X-ray crystallography and nuclear magnetic resonance measurements provide us with atomically resolved structures of an ever-growing number of biomolecules. These static structural snapshots are important to our understanding of biomolecular function, but real biomolecules are dynamic entities that often exploit conformational changes and transient molecular interactions to perform their tasks. Nuclear magnetic resonance methods can follow such structural changes, but only on millisecond timescales under non-equilibrium conditions. Time-resolved X-ray crystallography has recently been used to monitor the photodissociation of CO from myoglobin on a subnanosecond timescale, yet remains challenging to apply more widely. In contrast, two-dimensional infrared spectroscopy, which maps vibrational coupling between molecular groups and hence their relative positions and orientations, is now routinely used to study equilibrium processes on picosecond timescales. Here we show that the extension of this method into the non-equilibrium regime allows us to observe in real time in a short peptide the weakening of an intramolecular hydrogen bond and concomitant opening of a beta-turn. We find that the rate of this process is two orders of magnitude faster than the 'folding speed limit' established for contact formation between protein side chains.

We present an artificial photocatalytic model for photosystem I (PSI) using [ReBr(CO)(3)bipy)] (1) as a photosensitizer, [Co(dmgH)(2)] (2) as a hydrogen evolution reaction catalyst, and triethanolamine as an irreversible reductive quencher. Complex 1 is more robust in the long run, and turnover numbers were more than doubled in the present study as compared to the commonly used photosensitizer [Ru(bipy)(3)](2+). The quantum yield for hydrogen production with 1 was found to be 26 +/- 2% (H produced per absorbed photon). Forward electron transfer between 1(-) and 2 was found to occur at a rate close to diffusion control (k(1) = 2.5 +/- 0.1 x 10(8) M(-1) s(-1)). The rate of hydrogen production exhibited a linear dependence on the photon flux and a quadratic dependence on the total concentration of Co (k(obs) = 3.7 +/- 0.1 M(-1) s(-1)). Therefore, a second-order process in Co(III)-H is proposed. The process showed a complex dependence on [AcOH]. An excess of dimethylglyoxime was systematically added to the system to ensure the complete formation of 2 and reduce the portion of free [Co](solv)(2+), an efficient quencher of the excited state of 1.

Multidimensional spectroscopic experiments offer fascinating insights into molecular structure and dynamics in the field of NMR spectroscopy. With the introduction of ultrafast two-dimensional infrared spectroscopy (2D-IR), multidimensional concepts have entered the optical domain, measuring couplings and correlations between molecular vibrations with femtosecond time resolution. In the transient 2D-IR (T2D-IR) experiments described in this minireview we exploit the high time resolution of 2D-IR to study transient species during fast nonequilibrium processes in real time. Information on molecular structure and dynamics is obtained that is not available from one-dimensional spectroscopy. We discuss examples from chemistry, physics and biophysics.

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.