Intermolecular interaction of photoexcited Cu(TMpy-P4) with water studied by transient resonance Raman and picosecond absorption spectroscopies

photoinduced complex between Cu(TMpy-P4) and water molecules, reversibly axially coordinated to the central metal, was observed in picosecond transient absorption and nanosecond resonance Raman experiments. This complex is rapidly created (τ1 = 15 ± 5 ps) in the excited triplet (π, π*) state of Cu-porphyrin, and the subsequent relaxation is proposed to proceed via two parallel pathways. One is fast and efficient (≥90% of molecules), and presumably involves a (π, d) charge-transfer state. The second pathway is slow (τ2 >> 1 ns), has a low quantum yield (≤10%) and involves the excited (d, d) state which is responsible for transient Raman features at ≈ 1553 cm−1 (ν2*) and ≈ 1347 cm−1 (ν4*), and for low-intensity long-lived transient absorption features

Multiconformational analysis of tripeptides upon consideration of implicit and explicit hydration effects

International audienceDuring the last two decades, numerous observed data obtained by various physical techniques, also supported by molecular modeling approaches, have highlighted the structuring features of tripeptides, as well as their aggregation properties. Herein, we focus on the structural dynamics of four trimers, i.e., Gly-Gly-Gly, Gly-Ala-Gly, Ala-Ala-Ala and Ala-Phe-Ala, in an aqueous environment. Density functional theory calculations (DFT) were carried out to assess the stability of four types of secondary structures, i.e., β-strand, polyproline-II (pP-II), α-helix and γ-turn, of which the formation had been described in these tripeptides. Both implicit and explicit hydration effects were analyzed on the conformational and energetic features of trimers. It has been shown that the use of M062X functional (versus B3LYP) improve the stability of intramolecular H-bonds, especially in inverse γ-turn structures, as well as the energetic and conformational equilibrium in all tripeptides. Explicit hydration reflected by the presence of five water molecules around the backbone polar sites (NH3+, N-H, CO and NH2) considerably changes the conformational landscapes of the trimers. Characteristic intramolecular and intermolecular interactions evidenced by the calculations, were emphasized

Ultrafast heme dynamics in ferrous versus ferric cytochrome c studied by time-resolved resonance Raman and transient absorption spectroscopy.

International audienceCytochrome c (Cyt c) is a heme protein involved in electron transfer and also in apoptosis. Its heme iron is bisaxially ligated to histidine and methionine side chains and both ferric and ferrous redox states are physiologically relevant, as well as a ligand exchange between internal residue and external diatomic molecule. The photodissociation of internal axial ligand was observed for several ferrous heme proteins including Cyt c, but no time-resolved studies have been reported on ferric Cyt c. To investigate how the oxidation state of the heme influences the primary photoprocesses, we performed a comprehensive comparative study on horse heart Cyt c by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We found that in ferric Cyt c, in contrast to ferrous Cyt c, the photodissociation of an internal ligand does not take place, and relaxation dynamics is dominated by vibrational cooling in the ground electronic state of the heme. The intermolecular vibrational energy transfer was found to proceed in a single phase with a temperature decay of 7 ps in both ferric and ferrous Cyt c. For ferrous Cyt c, the instantaneous photodissociation of the methionine side chain from the heme iron is the dominant event, and its rebinding proceeds in two phases, with time constants of 5 and 16 ps. A mechanism of this process is discussed, and the difference in photoinduced coordination behavior between ferric and ferrous Cyt c is explained by an involvement of the excited electronic state coupled with conformational relaxation of the heme

Raman Tweezers Microspectroscopy of Functionalized 4.2 nm Diameter CdSe Nanocrystals in Water Reveals Changed Ligand Vibrational Modes by a Metal Cation

International audienceWe demonstrated the possibility of acquiring Raman spectra of colloidal quantum dots (QDs) at low concentration in water with a size as small as 2.5 nm in diameter using Raman tweezers microspectroscopy. We measured the spectra of CdSe QDs capped with thioglycerol and with L-cysteine. This technique was applied to probe the interaction between Co 2+ and Cys-CdSe QDs whose fluorescence emission is quenched in the presence of this metal cation. The quenching mechanism was so far hypothetical. The Raman spectra of Cys-CdSe QDs recorded in the absence and in the presence of Co 2+ demonstrated the binding of Co 2+ cations to the carboxylate groups of the L-cysteine ligand grafted on the surface of the 4.2 nm CdSe QDs. The frequency of modes for the grafted ligand is changed with respect to the free ligand in solution. Considering the vibrational coupling between the excitonic state and the ligand, we inferred that the binding of a metal cation to the grafted ligand modifies this coupling, so that exciton relaxation through crystal defects is favored. This result rationalizes the fluorescence quenching observed during the metal cation-QD interaction

Absorption band III kinetics probe the picosecond heme iron motion triggered by nitric oxide binding to hemoglobin and myoglobin.

International audienceTo study the ultrafast movement of the heme iron induced by nitric oxide (NO) binding to hemoglobin (Hb) and myoglobin (Mb), we probed the picosecond spectral evolution of absorption band III (∼760 nm) and vibrational modes (iron-histidine stretching, ν(4) and ν(7) in-plane modes) in time-resolved resonance Raman spectra. The time constants of band III intensity kinetics induced by NO rebinding (25 ps for hemoglobin and 40 ps for myoglobin) are larger than in Soret bands and Q-bands. Band III intensity kinetics is retarded with respect to NO rebinding to Hb and to Mb. Similarly, the ν((Fe-His)) stretching intensity kinetics are retarded with respect to the ν(4) and ν(7) heme modes and to Soret absorption. In contrast, band III spectral shift kinetics do not coincide with band III intensity kinetics but follows Soret kinetics. We concluded that, namely, the band III intensity depends on the heme iron out-of-plane position, as theoretically predicted ( Stavrov , S. S. Biopolymers 2004 , 74 , 37 - 40 )

Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy

The joint use of 3 complementary techniques, namely, nanoparticle tracking analysis (NTA), cryo-electron microscopy (Cryo-EM) and Raman tweezers microspectroscopy (RTM), is proposed for a rapid characterisation of extracellular vesicles (EVs) of various origins. NTA is valuable for studying the size distribution and concentration, Cryo-EM is outstanding for the morphological characterisation, including observation of vesicle heterogeneity, while RTM provides the global chemical composition without using any exogenous label. The capabilities of this approach are evaluated on the example of cell-derived vesicles of Dictyostelium discoideum, a convenient general model for eukaryotic EVs. At least 2 separate species differing in chemical composition (relative amounts of DNA, lipids and proteins, presence of carotenoids) were found for each of the 2 physiological states of this non-pathogenic microorganism, that is, cell growth and starvation-induced aggregation. These findings demonstrate the specific potency of RTM. In addition, the first Raman spectra of human urinary exosomes are reported, presumably constituting the primary step towards Raman characterisation of EVs for the purpose of human diseases diagnoses

Role of heme iron coordination and protein structure in the dynamics and geminate rebinding of nitric oxide to the H93G myoglobin mutant: Implications for nitric oxide sensors

International audienceThe influence of the heme iron coordination on nitric oxide binding dynamics was investigated for the myoglobin mutant H93G (H93G-Mb) by picosecond absorption and resonance Raman time-resolved spectroscopies. In the H93G-Mb, the glycine replacing the proximal histidine does not interact with the heme iron so that exogenous substituents like imidazole may coordinate to the iron at the proximal position. Nitrosylation of H93G-Mb leads to either 6- or 5-coordinate species depending on the imidazole concentration. At high concentrations, (imidazole)-(NO)-6-coordinate heme is formed, and the photoinduced rebinding kinetics reveal two exponential picosecond phases (~10 and ~100 ps) similar to those of wild type myoglobin. At low concentrations, imidazole is displaced by the trans effect leading to a (NO)-5-coordinate heme, becoming 4-coordinate immediately after photolysis as revealed from the transient Raman spectrum. In this case, NO rebinding kinetics remain bi-exponential with no change in time constant of the fast component whose amplitude increases with respect to the 6-coordinate species. Bi-exponential NO geminate rebinding in 5-coordinate H93G-Mb is in contrast with the single-exponential process reported for nitrosylated soluble guanylate cyclase (Negrerie, M., Bouzhir, L., Martin, J. L., and Liebl, U. (2001) J. Biol. Chem. 276, 46815-46821). Thus, our data show that the iron coordination state or the heme iron out-of-plane motion are not at the origin of the bi-exponential kinetics, which depends upon the protein structure, and that the 4-coordinate state favors the fast phase of NO geminate rebinding. Consequently, the heme coordination state together with the energy barriers provided by the protein structure control the dynamics and affinity for NO-binding enzymes. Cop. 2006 by The American Society for Biochemistry and Molecular Biology, Inc