Structure of a model salt bridge in solution investigated with 2D-IR spectroscopy

Salt bridges are known to be important for the stability of protein conformation, but up to now it has been difficult to study their geometry in solution. Here we characterize the spatial structure of a model salt bridge between guanidinium (Gdm+) and acetate (Ac-) using two-dimensional vibrational (2D-IR) spectroscopy. We find that as a result of salt bridging the infrared response of Gdm+ and Ac- change significantly, and in the 2D-IR spectrum, salt bridging of the molecules appears as cross peaks. From the 2D-IR spectrum we determine the relative orientation of the transition-dipole moments of the vibrational modes involved in the salt bridge, as well as the coupling between them. In this manner we reconstruct the geometry of the solvated salt bridge

The structure of salt bridges between Arg +

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. We use this probe to investigate a β-turn locked by a salt bridge, an α-helical peptide whose structure is stabilized by salt bridges, and a coiled coil that is stabilized by intra- and intermolecular salt bridges. We detect a bidentate salt bridge in the β-turn, a monodentate one in the α-helical peptide, and both salt-bridge geometries in the coiled coil. To our knowledge, this is the first time 2D-IR has been used to probe tertiary side chain interactions in peptides, and our results show that 2D-IR spectroscopy is a powerful method for investigating salt bridges in solution

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. We present our phenomenology-based analysis procedure and results in obtaining the excitonic energy levels, spectral linewidths, and transfer-rate matrix of Light-Harvesting Complex II directly from its 2D electronic spectra recorded at 77 K with waiting times between 100 fs to 100 ps. Due to the restriction of the models and complexity of the system, a unique EET network cannot be constructed. Nevertheless, a recurring pattern of energy transfer with very similar overall time scales between spectral components (excitons) is consistently obtained. The models identify a "bottleneck" state in the 664-668 nm region although with a relatively shorter lifetime (similar to 4-6 ps) of this state compared to previous studies. The model also determines three terminal exciton states at 675, 677-678, and 680-681 nm that are weakly coupled to each other. The excitation energy equilibration between the three termini is found to be independent of the initial excitation conditions, which is a crucial design for the light-harvesting complexes to ensure the energy flow under different light conditions and avoid excitation trapping. We proposed two EET schemes with tentative pigment assignments based on the interpretation of the modeling results together with previous structure-based calculations and spectroscopic observables. Published under license by AIP Publishing

Coupled vibrations in peptides and proteins: Structural information using 2D-IR spectroscopy

We describe experimental evidence of how detecting the coupling between vibrations can give access to structural information of proteins and peptides at the molecular level. We focus on the investigation of the folded and unfolded states of proteins and peptides in equilibrium. We investigate two types of vibrations: those involved in salt bridges, and backbone amide I vibrations, and the size of the systems than we study ranges from a dimer formed by a few atoms to proteins formed by several hundreds of atoms. The first three chapters are introductory. In Chapter 4 we present the characterization of the 2D-IR response of salt bridges in solution formed by guanidinium and acetate. Chapter 5 is closely related, and we show how the 2D-IR response of a salt bridge between arginine and glutamic acid can be detected in peptides in solution. In Chapter 6 we study the amide I vibrations of tripeptides, and discuss the conformational changes upon variation in the charges of the side groups and C-terminus. The next two chapters contain a study of the chemical-denaturation mechanism of guanidinium, which is a powerful denaturant. In Chapter 7 we investigate the guanidinium-induced denaturation of two well-known proteins, lysozyme and α-chymotrypsin. In Chapter 8 we study how guanidinium affects the stability of a designed mini-protein, a zinc-finger mutant, which has structural properties that differ from most natural proteins. Finally, in Chapter 9 we introduce the topic of amyloid-fibril formation, and show how the appearance of fibrils in lysozyme can be induced with a ‘temperature-shock’

Protein Denaturation with Guanidinium: A 2D-IR Study

[Image: see text] 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-(13)C(15)N(3)·Cl. We find that upon denaturation, the β-sheet protein shows a complete loss of β-sheet structure, whereas the α-helical protein maintains most of its secondary structure. These results suggest that Gdm(+) disrupts β-sheets much more efficiently than α-helices, possibly because in the former, hydrophobic interactions are more important and the number of dangling hydrogen bonds is larger

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.MOE (Min. of Education, S’pore