Investigation of peptide dynamics by means of time-resolved IR-laser spectroscopy

Abstract

β-Sheet-containing structures are ubiquitous for many misfolded protein states. In regard to the importance of aggregates and fibrils for the onset of neurodegenerative diseases like Parkinson and Alzheimer, a better understanding of the molecular driving forces for protein folding or misfolding could eventually offer new therapeutic approaches. Model peptides can help to understand the mechanisms that either establish a stable and functional β-sheet structure or result in misfolding and the formation of aggregates. In this work, different spectroscopic techniques were employed to unravel the folding properties of secondary structure elements. Measurements in thermodynamic equilibrium granted insight into structure and stability, while dynamics were monitored by time-resolved perturbation techniques. Although the spatial resolution of IR spectroscopy in general is limited, the specific contribution of single residues to structure formation could be identified via site-selective isotopic labeling.In order to access the folding kinetics of isotopically shifted modes, the existing laser-excited T-jump IR spectrometer was expanded with a set of tunable QCLs, which cover a large spectral range. Additionally, a Ho:YAG laser was incorporated in the setup to allow a direct excitation of a T-jump, thereby bypassing unfavorable side effects of the Raman shifter. Building on this, the impact of different perturbation techniques on relaxation kinetics was investigated. Laser-excited pH-jump as well as T-jump measurements were conducted on α- helical peptide, PGA, starting from different initial conditions and ending up in the very same final state. Whereas the rapid decrease in pH induced helix formation, a T-jump resulted in an unfolding of the helix. In both experiments similar kinetic steps were observed, thus independent from the perturbation technique, indicating the same mechanism for helix folding and unfolding.Ensuing these method-oriented studies, focus was put on β-structured peptides as a model for sheet formation. Particular emphasis was dedicated to three-stranded β-sheets, which are able to adopt a well-defined β-structure while depicting the complex properties of multi-stranded systems. However, the generation of small β-structures in aqueous solutions, i.e. in the absence of stabilizing tertiary contacts, is impeded by their high tendency for aggregation. There are different design strategies for the stabilization of these structures, which are either linking the strands together using strong cross-strand interactions or using geometrically constrained residues to favor the formation of a turn. It could be shown that formation of a β-structure is intrinsically rapid for a strong turn, as the folding process is driven by local forces at the turn. If hydrophobic cross-strand interactions are involved, the folding process must involve a hydrophobic collapse and is slowed down.A subsequent investigation of various three-stranded β-sheet peptides was focusing on intrastrand structure stabilization by aromatic side-chain interactions. The study revealed that the presence of an aromatic interaction in an otherwise folded peptide does not automatically result in an increased thermal stability. In these structures, which all had the rigid DPro – Gly turn sequences, the equilibrium properties seem to be dominated by general hydrophobic and hydrophilic contacts as well as by the turn, while they are only secondarily affected by a cross-strand aromatic interaction. These observations were supported by a comparison to the constituent hairpins, which represented either strands 1–2 or 2–3 of the three-stranded structures. For the N-terminal hairpins a higher stability was found than for the C-terminal hairpins. In contrast to equilibrium data, dynamics were more sensitive to the presence and location of an aromatic interaction. These deductions could already be drawn from the conformational dynamics of the global β-sheet and disordered structure. Additional sitespecific insight was obtained by using the unique band of DPro, which was built into the sequence to promote turn formation, as a site-specific probe. Differences to the β-sheet kinetics were small, reflecting the strong influence of the rigid turn sequences on the formation of the three-stranded structure. Furthermore, the three-stranded structures contain two DPro residues, one in each turn. As the two DPro bands were overlapping, different contributions to the dynamics could not be distinguished.Using isotopic labeling, it was possible to resolve the contribution of each mode. Labeling one of the Xxx – DPro linkages resulted in an isolated band at ~ 1570 cm-1, while the other one remained unchanged at ~ 1612 cm-1. This provided two distinct probes within one single peptide variant. T-jump measurements revealed a significantly slower relaxation for the first turn than for the second one. This was in very good agreement with two peptide variants which had the labels on the glycine residues in turn 1 or 2, respectively, and also differences in stability revealed by NMR structures. For the strand labels, a similar relationship between structural stability and dynamics could be observed, the most flexible strand showed the fastest relaxation. Hereby, spectral overlap of the modes of the 13C=O labels within the strands and the Xxx – DPro amide was resolved using a novel difference transient analysis approach. As all differently labeled peptide variants share the same turn sequence, and therefore also the contribution of the DPro, subtracting the kinetics of the unlabeled peptide from the combined kinetics isolates the dynamics of the label. This approach was supported by DFT calculations, which showed local, isotope-selected vibrations to be effectively uncoupled from other amide I modes. On the basis of our experimental data and supportive MD simulations a folding mechanism was proposed, where structure formation progresses from the turns, followed by the strands.While this demonstrates the possibilities of the isotopic labeling approach to gain site-specific information, it has to be noted that single isotopic labels may exhibit bands weak in intensity in comparison to a possibly large number of 12C amide oscillators in the peptide backbone. It was shown before that coupling of multiple labeled carbonyls within one β-sheet structure can alter the relative intensity of the modes and the specific coupling can be exploited to better determine local structure. A set of peptides with a varying number of cross-strand isotopic labels was created to study the effects of coupling emerging in dependence on the structure of the peptide. In contrast to the expectations for an ideally folded β-structure, multiple labels yielded resolved IR bands occurring at about the same frequencies as for single labeled variants. Although little impact of this weak coupling was observed in equilibrium IR spectra, the T-jump induced relaxation dynamics showed an enhanced sensitivity. The coupling of multiple labeled variants change the relaxation time in comparison to the corresponding single labeled variant significantly, reflecting different rates for folding and unfolding in the varioussegments of the peptide. Similarly, also the global β-sheet and disordered kinetics were altered. Removing one oscillator with a distinct contribution from the 12C=O oscillator system by 13C=O labeling changed the observed rates in an opposing manner, suggestive of an additive interaction among the C=O oscillators of the peptide backbone. These results reflect structural characteristics of the NMR data as well as the dynamics obtained by MD simulations and illustrate the high sensitivity of isotopes as vibrational probes.In conclusion, the studies performed in this thesis demonstrate the versatility of using nonequilibrium dynamics and isotopic labeling to explore the folding energy landscape of proteins and peptides. Insights gained on these model systems contribute to the understanding of complex processes like protein aggregation.