Samir Kumar Pal

Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution

Biological water at the interface of proteins is critical to their equilibrium structures and enzyme function and to phenomena such as molecular recognition and protein–protein interactions. To actually probe the dynamics of water structure at the surface, we must examine the protein itself, without disrupting the native structure, and the ultrafast elementary processes of hydration. Here we report direct study, with femtosecond resolution, of the dynamics of hydration at the surface of the enzyme protein Subtilisin Carlsberg, whose single Trp residue (Trp-113) was used as an intrinsic biological fluorescent probe. For the protein, we observed two well separated dynamical solvation times, 0.8 ps and 38 ps, whereas in bulk water, we obtained 180 fs and 1.1 ps. We also studied a covalently bonded probe at a separation of ≈7 Å and observed the near disappearance of the 38-ps component, with solvation being practically complete in (time constant) 1.5 ps. The degree of rigidity of the probe (anisotropy decay) and of the water environment (protein vs. micelle) was also studied. These results show that hydration at the surface is a dynamical process with two general types of trajectories, those that result from weak interactions with the selected surface site, giving rise to bulk-type solvation (≈1 ps), and those that have a stronger interaction, enough to define a rigid water structure, with a solvation time of 38 ps, much slower than that of the bulk. At a distance of ≈7 Å from the surface, essentially all trajectories are bulk-type. The theoretical framework for these observations is discussed.

Copper Quantum Clusters in Protein Matrix: Potential Sensor of Pb2+ Ion

A one-pot synthesis of extremely stable, water-soluble Cu quantum clusters (QCs) capped with a model protein, bovine serum albumin (BSA), is reported. From matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, we assign the clusters to be composed of Cu(5) and Cu(13) cores. The QCs also show luminescence properties having excitation and emission maxima at 325 and 410 nm, respectively, with a quantum yield of 0.15, which are found to be different from that of protein alone in similar experimental conditions. The quenching of luminescence of the protein-capped Cu QCs in the presence of very low hydrogen peroxide concentration (approximately nanomolar, or less than part-per-billion) reflects the efficacy of the QCs as a potential sensing material in biological environments. Moreover, as-prepared Cu QCs can detect highly toxic Pb(2+) ions in water, even at the part-per-million level, without suffering any interference from other metal ions.

Bright, NIR-Emitting Au23 from Au25: Characterization and Applications Including Biolabeling

A novel interfacial route has been developed for the synthesis of a bright-red-emitting new subnanocluster, Au(23), by the core etching of a widely explored and more stable cluster, Au(25)SG(18) (in which SG is glutathione thiolate). A slight modification of this procedure results in the formation of two other known subnanoclusters, Au(22) and Au(33). Whereas Au(22) and Au(23) are water soluble and brightly fluorescent with quantum yields of 2.5 and 1.3 %, respectively, Au(33) is organic soluble and less fluorescent, with a quantum yield of 0.1 %. Au(23) exhibits quenching of fluorescence selectively in the presence of Cu(2+) ions and it can therefore be used as a metal-ion sensor. Aqueous- to organic-phase transfer of Au(23) has been carried out with fluorescence enhancement. Solvent dependency on the fluorescence of Au(23) before and after phase transfer has been studied extensively and the quantum yield of the cluster varies with the solvent used. The temperature response of Au(23) emission has been demonstrated. The inherent fluorescence of Au(23) was used for imaging human hepatoma cells by employing the avidin-biotin interaction.

Water at DNA surfaces: Ultrafast dynamics in minor groove recognition

Water molecules at the surface of DNA are critical to its equilibrium structure, DNA–protein function, and DNA–ligand recognition. Here we report direct probing of the dynamics of hydration, with femtosecond resolution, at the surface of a DNA dodecamer duplex whose native structure remains unperturbed on recognition in minor groove binding with the bisbenzimide drug (Hoechst 33258). By following the temporal evolution of fluorescence, we observed two well separated hydration times, 1.4 and 19 ps, whereas in bulk water the same drug is hydrated with time constants of 0.2 and 1.2 ps. For comparison, we also studied calf thymus DNA for which the hydration exhibits similar time scales to that of dodecamer DNA. However, the time-resolved polarization anisotropy is very different for the two types of DNA and clearly elucidates the rigidity in drug binding and difference in DNA rotational motions. These results demonstrate that hydration at the surface of the groove is a dynamical process with two general types of trajectories; the slowest of them (≈20 ps) are those describing dynamically ordered water. Because of their ultrafast time scale, the “ordered” water molecules are the most weakly bound and are accordingly involved in the entropic (hydration/dehydration) process of recognition.

Ag7Au6: A 13-Atom Alloy Quantum Cluster†

An alloy cluster containing a 13-atom core, with a composition Ag 7 Au 6 (H 2 MSA) 10 (H 2 MSA=mercaptosuccinic acid) was synthesized from silver clusters by a galvanic exchange reaction. The clusters were characterized by several spectroscopic and microscopic methods. The alloy cluster shows luminescence with a quantum yield of 3.5×10 -2 at room temperature. Theoretical calculations for Ag 7 Au 6 (SCH 3 ) 10 suggest a distorted icosahedral core.

Hydration at the surface of the protein Monellin: Dynamics with femtosecond resolution

We have studied the femtosecond hydration dynamics of Monellin, a protein with a single tryptophan residue at its surface. Tryptophan was selectively used as a probe of the dynamics, and through monitoring of its fluorescence Stokes shift with time we obtained the hydration correlation function, which decays due to rotational and translational motions of water at the protein surface and in bulk. The decay exhibits a “bimodal” behavior with time constants of 1.3 and 16 ps, mirroring relaxation of the free/quasifree water molecules and surface-bound water layer (minimum binding energy of 1–2 kcal/mol). The observed slow decay of 16 ps for tryptophan in the native protein differs by more than an order of magnitude from that of bulk water because of the dynamical exchange in the layer. To examine the effect of unfolding, we also studied hydration dynamics when Monellin was denatured in a 6 M guanidine hydrochloride solution and obtained a totally different behavior: 3.5 and 56 ps. Comparing with the results of experiments on free tryptophan in the same concentration of the denaturing solution, we conclude that the fast component of 3.5 ps comes from bulk-type solvation in the 6 M guanidine hydrochloride. However, the absence of the 16-ps decay and appearance of the 56-ps component reflects a more “rigid solvation,” which is likely to involve the motions of the protein backbone in the random-coiled state. With the help of polymer theory, this time scale is reproduced in agreement with experimental observations.

Site- and sequence-selective ultrafast hydration of DNA

Water molecules in the DNA grooves are critical for maintaining structural integrity, conformational changes, and molecular recognition. Here we report studies of site- and sequence-specific hydration dynamics, using 2-aminopurine (Ap) as the intrinsic fluorescence probe and with femtosecond resolution. The dodecamer d[CGCA(Ap)ATTTGCG]2 was investigated, and we also examined the effect of a specific minor groove-binding drug, pentamidine, on hydration dynamics. Two time scales were observed: ≈1 ps (bulk-like) and 10–12 ps (weakly bound type), consistent with layer hydration observed in proteins and DNA. However, for denatured DNA, the cosolvent condition of 40% formamide hydration is very different: it becomes that of bulk (in the presence of formamide). Well known electron transfer between Ap and nearby bases in stacked assemblies becomes inefficient in the single-stranded state. The rigidity of Ap in the single strands is significantly higher than that in bulk water and that attached to deoxyribose, suggesting a unique role for the dynamics of the phosphate-sugar-base in helix formation. The disparity in minor and major groove hydration is evident because of the site selection of Ap and in the time scale observed here (in the presence and absence of the drug), which is different by a factor of 2 from that observed in the minor groove–drug recognition.

Photoreactivity of ZnO nanoparticles in visible light: Effect of surface states on electron transfer reaction

Wide band gap metal oxide semiconductors such as zinc oxide (ZnO) show visible band photolysis that has been employed, among others, to degrade harmful organic contaminants into harmless mineral acids. Metal oxides show enhanced photocatalytic activity with the increase in electronic defects in the crystallites. By introducing defects into the crystal lattice of ZnO nanoparticles, we observe a redshift in the optical absorption shifting from the ultraviolet region to the visible region (400-700 nm), which is due to the creation of intermediate defect states that inhibit the electron hole recombination process. The defects were introduced by fast nucleation and growth of the nanoparticles by rapid heating using microwave irradiation and subsequent quenching during the precipitation reaction. To elucidate the nature of the photodegradation process, picosecond resolved time correlated single photon count (TCSPC) spectroscopy was carried out to record the electronic transitions resulting from the de-excitation of the electrons to their stable states. Photodegradation and TCSPC studies showed that defect engineered ZnO nanoparticles obtained through fast crystallization during growth lead to a faster initial degradation rate of methylene blue as compared to the conventionally synthesized nanoparticles.

Femtosecond dynamics of rubredoxin: Tryptophan solvation and resonance energy transfer in the protein

We report here studies of tryptophan (Trp) solvation dynamics in water and in the Pyrococcus furiosus rubredoxin protein, including the native and its apo and denatured forms. We also report results on energy transfer from Trp to the iron-sulfur [Fe-S] cluster. Trp fluorescence decay with the onset of solvation dynamics of the chromophore in water was observed with femtosecond resolution (≈160 fs; 65% component), but the emission extended to the picosecond range (1.1 ps; 35% component). In contrast, the decay is much slower in the native rubredoxin; the Trp fluorescence decay extends to 10 ps and longer, reflecting the local rigidity imposed by residues and by the surface water layer. The dynamics of resonance energy transfer from the two Trps to the [Fe-S] cluster in the protein was observed to follow a temporal behavior characterized by a single exponential (15–20 ps) decay. This unusual observation in a protein indicates that the resonance transfer is to an acceptor of a well-defined orientation and separation. From studies of the mutant protein, we show that the two Trp residues have similar energy-transfer rates. The critical distance for transfer (R0) was determined, by using the known x-ray data, to be 19.5 Å for Trp-36 and 25.2 Å for Trp-3, respectively. The orientation factor (κ2) was deduced to be 0.13 for Trp-36, clearly indicating that molecular orientation of chromophores in the protein cannot be isotropic with κ2 value of 2/3. These studies of solvation and energy-transfer dynamics, and of the rotational anisotropy, of the wild-type protein, the (W3Y, I23V, L32I) mutant, and the fmetPfRd variant at various pH values reveal a dynamically rigid protein structure, which is probably related to the hyperthermophilicity of the protein.

Photoselective excited state dynamics in ZnO-Au nanocomposites and their implications in photocatalysis and dye-sensitized solar cells.

Improving the performance of photoactive solid-state devices begins with systematic studies of the metal-semiconductor nanocomposites (NCs) upon which such devices are based. Here, we report the photo-dependent excitonic mechanism and the charge migration kinetics in a colloidal ZnO-Au NC system. By using a picosecond-resolved Förster resonance energy transfer (FRET) technique, we have demonstrated that excited ZnO nanoparticles (NPs) resonantly transfer visible optical radiation to the Au NPs, and the quenching of defect-mediated visible emission depends solely on the excitation level of the semiconductor. The role of the gold layer in promoting photolytic charge transfer, the activity of which is dependent upon the degree of excitation, was probed using methylene blue (MB) reduction at the semiconductor interface. Incident photon-to-current efficiency measurements show improved charge injection from a sensitizing dye to a semiconductor electrode in the presence of gold in the visible region. Furthermore, the short-circuit current density and the energy conversion efficiency of the ZnO-Au NP based dye-sensitized solar cell (DSSC) are much higher than those of a DSSC comprised of only ZnO NP. Our results represent a new paradigm for understanding the mechanism of defect-state passivation and photolytic activity of the metal component in metal-semiconductor nanocomposite systems.