Interactions between molecules are fundamental in biology. They occur also between amyloidogenic peptides or proteins that are associated with different amyloid diseases, which makes it important to study the mutual influence of two polypeptides on each other's properties in mixed samples. However, addressing this research question with imaging techniques faces the challenge to distinguish different polypeptides without adding artificial probes for detection. Here, we show that nanoscale infrared spectroscopy in combination with C-13, N-15-labeling solves this problem. We studied aggregated amyloid-& beta; peptide (A & beta;) and its interaction with an inhibitory peptide (NCAM1-PrP) using scattering-type scanning near-field optical microscopy. Although having similar secondary structure, labeled and unlabeled peptides could be distinguished by comparing optical phase images taken at wavenumbers characteristic for either the labeled or the unlabeled peptide. NCAM1-PrP seems to be able to associate with or to dissolve existing A & beta; fibrils because pure A & beta; fibrils were not detected after mixing. Interactions of proteins or polypeptides with different secondary structures can be studied in a mixture by nanoscale infrared spectroscopy, however, this technique remains challenging for polypeptides with similar secondary structures. Here, the authors demonstrate clear discrimination of two polypeptides from a mixture by scattering-type scanning near-field optical microscopy when one of the components is labeled with C-13- and N-15-isotopes.

The amyloid-& beta;(A & beta;) peptide is associated with the developmentof Alzheimer's disease and is known to form highly neurotoxicprefibrillar oligomeric aggregates, which are difficult to study dueto their transient, low-abundance, and heterogeneous nature. To obtainhigh-resolution information about oligomer structure and dynamicsas well as relative populations of assembly states, we here employa combination of native ion mobility mass spectrometry and moleculardynamics simulations. We find that the formation of A & beta; oligomersis dependent on the presence of a specific & beta;-hairpin motif inthe peptide sequence. Oligomers initially grow spherically but startto form extended linear aggregates at oligomeric states larger thanthose of the tetramer. The population of the extended oligomers couldbe notably increased by introducing an intramolecular disulfide bond,which prearranges the peptide in the hairpin conformation, therebypromoting oligomeric structures but preventing conversion into maturefibrils. Conversely, truncating one of the & beta;-strand-formingsegments of A & beta; decreased the hairpin propensity of the peptideand thus decreased the oligomer population, removed the formationof extended oligomers entirely, and decreased the aggregation propensityof the peptide. We thus propose that the observed extended oligomerstate is related to the formation of an antiparallel sheet state,which then nucleates into the amyloid state. These studies provideincreased mechanistic understanding of the earliest steps in A & beta;aggregation and suggest that inhibition of A & beta; folding into thehairpin conformation could be a viable strategy for reducing the amountof toxic oligomers.

The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH.

Uranium (U) is naturally present in ambient air, water, and soil, and depleted uranium (DU) is released into the environment via industrial and military activities. While the radiological damage from U is rather well understood, less is known about the chemical damage mechanisms, which dominate in DU. Heavy metal exposure is associated with numerous health conditions, including Alzheimer’s disease (AD), the most prevalent age-related cause of dementia. The pathological hallmark of AD is the deposition of amyloid plaques, consisting mainly of amyloid-β (Aβ) peptides aggregated into amyloid fibrils in the brain. However, the toxic species in AD are likely oligomeric Aβ aggregates. Exposure to heavy metals such as Cd, Hg, Mn, and Pb is known to increase Aβ production, and these metals bind to Aβ peptides and modulate their aggregation. The possible effects of U in AD pathology have been sparsely studied. Here, we use biophysical techniques to study in vitro interactions between Aβ peptides and uranyl ions, UO₂²⁺, of DU. We show for the first time that uranyl ions bind to Aβ peptides with affinities in the micromolar range, induce structural changes in Aβ monomers and oligomers, and inhibit Aβ fibrillization. This suggests a possible link between AD and U exposure, which could be further explored by cell, animal, and epidemiological studies. General toxic mechanisms of uranyl ions could be modulation of protein folding, misfolding, and aggregation. 

Cell-penetrating peptides (CPPs) are highly promising transfection agents that can deliver various compounds into living cells, including nucleic acids (NAs). Positively charged CPPs can form non-covalent complexes with negatively charged NAs, enabling simple and time-efficient nanoparticle preparation. However, as CPPs have substantially different chemical and physical properties, their complexation with the cargo and characteristics of the resulting nanoparticles largely depends on the properties of the surrounding environment, i.e., solution. Here, we show that the solvent used for the initial dissolving of a CPP determines the properties of the resulting CPP particles formed in an aqueous solution, including the activity and toxicity of the CPP–NA complexes. Using different biophysical methods such as dynamic light scattering (DLS), atomic force microscopy (AFM), transmission and scanning electron microscopy (TEM and SEM), we show that PepFect14 (PF14), a cationic amphipathic CPP, forms spherical particles of uniform size when dissolved in organic solvents, such as ethanol and DMSO. Water-dissolved PF14, however, tends to form micelles and non-uniform aggregates. When dissolved in organic solvents, PF14 retains its α-helical conformation and biological activity in cell culture conditions without any increase in cytotoxicity. Altogether, our results indicate that by using a solvent that matches the chemical nature of the CPP, the properties of the peptide–cargo particles can be tuned in the desired way. This can be of critical importance for in vivo applications, where CPP particles that are too large, non-uniform, or prone to aggregation may induce severe consequences.

Oligomerization of antimicrobial peptides (AMPs) is critical in their effects on pathogens. LL-37 and its truncated fragments are widely investigated regarding their structures, antimicrobial activities, and application, such as developing new antibiotics. Due to the small size and weak intermolecular interactions of LL-37 fragments, it is still elusive to establish the relationship between oligomeric states and antimicrobial activities. Here, an α-hemolysin nanopore, mass spectrometry (MS), and molecular dynamic (MD) simulations are used to characterize the oligomeric states of two LL-37 fragments. Nanopore studies provide evidence of trapping events related to the oligomer formation and provide further details on their stabilities, which are confirmed by MS and MD simulations. Furthermore, simulation results reveal the molecular basis of oligomer dynamics and states of LL-37 fragments. This work provides unique insights into the relationship between the oligomer dynamics of AMPs and their antimicrobial activities at the single-molecule level. The study demonstrates how integrating methods allows deciphering single molecule level understanding from nanopore sensing approaches. 

Misfolding of the cellular prion protein (PrP^C) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in PrP^C misfolding. PrP^C is a combined Cu(II) and Zn(II) metal-binding protein, where the main metal-binding site is located in the octarepeat (OR) region. Thus, the biological function of PrP^C may involve the transport of divalent metal ions across membranes or buffering concentrations of divalent metal ions in the synaptic cleft. Recent studies have shown that an excess of Cu(II) ions can result in PrP^C instability, oligomerization, and/or neuroinflammation. Here, we have used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region of PrP^C. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Binding of the first metal ion results in a structural transition from the polyproline II helix to the β-turn structure, while the binding of additional metal ions induces the formation of β-sheet structures. Fluorescence spectroscopy data indicate that the OR region can bind both Cu(II) and Zn(II) ions at neutral pH, but under acidic conditions, it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of either metal ion to the OR region results in the formation of β-hairpin structures. As the formation of β-sheet structures can be a first step toward amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSE diseases.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. AD brains display deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-beta (A beta) peptides, and A beta oligomers are likely a toxic species in AD pathology. AD patients display altered metal homeostasis, and AD plaques show elevated concentrations of metals such as Cu, Fe, and Zn. Yet, the metal chemistry in AD pathology remains unclear. Ni(II) ions are known to interact with A beta peptides, but the nature and effects of such interactions are unknown. Here, we use numerous biophysical methods-mainly spectroscopy and imaging techniques-to characterize A beta/Ni(II) interactions in vitro, for different A beta variants: A beta(1-40), A beta(1-40)(H6A, H13A, H14A), A beta(4-40), and A beta(1-42). We show for the first time that Ni(II) ions display specific binding to the N-terminal segment of full-length A beta monomers. Equimolar amounts of Ni(II) ions retard A beta aggregation and direct it towards non-structured aggregates. The His6, His13, and His14 residues are implicated as binding ligands, and the Ni(II)center dot A beta binding affinity is in the low mu M range. The redox-active Ni(II) ions induce formation of dityrosine cross-links via redox chemistry, thereby creating covalent A beta dimers. In aqueous buffer Ni(II) ions promote formation of beta sheet structure in A beta monomers, while in a membrane-mimicking environment (SDS micelles) coil-coil helix interactions appear to be induced. For SDS-stabilized A beta oligomers, Ni(II) ions direct the oligomers towards larger sizes and more diverse (heterogeneous) populations. All of these structural rearrangements may be relevant for the A beta aggregation processes that are involved in AD brain pathology.

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD β-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first β-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-β (Aβ42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with Aβ, the CTD constructs instead bind to Aβ42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two β-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD.

Amyloid β-peptide (Aβ) misfolding into β-sheet structures triggers neurotoxicity inducing Alzheimer’s disease (AD). Molecules able to reduce or to impair Aβ aggregation are highly relevant as possible AD treatments since they should protect against Aβ neurotoxicity. We have studied the effects of the interaction of dynorphins, a family of opioid neuropeptides, with Aβ₄₀ the most abundant species of Aβ. Biophysical measurements indicate that Aβ₄₀ interacts with Big Dynorphin (BigDyn), lowering the amount of hydrophobic aggregates, and slowing down the aggregation kinetics. As expected, we found that BigDyn protects against Aβ₄₀ aggregates when studied in human neuroblastoma cells by cell survival assays. The cross-interaction between BigDyn and Aβ₄₀ provides insight into the mechanism of amyloid pathophysiology and may open up new therapy possibilities.