Michael Schleeger

The Polyphenol EGCG Inhibits Amyloid Formation Less Efficiently at Phospholipid Interfaces than in Bulk Solution

Age-related diseases, like Alzheimers disease and type 2 diabetes mellitus, are characterized by protein misfolding and the subsequent pathological deposition of fibrillized protein, also called amyloid. Several classes of amyloid-inhibitors have recently been tested, traditionally under bulk conditions. However, it has become apparent that amyloid fibrils and oligomers assemble and exert their cytotoxic effect at cellular membranes, rather than in bulk solution. Knowledge is therefore required of inhibitor activity specifically at the phospholipid membrane interface. Here we show, using surface-specific sum-frequency generation (SFG) spectroscopy and atomic force microscopy (AFM), that the commonly used (-)-epigallocatechin gallate (EGCG) is a much less efficient amyloid inhibitor at a phospholipid interface than in bulk solution. Moreover, EGCG is not able to disaggregate existing amyloid fibrils at a phospholipid interface, in contrast to its behavior in bulk. Our results show that interfaces significantly affect the efficiency of inhibition by EGCG inhibitors and should therefore be considered during the design and testing of amyloid inhibitors.

Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip-Enhanced Raman Spectroscopy

Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by β-sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy. However, with these techniques it is not possible to distinguish between the core and the surface structure of the fibrils. Since the fibril surface crucially affects amyloid toxicity, it is essential to know its structure. Here the surface molecular structure and amino acid residue composition of hIAPP fibrils are specifically probed with nanoscale resolution using tip-enhanced Raman spectroscopy (TERS). The fibril surface mainly contains unordered or α-helical structures, in contrast to the β-sheet-rich core. This experimentally validates recent models of hIAPP amyloids based on NMR measurements. Spatial mapping of the surface structure reveals a highly heterogeneous surface structure. Finally, TERS can probe fibrils formed on a lipid interface, which is more representative of amyloids in vivo.

Quantifying Surfactant Alkyl Chain Orientation and Conformational Order from Sum Frequency Generation Spectra of CH Modes at the Surfactant-Water Interface.

We combine second-order nonlinear vibrational spectroscopy and quantum-chemical calculations to quantify the molecular tilt angle and the structural variation of a decanoic acid surfactant monolayer on water. We demonstrate that there is a remarkable degree of delocalization of the vibrational modes along the backbone of the amphiphilic molecule. A simulation-based on modeled sum frequency generation (SFG) spectra offers quantitative insights into the disorder of surfactant monolayers at the water-air interface. It is shown that an average of one gauche defect in the alkyl chain suffices to give rise to the methylene stretch intensity similar in magnitude to the methyl stretch.

Formation of Lysozyme Oligomers at Model Cell Membranes Monitored with Sum Frequency Generation Spectroscopy

A growing number of studies suggest that the formation of toxic oligomers, precursors of amyloid fibrils, is initiated at the cell membrane and not in the cytosolic compartments of the cell. Studies of membrane-induced protein oligomerization are challenging due to the difficulties of probing small numbers of proteins present at membrane surfaces. Here, we employ surface-sensitive vibrational sum frequency generation (VSFG) to investigate the secondary structure of lysozyme at the surface of lipid monolayers. We investigate lysozyme aggregation at negatively charged 1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-1-glycerol) (DPPG) lipid monolayers under different pH conditions. The changes in the molecular vibrations of lipids, proteins, and water as a function of pH and surface pressure allow us to simultaneously monitor details of the conformation state of lysozyme, the organization of lipids, and the state of lipid-bound water. At pH = 6 lysozyme induces significant disordering of the lipid layer, and it exists in two states: a monomeric state with a predominantly α-helix content and an oligomeric (za-mer) state. At pH ≤ 3, all membrane-bound lysozyme self-associates into oligomers characterized by an antiparallel β-sheet structure. This is different from the situation in bulk solution, for which circular dichroism (CD) shows that the protein maintains an α-helix conformation, under both neutral and acidic pH conditions. The transition from monomers to oligomers is also associated with a decreased hydration of the lipid monolayer resulting in an increase of the lipid acyl chains ordering. The results indicate that oligomerization requires cooperative action between lysozyme incorporated into the lipid membrane and peripherally adsorbed lysozyme and is associated with the membrane dehydration and lipid reorganization. Membrane-bound oligomers with antiparallel β-sheet structure are found to destabilize lipid membranes.

Model peptides uncover the role of the β-secretase transmembrane sequence in metal ion mediated oligomerization.

The β-secretase or β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the enzyme responsible for the formation of amyloid-β peptides, which have a major role in Alzheimer pathogenesis. BACE1 has a transmembrane sequence (TMS), which makes it unique among related proteases. We noticed that the BACE1 TMS contains an uncommon sulfur-rich motif. The sequence MxxxCxxxMxxxCxMxC spans the entire TMS, resembles metal ion binding motifs, and is highly conserved among homologues. We used a synthetic 31-mer model peptide comprising the TMS to study metal ion binding and oligomerization. Applying diverse biochemical and biophysical techniques, we detected dimer and trimer formation of the TMS peptide with copper ions. Replacement of the central Cys466 by Ala essentially abolished these effects. We show that the peptide undergoes a redox reaction with copper ions resulting in a disulfide bridge involving Cys466. Further, we find peptide trimerization that depends on the presence of monovalent copper ions and the sulfhydryl group of Cys466. We identified Cys466 as a key residue for metal ion chelation and to be the core of an oligomerization motif of the BACE1-TMS peptide. Our results demonstrate a novel metal ion controlled oligomerization of the BACE1 TMS, which could have an enormous therapeutic importance against Alzheimer disease.

Multimodal Spectroscopic Study of Amyloid Fibril Polymorphism

Amyloid fibrils are a large class of self-assembled protein aggregates that are formed from unstructured peptides and unfolded proteins. The fibrils are characterized by a universal β-sheet core stabilized by hydrogen bonds, but the molecular structure of the peptide subunits exposed on the fibril surface is variable. Here we show that multimodal spectroscopy using a range of bulk- and surface-sensitive techniques provides a powerful way to dissect variations in the molecular structure of polymorphic amyloid fibrils. As a model system, we use fibrils formed by the milk protein β-lactoglobulin, whose morphology can be tuned by varying the protein concentration during formation. We investigate the differences in the molecular structure and composition between long, straight fibrils versus short, wormlike fibrils. We show using mass spectrometry that the peptide composition of the two fibril types is similar. The overall molecular structure of the fibrils probed with various bulk-sensitive spectroscopic techniques shows a dominant contribution of the β-sheet core but no difference in structure between straight and wormlike fibrils. However, when probing specifically the surface of the fibrils with nanometer resolution using tip-enhanced Raman spectroscopy (TERS), we find that both fibril types exhibit a heterogeneous surface structure with mainly unordered or α-helical structures and that the surface of long, straight fibrils contains markedly more β-sheet structure than the surface of short, wormlike fibrils. This finding is consistent with previous surface-specific vibrational sum-frequency generation (VSFG) spectroscopic results ( VandenAkker et al. J. Am. Chem. Soc. , 2011 , 133 , 18030 - 18033 , DOI: 10.1021/ja206513r ). In conclusion, only advanced vibrational spectroscopic techniques sensitive to surface structure such as TERS and VSFG are able to reveal the difference in structure that underlies the distinct morphology and rigidity of different amyloid fibril polymorphs that have been observed for a large range of food and disease-related proteins.

Time-resolved FT-IR Spectroscopy of Membrane Proteins

Time-resolved Fourier transform infrared spectroscopy (FT-IR) offers distinct advantages concerning restrictions pertinent to biomolecules. In particular, it is possible to monitor the temporal evolution of the reaction mechanism of complex machineries as membrane proteins, where other techniques encounter significant experimental difficulties. Here, we present the classical principles and experimental realizations of time-resolved FT-IR spectroscopy together with recent developments employed in our laboratory. Examples from applications to retinal proteins are reviewed that underline the impact of time-resolved FT-IR spectroscopy on the understanding of protein reactions on the level of single bonds.

Background-Free Fourth-Order Sum Frequency Generation Spectroscopy.

The recently developed 2D sum frequency generation spectroscopy offers new possibilities to analyze the structure and structural dynamics of interfaces in a surface-specific manner. Its implementation, however, has so far remained limited to the pump-probe geometry, with its inherent restrictions. Here we present 2D SFG experiments utilizing a novel noncollinear geometry of four incident laser pulses generating a 2D SFG response, analogous to the triangle geometry applied in bulk-sensitive 2D infrared spectroscopy. This approach allows for background-free measurements of fourth-order nonlinear signals, which is demonstrated by measuring the fourth-order material response from a GaAs (110) surface. The implementation of phase-sensitive detection and broadband excitation pulses allows for both highest possible time resolution and high spectral resolution of the pump axis of a measured 2D SFG spectrum. To reduce the noise in our spectra, we employ a referencing procedure, for which we use noncollinear pathways and individual focusing for the signal and local oscillator beams. The 2D spectra recorded from the GaAs (110) surface show nonzero responses for the real and imaginary component, pointing to contributions from resonant electronic pathways to the χ((4)) response.

Structural Basis for the Polymorphism of β-Lactoglobulin Amyloid-Like Fibrils

Abstract The whey protein β-lactoglobulin (β-lg) forms amyloid fibrils upon hydrolysis or partial unfolding. The morphology of these fibrils depends on the environmental conditions during formation, including pH, ionic strength, protein concentration and reaction time. Long and straight, as well as short, worm-like β-lg fibrils are observed. The molecular basis of this polymorphism remains poorly understood. In this chapter we review the relationship between the fibril morphology and the peptide molecular structure of amyloids formed from β-lg. Fibril morphologies are typically measured by atomic force microscopy and electron microscopy, and the molecular structure is measured by a variety of spectroscopic techniques, such as Raman and FT-IR spectroscopy. Recent studies have begun to combine spectroscopy with imaging techniques to correlate fibril morphology with the underlying molecular structure. It was shown that straight and rigid fibrils have a substantially higher β-sheet content compared to worm-like fibrils. However, in the future, to elucidate the local molecular structure of peptides within fibrils, newly developed techniques, such as tip-enhanced Raman spectroscopy, will be necessary.

Time-Resolved FT-IR Spectroscopy for the Elucidation of Protein Function

Time-resolved Fourier transform infrared spectroscopy (FT-IR) has been proven to be an excellent method with important applications in bioscience. In particular, it is possible to monitor the temporal evolution of the reaction mechanism of complex machineries as membrane proteins, where other techniques encounter significant experimental difficulties. Here, we summarize the classical principles and experimental realizations of time-resolved FT-IR spectroscopy together with new developments realized in our laboratory. Examples from applications to retinal proteins are reviewed that showcase the impact of time-resolved FT-IR spectroscopy on the understanding of protein reactions on the level of single bonds.