Revealing the excitation energy transfer network of Light-Harvesting Complex II by a phenomenological analysis of two-dimensional electronic spectra at 77 K.

Energy equilibration in light-harvesting antenna systems normally occurs before energy is transferred to a reaction center. The equilibration mechanism is a characteristic of the excitation energy transfer (EET) network of the antenna. Characterizing this network is crucial in understanding the first step of photosynthesis.

Temperature Dependence of the Energy Transfer in LHCII Studied by Two-Dimensional Electronic Spectroscopy.

We measured two-dimensional electronic spectra of light-harvesting complex II (LHCII) at various temperatures (77, 110, 150, 230, and 295 K) under conditions free from singlet-singlet annihilation. We elucidated the temperature-dependent excitation energy transfer dynamics in the Chl manifold of LHCII. Global analysis revealed that the dynamics can be summarized in distinct time scales from 200 fs up to 15 ps.

Glass formation of a DMSO-water mixture probed with a photosynthetic pigment.

Despite their extensive industrial usage, glass-forming liquids are not fully understood, and methods to investigate their dynamical heterogeneity are sought after. Here we show how the appearance of a second component in the visible absorption spectrum of a photosynthetic pigment upon cooling can be used to probe the glass transition of a dimethylsulfoxide-water mixture. The changes in the relative ratio of the two components with respect to temperature follow a sigmoid curve, and we show that the second component arises due to protonation of the pigment at low temperatures.

Two-dimensional electronic-Raman spectroscopy.

We present a new technique, two-dimensional electronic-Raman spectroscopy (2DER), which combines femtosecond stimulated Raman spectroscopy and a pulse-shaper-assisted 2D spectroscopic scheme for the actinic pump. The 2DER spectrum presents the initial actinic excitation wavelength with nanometer spectral resolution in the first axis and the detected stimulated Raman spectra in the second axis. We measured the correlation of the electronic and vibrational states in the photosynthetic accessory pigment β-carotene and reveal its photoexcited state manifold.

Transient two-dimensional vibrational spectroscopy of an operating molecular machine.

Synthetic molecular machines are promising building blocks for future nanoscopic devices. However, the details of their mechanical behaviour are in many cases still largely unknown. A deeper understanding of mechanics at the molecular level is essential for the design and construction of complex nanodevices.

Sample exchange by beam scanning with applications to noncollinear pump-probe spectroscopy at kilohertz repetition rates.

In laser spectroscopy, high photon flux can perturb the sample away from thermal equilibrium, altering its spectroscopic properties. Here, we describe an optical beam scanning apparatus that minimizes repetitive sample excitation while providing shot-to-shot sample exchange for samples such as cryostats, films, and air-tight cuvettes. In this apparatus, the beam crossing point is moved within the focal plane inside the sample by scanning both tilt angles of a flat mirror.

Interferometrically stable, enclosed, spinning sample cell for spectroscopic experiments on air-sensitive samples.

In experiments with high photon flux, it is necessary to rapidly remove the sample from the beam and to delay re-excitation until the sample has returned to equilibrium. Rapid and complete sample exchange has been a challenge for air-sensitive samples and for vibration-sensitive experiments. Here, a compact spinning sample cell for air and moisture sensitive liquid and thin film samples is described.

The structure of salt bridges between Arg(+) and Glu(-) in peptides investigated with 2D-IR spectroscopy: Evidence for two distinct hydrogen-bond geometries.

Salt bridges play an important role in protein folding and in supramolecular chemistry, but they are difficult to detect and characterize in solution. Here, we investigate salt bridges between glutamate (Glu(-)) and arginine (Arg(+)) using two-dimensional infrared (2D-IR) spectroscopy. The 2D-IR spectrum of a salt-bridged dimer shows cross peaks between the vibrational modes of Glu(-) and Arg(+), which provide a sensitive structural probe of Glu(-)⋯Arg(+) salt bridges.

A salt-bridge structure in solution revealed by 2D-IR spectroscopy.

Salt bridges are important interactions for the stability of protein conformations, but up to now it has been difficult to determine salt-bridge geometries in solution. Here we characterize the spatial structure of a salt bridge between guanidinium (Gdm(+)) and acetate (Ac(-)) using two-dimensional vibrational (2D-IR) spectroscopy. We find that as a result of salt bridge formation there is a significant change in the infrared response of Gdm(+) and Ac(-), and cross peaks between them appear in the 2D-IR spectrum.

Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding.

Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu and Arg are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding.

Amplified vibrational circular dichroism as a probe of local biomolecular structure.

We show that the VCD signal intensities of amino acids and oligopeptides can be enhanced by up to 2 orders of magnitude by coupling them to a paramagnetic metal ion. If the redox state of the metal ion is changed from paramagnetic to diamagnetic the VCD amplification vanishes completely. From this observation and from complementary quantum-chemical calculations we conclude that the observed VCD amplification finds its origin in vibronic coupling with low-lying electronic states.

Protein Denaturation with Guanidinium: A 2D-IR Study.

Guanidinium (Gdm) is a widely used denaturant, but it is still largely unknown how it operates at the molecular level. In particular, the effect of guanidinium on the different types of secondary structure motifs of proteins is at present not clear. Here, we use two-dimensional infrared spectroscopy (2D-IR) to investigate changes in the secondary structure of two proteins with mainly α-helical or β-sheet content upon addition of Gdm-CN·Cl.

Folding dynamics of the Trp-cage miniprotein: evidence for a native-like intermediate from combined time-resolved vibrational spectroscopy and molecular dynamics simulations.

Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding mechanisms. However, Trp-cage's exact folding mechanism is still a matter of debate.

pH dependence of the conformation of small peptides investigated with two-dimensional vibrational spectroscopy.

We investigate how the conformation of small peptides is influenced by the presence or absence of charge on the C-terminus and on the side groups. To this purpose, the conformations of two tripeptides, with acidic and basic side groups, is determined at several pD values using two-dimensional infrared (2DIR) spectroscopy. The investigated pD values are chosen relative to the C-terminal and side-chain pK(a) values in such a way that the C-terminus and side groups are in well-defined protonation states.