5 research outputs found
Structure and Composition of Insulin Fibril Surfaces Probed by TERS
Amyloid fibrils associated with many neurodegenerative
diseases
are the most intriguing targets of modern structural biology. Significant
knowledge has been accumulated about the morphology and fibril-core
structure recently. However, no conventional methods could probe the
fibril surface despite its significant role in the biological activity.
Tip-enhanced Raman spectroscopy (TERS) offers a unique opportunity
to characterize the surface structure of an individual fibril due
to a high depth and lateral spatial resolution of the method in the
nanometer range. Herein, TERS is utilized for characterizing the secondary
structure and amino acid residue composition of the surface of insulin
fibrils. It was found that the surface is strongly heterogeneous and
consists of clusters with various protein conformations. More than
30% of the fibril surface is dominated by β-sheet secondary
structure, further developing Dobson’s model of amyloid fibrils
(Jimenez et al. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 9196–9201). The propensity
of various amino acids to be on the fibril surface and specific surface
secondary structure elements were evaluated. β-sheet areas are
rich in cysteine and aromatic amino acids, such as phenylalanine and
tyrosine, whereas proline was found only in α-helical and unordered
protein clusters. In addition, we showed that carboxyl, amino, and
imino groups are nearly equally distributed over β-sheet and
α-helix/unordered regions. Overall, this study provides valuable
new information about the structure and composition of the insulin
fibril surface and demonstrates the power of TERS for fibril characterization
Protein Handshake on the Nanoscale: How Albumin and Hemoglobin Self-Assemble into Nanohybrid Fibers
Creating
and establishing proof of hybrid protein nanofibers (hPNFs), <i>i</i>.<i>e</i>., PNFs that contain more than one protein,
is a currently unsolved challenge in bioinspired materials science.
Such hPNFs could serve as universal building blocks for the bottom-up
preparation of functional materials with bespoke properties. Here,
inspired by the protein assemblies occurring in nature, we introduce
hPNFs created <i>via</i> a facile self-assembly route and
composed of human serum albumin (HSA) and human hemoglobin (HGB) proteins.
Our circular dichroism results shed light on the mechanism of the
proteins’ self-assembly into hybrid nanofibers, which is driven
by electrostatic/hydrophobic interactions between similar amino acid
sequences (protein handshake) exposed to ethanol-triggered protein
denaturation. Based on nanoscale characterization with tip-enhanced
Raman spectroscopy (TERS) and immunogold labeling, our results demonstrate
the existence and heterogenic nature of the hPNFs and reveal the high
HSA/HGB composition ratio, which is attributed to the fast self-assembling
kinetics of HSA. The self-assembled hPNFs with a high aspect ratio
of over 100 can potentially serve as biocompatible units to create
larger bioactive structures, devices, and sensors
Mechanism of Plasmon-Induced Catalysis of Thiolates and the Impact of Reaction Conditions
The conversion of the thiols 4-aminothiophenol (ATP)
and 4-nitrothiophenol
(NTP) can be considered as one of the standard reactions of plasmon-induced
catalysis and thus has already been the subject of numerous studies.
Currently, two reaction pathways are discussed: one describes a dimerization
of the starting material yielding 4,4′-dimercaptoazobenzene
(DMAB), while in the second pathway, it is proposed that NTP is reduced
to ATP in HCl solution. In this combined experimental and theoretical
study, we disentangled the involved plasmon-mediated reaction mechanisms
by carefully controlling the reaction conditions in acidic solutions
and vapor. Motivated by the different surface-enhanced Raman scattering
(SERS) spectra of NTP/ATP samples and band shifts in acidic solution,
which are generally attributed to water, additional experiments under
pure gaseous conditions were performed. Under such acidic vapor conditions,
the Raman data strongly suggest the formation of a hitherto not experimentally
identified stable compound. Computational modeling of the plasmonic
hybrid systems, i.e., regarding the wavelength-dependent character
of the involved electronic transitions of the detected key intermediates
in both reaction pathways, confirmed the experimental finding of the
new compound, namely, 4-nitrosothiophenol (TP*). Tracking the reaction
dynamics via time-dependent SERS measurements allowed us to establish
the link between the dimer- and monomer-based pathways and to suggest
possible reaction routes under different environmental conditions.
Thereby, insight at the molecular level was provided with respect
to the thermodynamics of the underlying reaction mechanism, complementing
the spectroscopic results
Mechanism of Plasmon-Induced Catalysis of Thiolates and the Impact of Reaction Conditions
The conversion of the thiols 4-aminothiophenol (ATP)
and 4-nitrothiophenol
(NTP) can be considered as one of the standard reactions of plasmon-induced
catalysis and thus has already been the subject of numerous studies.
Currently, two reaction pathways are discussed: one describes a dimerization
of the starting material yielding 4,4′-dimercaptoazobenzene
(DMAB), while in the second pathway, it is proposed that NTP is reduced
to ATP in HCl solution. In this combined experimental and theoretical
study, we disentangled the involved plasmon-mediated reaction mechanisms
by carefully controlling the reaction conditions in acidic solutions
and vapor. Motivated by the different surface-enhanced Raman scattering
(SERS) spectra of NTP/ATP samples and band shifts in acidic solution,
which are generally attributed to water, additional experiments under
pure gaseous conditions were performed. Under such acidic vapor conditions,
the Raman data strongly suggest the formation of a hitherto not experimentally
identified stable compound. Computational modeling of the plasmonic
hybrid systems, i.e., regarding the wavelength-dependent character
of the involved electronic transitions of the detected key intermediates
in both reaction pathways, confirmed the experimental finding of the
new compound, namely, 4-nitrosothiophenol (TP*). Tracking the reaction
dynamics via time-dependent SERS measurements allowed us to establish
the link between the dimer- and monomer-based pathways and to suggest
possible reaction routes under different environmental conditions.
Thereby, insight at the molecular level was provided with respect
to the thermodynamics of the underlying reaction mechanism, complementing
the spectroscopic results
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