromolar concentration range. The Trp-Cu NC possesses excellent biocompatibility, making it a suitable nanoprobe for cell imaging; thus, it can act as an in vivo biomarker. The nanocluster showed a significant spectral overlap with anticancer drug doxorubicin and thus can be used as an effective fluorescence resonance energy transfer (FRET) pair. FRET results can reveal important information regarding the attachment of the drug to the nanocluster and hence its role as a potential drug carrier for targeted drug delivery within the human body.

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

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 ß-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.

In what time scale proton transfer takes place in a live CHO cell?

Excited state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6-trisulfonate, HPTS) in a live Chinese hamster ovary (CHO) cell is studied by time resolved confocal microscopy. The cytoplasm region of the cell is stained by a photoacid, HPTS (HA). The time constant of initial proton transfer (τ(PT)) in the cell is found to be ~10 times longer than that in bulk water, while the time constants of recombination (τ(rec)) and dissociation (τ(diss)) in the cell are ~3 times and ~2 times longer, respectively. The slower rate of proton transfer (~10 times) inside the CHO cell compared to that in bulk water is ascribed to slower solvation dynamics, lower availability of free water molecules, and disruption of hydrogen-bond network inside the cell. Translational and rotational diffusion of HPTS inside a single CHO cell have been investigated by fluorescence correlation spectroscopy (FCS) and picosecond anisotropy measurement, respectively. Both the translational and rotational diffusion slow down inside the live cell. FCS studies indicate that HPTS remains tightly bound to a macromolecule inside the cell.

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 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 (r(H)) 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, r(H)) 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.

Effect of ionic liquid on the native and denatured state of a protein covalently attached to a probe: solvation dynamics study.

Effect of a room temperature ionic liquid (RTIL, [pmim][Br]) on the solvation dynamics of a probe covalently attached to a protein (human serum albumin (HSA)) has been studied using femtosecond up-conversion. For this study, a solvation probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) has been covalently attached to the lone cysteine group (cys-34) of the protein HSA. Addition of 1.5 M RTIL or 6 M GdnHCl causes a red shift of the emission maxima of CPM bound to HSA by 3 nm and 12 nm, respectively. The average solvation time 〈τ(s)〉 decreases from 650 ps (in native HSA) to 260 ps (~2.5 times) in the presence of 1.5 M RTIL and to 60 ps (~11 times) in the presence of 6 M GdnHCl. This is ascribed to unfolding of the protein by RTIL or GdnHCl and therefore making the probe CPM more exposed. When 1.5 M RTIL is added to the protein denatured by 6 M GdnHCl in advance, a further ~5 nm red shift along with further ~2 fold faster solvent relaxation (<τ> ~30 ps) is observed. Our previous fluorescence correlation spectroscopy study [D. K. Sasmal, T. Mondal, S. Sen Mojumdar, A. Choudhury, R. Banerjee, and K. Bhattacharyya, J. Phys. Chem. B 115, 13075 (2011)] suggests that addition of RTIL to the protein denatured by 6 M GdnHCl causes a reduction in hydrodynamic radius (r(h)). It is demonstrated that in the presence of RTIL and GdnHCl, though the protein is structurally more compact, the local environment of CPM is very different from that in the native state.

Solvation dynamics under a microscope: single giant lipid vesicle.

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.

Deoxycholate induced tetramer of αA-crystallin and sites of phosphorylation: fluorescence correlation spectroscopy and femtosecond solvation dynamics.

Structure and dynamics of acrylodan labeled αA-crystallin tetramer formed in the presence of a bile salt (sodium deoxycholate, NaDC) has been studied using fluorescence correlation spectroscopy (FCS) and femtosecond up-conversion techniques. Using FCS it is shown that, the diffusion constant (D(t)) of the αA-crystallin oligomer (mass ~800 kDa) increases from ~35 µm(2) s(-1) to ~68 µm(2) s(-1). This corresponds to a decrease in hydrodynamic radius (r(h)) from ~6.9 nm to ~3.3 nm. This corresponds to about 10-fold decrease in molecular mass to ~80 kDa and suggests formation of a tetramer (since mass of αA-crystallin monomer is ~20 kDa). The steady state emission maximum and average solvation time (<τ(s)>) of acrylodan labeled at cysteine 131 position of αA-crystallin is markedly affected on addition of NaDC, while the tryptophan (trp-9) becomes more exposed. This suggests that NaDC binds near the cys-131 and makes the terminal region of αA-crystallin exposed. This may explain the enhanced auto-phosphorylation activity of αA-crystallin near the terminus of the 173 amino acid protein (e.g., at the threonine 13, serine 45, or serine 169 and 172) and suggests that phosphorylation at ser-122 (close to cys-131) is relatively less important.

Effect of ionic liquid on diffusion in P123 gel: fluorescence correlation spectroscopy.

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 (D(t)) of the hydrophobic dye DCM (1 µm(2) s(-1) at the core) is smaller than that of the other two hydrophilic dyes (7 µm(2) s(-1) for C480 and C343). On addition of RTIL, the D(t) values of all of the dyes increase, indicating a decrease in local viscosity (η(eff)). The η(eff) 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.

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

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 (D(t)) decreases and the hydrodynamic radius (R(h)) 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 D(t) 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.

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

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 (K(b)) 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 τ(off) and τ(on), 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.

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

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.

Diffusion of organic dyes in immobilized and free catanionic vesicles.

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 (D(t)) 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 µm(2)/s, respectively, which are 280, 120, and 55 times slower compared to those for the same dyes in bulk water. The magnitude of D(t) 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 D(t) = 0.5 ± 0.1 µm(2)/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 D(t) (0.5 ± 0.1 µm(2)/s) corresponds to the diffusion of the vesicle as a whole and the large D(t) value (300 and 550 µm(2)/s for DCM and C343, respectively) corresponds to the free dye molecules in bulk water.

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

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] [BF(4)]) 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] [BF(4)]). 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 H(2)O by D(2)O causes an increase in the time constant of the ultraslow rise to 2350 ps.

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

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 approximately 100 nm) is smaller than the diffraction limited spot size (approximately 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 degrees C, respectively) were studied. The diffusion coefficient of the dye DCM in bulk water is approximately 300 microm(2)/s. In the lipid vesicle, the average D(t) decreases markedly to approximately 5 microm(2)/s (approximately 60 times) in the gel phase (for DPPC at 20 degrees C) and 40 microm(2)/s ( approximately 8 times) in the fluid phase (for DLPC at 20 degrees C). This clearly demonstrates higher mobility in the fluid phase compared with the gel phase of a lipid. It is observed that the D(t) values vary from lipid to lipid and there is a distribution of D(t) values. The diffusion of the hydrophobic dye DCM (D(t) approximately 5 microm(2)/s) in the DPPC vesicle is found to be 8 times smaller than that of a hydrophilic anioinic dye C343 (D(t) approximately 40 microm(2)/s). This is attributed to different locations of the hydrophobic (DCM) and hydrophilic (C343) dyes.