Thioflavin T (ThT) is widely used as a fluorescent probe for amyloid fibril detection. Yet the exact kinetic mechanism of ThT binding onto amyloid fibril remains elusive. Previously reported kinetic studies using ThT-fluorescence-detected kinetic design suggested two completely different ThT-binding mechanisms. In one study, a multistep sequential binding mechanism onto a single ThT-binding site was suggested. In another study, a one-step parallel binding mechanism onto multiple ThT-binding sites was suggested. The discrepancy is likely due to the incapability of ThT-fluorescence-detected kinetic design to differentiate the two above-mentioned mechanisms. Considering the weakness of the ThT-fluorescence-detected approach, we investigated the ThT-binding mechanism onto the amyloid fibril of hen egg white lysozyme (HEWL) using a new approach, ThT-absorbance-detected kinetic design. Our new results suggest that ThT binds to HEWL fibril through the one-step parallel binding mechanism. We hope our work can offer some new insights into the interactions between dye molecules and amyloid fibrils.

In this study, we performed the first comparative study of the antibacterial mechanisms of silver ion (Ag+) and silver nanoparticles (AgNPs) on Escherichia coli (E. coli) using Fourier transform infrared (FTIR) spectroscopy. Through a thorough analysis of the FTIR spectra of E. coli after silver treatment in the spectral regions corresponding to thiol group, protein, lipopolysaccharide (LPS), and DNA, we were able to reveal a multifaceted antibacterial mechanism of silver at the molecular level for both Ag+ and AgNPs. Features of such mechanism include: (1) silver complexes with thiol group; (2) silver induces protein misfolding; (3) silver causes loss of LPS from bacterial membrane; (4) silver changes the overall conformation of DNA. Despite the similarities between Ag+ and AgNPs with respect to their antibacterial mechanisms, we further revealed that Ag+ and AgNPs display quite different kinetics for silver-thiol complexation and loss of LPS, with Ag+ displaying fast kinetics and AgNPs displaying slow kinetics. At last, we proposed a hypothesis to interpret the observed different behaviors between Ag+ and AgNPs when interacting with E. coli.

Amyloid fibrils are unique fibrous polypeptide aggregates. They have been associated with more than 20 serious human diseases including Alzheimer’s disease and Parkinson’s disease. Besides their pathological significance, amyloid fibrils are also gaining increasing attention as emerging nanomaterials with novel functions. Structural characterization of amyloid fibril is no doubt fundamentally important for the development of therapeutics for amyloid-related diseases and for the rational design of amyloid-based materials. In this study, we explored to use side-chain-based infrared (IR) probe to gain detailed structural insights into the amyloid fibril by a 21-residue model amyloidogenic peptide, Aβ(8–28). We first proposed an approach to incorporate thiocyanate (SCN) IR probe in a site-specific manner into amyloidogenic peptide using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate as cyanylating agent. Using this approach, we obtained three Aβ(8–28) variants, labeled with SCN probe at three different positions. We then showed with thioflavin T fluorescence assay, Congo red assay, and atomic force microscopy that the three labeled Aβ(8–28) peptides can quickly form amyloid fibrils under high concentration and high salt conditions. Finally, we performed a detailed IR spectral analysis of the Aβ(8–28) fibril in both amide I and probe regions and proposed a millipede-like structure for the Aβ(8–28) fibril.

Hen egg white lysozyme (HEWL) is widely used in the mechanistic study of amyloid fibril formation. Yet, the fibrillation mechanism of HEWL is not well understood. In particular, in situ structural evidence for the on-pathway oligomeric intermediate has never been captured. Such evidence is crucial for confirming nucleated conformational conversion mechanism. Herein, we attempt to use a two-step temperature-dependent Fourier transform infrared (FTIR) approach to capture the in situ evidence for the on-pathway oligomeric intermediate and the oligomer-to-fibril transition during HEWL fibrillation. Key features of this approach include using lower temperature to generate the on-pathway oligomeric intermediate, using elevated temperature to eliminate the interference from the off-pathway oligomer and to facilitate the oligomer-to-fibril transition, and using FTIR difference spectroscopy and atomic force microscopy to tackle structure and morphology. Using such an approach, we reveal that the on-pathway oligomeric intermediate is in parallel β-sheet configuration featuring a frequency at 1622 cm–1 and the oligomer-to-fibril transition is accompanied by a spectral transition from 1622 to 1618 cm–1. We also discover the beneficial role of the off-pathway oligomer in the capturing of the transient on-pathway oligomeric intermediate by serving as a monomer-releasing reservoir. This approach should also be useful in other amyloidogenic systems.

Amyloid formation of hen egg white lysozyme (HEWL) usually requires elevated temperature, while biophysical characterizations on the incubation solution are often performed at room temperature. Whether maintaining the incubation solution at room temperature results in further structural changes is a significantly important issue that has never been explored. Herein, we use FTIR spectroscopy to assess this issue and reveal that the hot incubation solution of HEWL after cooling to room temperature is in a dynamically evolving state and forms β-sheet aggregates continuously over time. Combined with AFM, we show that these aggregates are non-fibrillar β-sheet aggregates and have vibrational signature distinct from that of fibrillar aggregates. Using FTIR difference spectroscopy, we demonstrate that these non-fibrillar aggregates are in an anti-parallel β-sheet configuration. We also provide a detailed discussion on the spectral assignments for protein aggregates in anti-parallel and parallel β-sheet configurations. With FTIR second derivative technique, we show that these non-fibrillar aggregates are in fact present along with fibrillar aggregates during incubation under elevated temperature but are less stable compared with that at room temperature. Implications from the current work are discussed.

The construction of a convenient FTIR spectrometer purging setup for the undergraduate teaching laboratory is described. This simple setup consists of an inexpensive aquarium air pump and a drying-tube assembly. This setup can efficiently suppress atmospheric water-vapor interference, thus producing good-quality FTIR spectra.Keywords: Analytical Chemistry; First-Year Undergraduate/General; IR Spectroscopy; Laboratory Equipment/Apparatus; Organic Chemistry; Physical Chemistry; Second-Year Undergraduate; Upper-Division Undergraduate;

Hen egg white lysozyme (HEWL) is widely used as a model protein for amyloid research. In this study, we aim to use Fourier transform infrared (FTIR) spectroscopy to gain new structural insights into amyloid formation of HEWL under heat and acidic condition. We reveal that the fibril-forming solution of HEWL has the capability to form fibril and oligomer with distinct β-sheet configurations under different temperatures. Amyloid fibril with parallel β-sheet configuration is formed at elevated temperature, while oligomer with antiparallel β-sheet configuration is formed at room temperature. The interplay between fibrillation and oligomerization suggests that the two β-sheet aggregates consume the same amyloidogenic materials such as peptide fragments and nicked HEWL due to lysozyme hydrolysis under heat and acidic condition. Temperature-dependent FTIR reveals that the oligomer is unstable at elevated temperature, demonstrating its off-pathway nature. The temperature-dependent formation of parallel and antiparallel β-sheet configurations discovered in lysozyme system is compared with that of amyloid-β and α-synuclein systems and the implication is discussed.