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Transient absorption measurements on cis-and trans-azobenzene after UV excitation in the ππ* band are presented and compared to data obtained after VIS excitation in the nπ* band. The data show a two-step process, where after a fast motion on the S 2 potential energy surface the molecule relaxes to the S 1 state and in both cases (trans f cis and cis f trans) the isomerization reaction involves large-amplitude motion on the S 1 potential energy surface.

Intrachain loop formation allows unfolded polypeptide chains to search for favorable interactions during protein folding. We applied triplet-triplet energy transfer between a xanthone moiety and naphthylalanine to directly measure loop formation in various unfolded polypeptide chains with loop regions consisting of polyserine, poly(glycine-serine) or polyproline. By combination of femtosecond and nanosecond laserflash experiments loop formation could be studied over many orders of magnitude in time from picoseconds to microseconds. The results reveal processes on different time scales indicating motions on different hierarchical levels of the free energy surface. A minor (<15%) very fast reaction with a time constant of Ϸ3 ps indicates equilibrium conformations with donor and acceptor in contact at the time of the laserflash. Complex kinetics of loop formation were observed on the 50-to 500-ps time scale, which indicate motions within a local well on the energy landscape. Conformations within this well can form loops by undergoing local motions without having to cross major barriers. Exponential kinetics observed on the 10-to 100-ns time scale are caused by diffusional processes involving large-scale motions that allow the polypeptide chain to explore the complete conformational space. These results indicate that the free energy landscape for unfolded polypeptide chains and native proteins have similar properties. The presence of local energy minima reduces the conformational space and accelerates the conformational search for energetically favorable local intrachain contacts.conformational substates ͉ femtoseconds spectroscopy ͉ peptide dynamics ͉ protein folding ͉ triplet-triplet energy transfer U nderstanding the protein folding process requires the characterization of the structure and dynamics of all states along the reaction coordinate and the transition states separating them. Properties of native proteins have been extensively characterized by x-ray crystallography and NMR spectroscopy. Detailed structural information on partially folded states has been obtained by hydrogen exchange and NMR techniques. The properties of unfolded proteins are much less understood, although a detailed characterization of the structure and dynamics of the unfolded state is essential for a better understanding of the early steps in protein folding and the free energy landscape of the folding reaction. A major problem in the characterization of unfolded proteins is the nonphysiological solvent conditions that are required to populate the unfolded state in equilibrium. NMR studies (1-9) and the analysis of the effect of mutations on the solvent accessibility of the unfolded state (10) have revealed the presence of both native and non-native interactions in unfolded states of several proteins. In addition, steric constraints and intramolecular hydrogen bonding were suggested to promote the folding reaction by restricting the conformational space of unfolded polypeptide chains (11,12). Recently, the dynamics of the unfolded stat...

The formation and quenching of the triplet state of xanthone are studied by femtosecond techniques. As revealed by femtosecond fluorescence spectroscopy, the primarily excited 1ππ* state decays within 1.5 ps. In a transient absorption experiment, this time constant is associated with a partial rise of a triplet signature. This rise has a second and slower component with a time constant of 12 ps. In the presence of high concentrations of the quencher 1-methylnaphthalene, the slow 12 ps rise component is absent. This finding gives strong evidence that the biphasic rise of the triplet absorption of xanthone is due to a sequential mechanism, namely, a 1ππ* → 3 nπ* with fast intersystem crossing followed by a 3 nπ* → 3ππ* internal conversion. Furthermore, an analysis of the concentration dependence of the quenching kinetics allows one to pin down the intrinsic transfer time of the triplet energy from xanthone to 1-methylnaphthalene to ∼1 ps.

Photo-excited xanthone is known to undergo ultrafast intersystem crossing (ISC) in the 1 ps time domain. Correspondingly, its fluorescence quantum yield in most solvents is very small ( approximately 10(-4)). Surprisingly, the quantum yield in water is 100 times larger, while ISC is still rapid ( approximately 1 ps), as seen by ultrafast pump probe absorption spectroscopy. Temperature dependent steady state and time resolved fluorescence experiments point to a delayed fluorescence mechanism, where the triplet (3)npi* state primarily accessed by ISC is nearly isoenergetic with the photo-excited (1)pipi* state. The delayed fluorescence of xanthone in water decays with a time constant of 700 ps, apparently by internal conversion between the (3)npi* state and the lowest lying triplet state (3)pipi*.