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The far-UV spectral window widely used for the conformational analysis of biomolecules is not easily covered with broad-band lasers. This has made it difficult to use circular dichroism (CD) spectroscopy to directly follow fast structure changes. By combining transient CD spectroscopy in the deep-UV with thioamide substitution, we demonstrate a method to overcome this difficulty. We investigated a dipeptide whose two carbonyl oxygen atoms were replaced by sulfur, red-shifting the strong lowest-lying pi pi* transitions into the more accessible 250-370 nm spectral window. Coupling of the two thioamide units cannot be resolved by achiral 2D-UV spectroscopy, but it gives rise to a pronounced bisignate CD spectrum. The transient CD spectra reveal weakening of this coupling in the electronically excited state, where conformational constraints are released. Our results show that direct local probing of fast backbone conformational change via CD spectroscopy is possible in combination with site-selective thio substitution in peptides and proteins.

When azide binds to ferric Myoglobin it forms either a low-spin or a high-spin complex, which give rise to two well-separated asymmetric stretch bands of the ligand. Both electronic excitation of the Q-band and vibrational excitation of N 3 - in the mid-IR lead to a similar ultrafast population redistribution in favor of the high spin configuration, which is characterized by a 8° reorientation of the ligand transition dipole moment. The more stable low spin complex subsequently re-emerges with a 18 ps time-constant. It is argued that the observed spin state changes are caused by the participation of low-lying electronic excitations in the cooling process of heme.

The monosubstituted thioxopeptide Boc-Ala-Pro-ψ(SC—NH)-Aib-Ala-OMe is investigated by time-resolved UV-pump/IR-probe and IR-pump/IR-probe spectroscopy, steady-state FTIR-spectroscopy, and NMR-techniques. The compound has a high propensity to adopt a i → i + 3 hydrogen-bonded conformation. Time-resolved infrared measurements reveal the opening of this β-turn structure upon trans → cis photo isomerization of the thioamide bond. Comparison is made with three protected tripeptides containing the -SC—NH-Aib- moiety with different thio-substituted residues. Very similar photo isomerization dynamics and comparable quantum efficiencies are found. Differences are seen for the thermally activated cis → trans relaxation in the electronic ground state, where thioxopeptides with larger residues next to the thioamide moiety exhibit subsecond isomerization times. Anisotropy measurements indicate a very rigid Aib-containing core structure for all four thioxopeptides in acetonitrile solution.

Two cyclic disulfide-bridged tetrapeptides [cyclo(Boc-Cys-Pro-Aib-Cys-OMe) (1) and cyclo(Boc-Cys-Pro-Phe-Cys-OMe) (2)] have been monitored by time-resolved mid-IR spectroscopy in the C=O vibrational range. A conformational change is induced by cleavage of the intramolecular disulfide bridge upon UV excitation (lambda(exc) = 260 nm), giving rise to a pair of cysteinyl radicals (thiyl radicals), which diffuse apart allowing the peptide to change conformation before they undergo quenching. The amide I band reports on the dynamics of the peptide backbone, which evolves on a 100 ps time scale and then stays constant up to 10 micros at low enough concentrations ( approximately 100 mM). To probe specifically the lifetime of the free cysteinyl radicals, time-resolved UV laser flash photolysis has been applied. The concentration of the cysteinyl radical decays nonexponentially, but about 50% are still present after 1 ms. The photocleavable disulfide bridge hence may serve as an intrinsic, naturally occurring phototrigger to study peptide dynamics that opens a wide time-window from a few picoseconds to many hundreds of microseconds.

Time-resolved infrared spectroscopy was used to study the photoisomerization of N-Methylthioacetamide (NMTAA) in D2O in both the cis--trans and the trans--cis direction upon selective excitation of the n-pi (S1) and pi-pi (S2) electronic transitions. While isomerization and the return to the ground state takes place on two distinct time scales (or=8 ps, approximately 250 ps) upon pi-pi excitation of both cis- and trans-NMTAA in D2O, ground state recovery is only observed on the slower time scale upon n-pi excitation. The quantum efficiency for trans--cis isomerization is 30-40%, independent of the electronic state excited, while the cis--trans isomerization proceeds with a 60-70% quantum efficiency. These results support a mechanism by which isomerization takes place via one common intermediate state independent of electronic excitation energy and initial conformation.

International audience; The dynamics of the ferric CN complexes of the heme proteins Myoglobin and Hemoglobin I from the clam Lucina pectinata upon Soret band excitation is monitored using infrared and broad band visible pump-probe spectroscopy. The transient response in the UV-vis spectral region does not depend on the heme pocket environment and is very similar to that known for ferrous proteins. The main feature is an instantaneous, broad, short-lived absorption signal that develops into a narrower red-shifted Soret band. Significant transient absorption is also observed in the 360 - 390 nm range. At all probe wavelengths the signal decays to zero with a longest time constant of 3.6 ps. The infrared data on MbCN reveal a bleaching of the C = N stretch vibration of the heme-bound ligand, and the formation of a five-times weaker transient absorption band, 28 cm(-1) lower in energy, within the time resolution of the experiment. The MbC = N stretch vibration provides a direct measure for the return of population to the ligated electronic ( and vibrational) ground state with a 3 - 4 ps time constant. In addition, the CN-stretch frequency is sensitive to the excitation of low frequency heme modes, and yields independent information about vibrational cooling, which occurs on the same timescale.

Femtosecond fluorescence up-conversion, UV-Vis and IR transient absorption spectroscopy are used to study the photo-isomerization dynamics of a new type of zwitterionic photoswitch based on a N-alkylated indanylidene pyrroline Schiff base framework (ZW-NAIP). The system is biomimetic, as it mimics the photophysics of retinal, in coupling excited state charge translocation and isomerization. While the fluorescence lifetime is 140 fs, excited state absorption persists over 230 fs in the form of a vibrational wavepacket according to twisting of the isomerizing double bond. After a short "dark'' time window in the UV-visible spectra, which we associate with the passage through a conical intersection (CI), the wavepacket appears on the ground state potential energy surface, as evidenced by the transient mid-IR data. This allows for a precise timing of the photoreaction all the way from the initial Franck-Condon region, through the CI and into both ground state isomers, until incoherent vibrational relaxation dominates the dynamics. The photo-reaction dynamics remarkably follow those observed for retinal in rhodopsin, with the additional benefit that in ZW-NAIP the conformational change reverses the zwitterion dipole moment direction. Last, the pronounced low-frequency coherences make these molecules ideal systems for investigating wavepacket dynamics in the vicinity of a CI and for coherent control experiments.

Difference two-dimensional-infrared (2D-IR) spectroscopy has been employed to measure the 2D-IR spectrum of the C=C and C=ND chromophore bands in bacteriorhodopsin, demonstrating that couplings between individual bands in a mid-size protein can be measured.

Femtosecond UV-VIS and mid-IR experiments show that a new class of biomimetic photoswitches photo-isomerizes in less than 300 fs. In close analogy to rhodopsin, the isomerization is driven by ultrafast motion along the stretch and the torsional coordinates.

Transient 2D-IR spectra have been recorded during the photoreaction of the thioxopeptide Boc-Ala-Gly(S)-Ala-Aib-OMe. We demonstrate the potential of transient 2D-IR spectroscopy to resolve vibrational bands hidden in conventional pump-probe spectra. Different types of transient cross-peak signals are observed, and their information content is discussed by comparison with model spectra.

The photoisomerization of the protected tetrathioxopeptide Boc-Ala-Gly(=S)-Ala-Aib-OMe was followed using time-resolved infrared spectroscopy in the amide I region in combination with isotope labeling. In acetonitrile at room temperature, approximately half of the molecules are found in a loop conformation, restrained by an intramolecular hydrogen bond, while the other half adopts more extended conformations. UV-excitation of the thioxopeptide unit immediately weakens the intramolecular hydrogen bond. After the molecules have relaxed to the electronic ground state with a 130 ps time-constant, a delayed re-formation of the intramolecular hydrogen bond is observed for molecules returning to the initial trans conformation of the thioamide bond, while the loop structure is permanently broken when the molecules isomerize to the cis conformation.

Photo-triggered α-helix formation of a 16-residue peptide featuring a built-in conformational photoswitch is monitored by time-resolved IR spectroscopy. An experimental approach with 2-ps time resolution and a scanning range up to 30 μs is used to cover all time scales of the peptide dynamics. Experiments are carried out at different temperatures between 281 and 322 K. We observe single-exponential kinetics of the amide I′ band at 322 K on a time scale comparable to a recent temperature-jump folding experiment. When lowering the temperature, the kinetics become slower and nonexponential. The transition is strongly activated. Spectrally dispersed IR measurements provide multiple spectroscopic probes simultaneously in one experiment by resolving the amide I′ band, isotope-labeled amino acid residues, and side chains. We find differing relaxation dynamics at different spectral positions.

Thio amino acids can be integrated into the backbone of peptides without significantly perturbing their structure. In this contribution we use ultrafast infrared and visible spectroscopy as well as state-of-the-art ab initio computations to investigate the photoisomerization of the trans form of N-methylthioacetamide (NMTAA) as a model conformational photoswitch. Following the S2 excitation of trans-NMTAA in water, the return of the molecule into the trans ground state and the formation of the cis isomer is observed on a dual time scale, with a fast component of 8-9 ps and a slow time constant of approximately 250 ps. On both time scales the probability of isomerization to the cis form is found to be 30-40%, independently of excitation wavelength. Ab initio CASPT2//CASSCF photochemical reaction path calculations indicate that, in vacuo, the trans--cis isomerization event takes place on the S1 and/or T1 triplet potential energy surfaces and is controlled by very small energy barriers, in agreement with the experimentally observed picosecond time scale. Furthermore, the calculations identify one S2/S1 and four nearly isoenergetic S1/S0 conical intersection decay channels. In line with the observed isomerization probability, only one of the S1/S0 conical intersections yields the cis conformation upon S1--S0 decay. A substantially equivalent excited-state relaxation results from four T1/S0 intersystem crossing points.

Hb I (HbI) is an invertebrate monomeric hemeprotein from Lucina pectinata that contains 142 amino acid residues. The HbI heme pocket structural compn. shows an arrangement of phenylalanyl residues (Phe (29), Phe (43) and Phe (68)) that has not been obsd. in the globin families. The photochem. of HbI ligand complexes can yield relevant information about invertebrate globins in terms of ligands rebinding and photophys. processes. We have performed ultrafast measurements to det. the extent of O2 and CO recombination subsequent to their photodissocn. from the HbI moiety. To sep. the contributions of electronic, thermal and conformational relaxation, from ligand rebinding in the spectroscopic signals, measurements of the deoxyHbI were also performed. The transient absorption signals in the Soret region of HbICO and HIO2 are composed of similar time consts. as obsd. for other heme proteins. The fast component (300 fs) is due to the formation of the unligated deoxy species from the excited heme state HbI*. The second time const. (2.2-2.5 ps) corresponds to the decay of the excited heme state HbII*. The contribution of this component to the transient signal near the Soret band max. appears to be greater in HbI complexes than in other myoglobins. Its presence in the decay of the Soret bleaching signals has been interpreted as a signature of fast geminate recombination. Thus, these results could suggest that the differences in the transient signal between HbI and Mb are influenced by the different heme-pocket structures. Photoexcitation of the HbI complexes led to the formation of a short-lived species in the 630 nm region. Formation of this species appears to be instantaneously, while it decays with a 300 fs time const. This new transient is related to an iron-to-porphyrin charge transfer state. [on SciFinder (R)]

Single molecules that act as light-energy transducers (e.g., converting the energy of a photon into atomic-level mechanical motion) are examples of minimal molecular devices. Here, we focus on a molecular switch designed by merging a conformationally locked diarylidene skeleton with a retinal-like Schiff base and capable of mimicking, in solution, different aspects of the transduction of the visual pigment Rhodopsin. Complementary ab initio multiconfigurational quantum chemistry-based computations and time-resolved spectroscopy are used to follow the light-induced isomerization of the switch in methanol. The results show that, similar to rhodopsin, the isomerization occurs on a 0.3-ps time scale and is followed by

The photochem. of ICN isolated in Ar matrixes has been investigated using UV absorption and laser spectroscopy under a variety of exptl. conditions. Laser excitation of the repulsive A continuum gives rise to long-lived near-IR emission from stabilized I and CN fragments in the shallow excited 3P2 potential and to the formation of the INC isomer. The latter is identified by its UV absorption band, which overlaps the ICN UV absorption. A photoinduced equil. is rapidly established between the two species under 250-nm irradn., in line with the interpretation of previously published IR data (Samuni, U. et al. Chem. Phys. Lett. 1994, 225, 391). This equil. can be shifted in favor of either species by the choice of irradn. wavelength. Permanent dissocn. and the stabilization of CN fragments under UV irradn. is also reported, but the dissocn. efficiency depends strongly on exptl. conditions. This observation allows for the reconciliation of conflicting views in the literature on the photostability of ICN in solid argon. [on SciFinder (R)]

The photophysics of ferricyanide in H2O, D2O and ethylene glycol was studied upon excitation of ligand-to-metal charge transfer (LMCT) transitions by combining ultrafast photoelectron spectroscopy (PES) of liquids and transient vibrational spectroscopy. Upon 400 nm excitation in water, the PES results show a prompt reduction of the Fe3+ to Fe2+ and a back electron transfer in similar to 0.5 ps concomitant with the appearance and decay of a strongly broadened infrared absorption at similar to 2065 cm(-1). In ethylene glycol, the same IR absorption band decays in similar to 1 ps, implying a strong dependence of the back electron transfer on the solvent. Thereafter, the ground state ferric species is left vibrationally hot with significant excitation of up to two quanta of the CN-stretch modes, which completely decay on a 10 ps time scale. Under 265 nm excitation even higher CN-stretch levels are populated. Finally, from a tiny residual transient IR signal, we deduce that less than 2% of the excited species undergo photoaquation, in line with early flash photolysis experiments. The latter is more significant at 265 nm compared to 400 nm excitation, which suggests photodissociation in this system is an unlikely statistical process related to the large excess of vibrational energy.