The reversible photoswitching between the 'on' and 'off' states of the fluorescent protein Dronpa involves photoisomerization as well as protein side-chain rearrangements, but the process of interconversion remains poorly characterized. Here we use time-resolved infrared measurements to monitor the sequence of these structural changes, but also of proton transfer events, which are crucial to the development of spectroscopic contrast. Light-induced deprotonation of the chromophore phenolic oxygen in the off state is a thermal ground-state process, which follows ultrafast (9 ps) trans-cis photoisomerization, and so does not involve excited-state proton transfer. Steady-state infrared difference measurements exclude protonation of the imidazolinone nitrogen in both the on and off states. Pump-probe infrared measurements of the on state reveal a weakening of the hydrogen bonding between Arg66 and the chromophore C ¼ O, which could be central to initiating structural rearrangement of Arg66 and His193 coinciding with the low quantum yield cis-trans photoisomerization

Insulin homodimer associates through the coupled folding and binding of two partially disordered monomers. We aim to understand this dynamics by observing insulin dimer dissociation initiated with a nanosecond temperature jump using transient two-dimensional infrared spectroscopy (2D IR) of amide I vibrations. With the help of equilibrium FTIR and 2D IR spectra, and through a systematic study of the dependence of dissociation kinetics on temperature and insulin concentration, we are able to decompose and analyze the spectral evolution associated with different secondary structures. We find that the dissociation under all conditions is characterized by two processes whose influence on the kinetics varies with temperature: the unfolding of the β sheet at the dimer interface observed as exponential kinetics between 250 and 1000 μs and nonexponential kinetics between 5 and 150 μs that we attribute to monomer disordering. Microscopic reversibility arguments lead us to conclude that dimer association requires significant conformational changes within the monomer in concert with the folding of the interfacial β sheet. While our data indicates a more complex kinetics, we apply a two-state model to the β-sheet unfolding kinetics to extract thermodynamic parameters and kinetic rate constants. The association rate constant, ka (23 °C) = 8.8 × 10(5) M(-1) s(-1) (pH 0, 20% EtOD), is approximately 3 orders of magnitude slower than the calculated diffusion limited association rate, which is explained by the significant destabilizing effect of ethanol on the dimer state and the highly positive charge of the monomers at this pH.

The quantum yield of photoisomerisation of the photoactive yellow protein (PYP) strongly depends on peak power and wavelength with femtosecond optical excitation. Using systematic power titrations and addition of second order dispersion resulting in 140, 300 and 600 fs pulse durations, the one and multi-photon cross-sections at 400, 450 and 490 nm have been assessed from transient absorption spectroscopy and additionally the Z-scan technique. Applying a target model that incorporates photoselection theory, estimates for the cross-sections for stimulated emission and absorption of the first excited state, the amount of ultrafast internal conversion and the underlying species associated dynamics have been determined. The final quantum yields for photoisomerisation were found to be 0.06, 0.14-0.19 and 0.02 for excitation wavelengths 400, 450 and 490 nm and found to increase with increasing pulse durations. Transient absorption measurements and Z-scan measurements at 450 nm, coinciding with the maximum wavelength of the ground state absorption, indicate that the photochemical quantum yield is intrinsically limited by an ultrafast internal conversion reaction as well as by stimulated emission cross-section. With excitation at 400 nm photoisomerisation quantum yield is further significantly limited by competing multi-photon excitation into excited state absorption at 385 nm previously proposed to result in photoionisation. With excitation at 490 nm the photoisomerisation quantum yield is predominantly limited further by the significantly higher stimulated emission cross-section compared to ground state cross-section as well as multi-photon processes. In addition to photoionisation, a second product of multi-photon excitation is identified and characterised by an induced absorption at 500 nm and a time constant of 2 ps for relaxation. With power densities up to 138 GW cm(-2) the measurements have not provided indication for coherent multi-photon absorption of PYP. In the saturation regime with 450 nm excitation, the limit for the photoisomerisation quantum yield was found to be 0.14-0.19 and the excited state absorption cross-section 6.1 × 10(-17) cm(2) or 0.36 times the ground state cross-section of 1.68 × 10(-16) cm(2) per molecule. This places a fundamental restriction on the maximum populations and sample penetration that may be achieved for instance in femtosecond pump-probe experiments with molecular crystals of PYP.

The primary photoreactions of the red absorbing ground state (Pr) of the cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 involve C15═C16 Z-E photoisomerization of its phycocyanobilin chromophore. The first observable product intermediate in pump-probe measurements of the photocycle, "Lumi-R", is formed with picosecond kinetics and involves excited state decay reactions that have 3 and 14 ps time constants. Here, we have studied the photochemical formation of the Lumi-R intermediate using multipulse picosecond visible spectroscopy. Pump-dump-probe (PDP) and pump-repump-probe (PRP) experiments were carried out by employing two femtosecond visible pulses with 1, 14, and 160 ps delays, together with a broadband dispersive visible probe. The time delays between the two excitation pulses have been selected to allow interaction with the dominant (3 and 14 ps) kinetic phases of Lumi-R formation. The frequency dependence of the PDP and PRP amplitudes was investigated at 620, 640, 660, and 680 nm, covering excited state absorption (λ(max) = 620 nm), ground state absorption (λ(max) = 660 nm), and stimulated emission (λ(max) = 680 nm) cross sections. Experimental double difference transient absorbance signals (ΔΔOD), from the PDP and PRP measurements, required corrections to remove contributions from ground state repumping. The sensitivity of the resulting ΔΔOD signals was systematically investigated for possible connectivity schemes and photochemical parameters. When applying a homogeneous (sequentially decaying) connectivity scheme in both the 3 and 14 ps kinetic phases, evidence for repumping of an intermediate that has an electronic ground state configuration (GSI) is taken from the dump-induced S1 formation with 620, 640, and 660 nm wavelengths and 1 and 14 ps repump delays. Evidence for repumping a GSI is also seen, for the same excitation wavelengths, when imposing a target connectivity scheme proposed in the literature for the 1 ps repump delay. In contrast, using a 680 nm dump pulse, ground state formation is observed for all models examined. The ΔΔOD signals were dominated by stimulated emission, at both 1 and 14 ps delays for the longer wavelength excitation. The GSI, which is revealed by the PRP measurements and not resolved from pump-probe measurements, is found to be directly formed from the excited state of Pr, and its formation is considered using heterogeneous, homogeneous, and target models to globally fit the data.