LL-37 and its variants with amphiphilic structure can modulate amyloid-β (Aβ) fibril formation, but the detailed mechanism behind it is still unclear. By using four different peptides (LL-37, LL-379–32, LL-3718–29, LL-3719–28), we found these peptides affect Aβ40 aggregation differently. Nanoscale analysis showed that all LL-37 peptides form hetero-oligomers and nanoclusters with Aβ40, but LL-37 and LL-3719–28, which exhibit the strongest inhibition of Aβ fibrillation, form more hetero-oligomers and smaller nanoclusters. This suggests that hetero-oligomers and small nanoclusters may represent an off-pathway, preventing the formation of productive aggregates. At the microscale, all LL-37 peptides were found to promote Aβ cluster formation, but LL-37 and LL-3719–28 can form larger clusters with Aβ rapidly, emphasizing that smaller nanoclusters can assemble to macroscale clusters easier, inducing more toxic aggregates. Both nanoscopic and microscopic mechanisms revealed inhibition of Aβ fibrillation by all LL-37 peptides, impacting Aβ primary and secondary nucleation, while only LL-37 and LL-3719–28 affected Aβ elongation. Our findings highlight the role of LL-37 and its synthetic fragments in Aβ40 aggregation across different scales, particularly focusing on cluster formation at the nanoscale and microscale to fill the knowledge gap between oligomerization and fibrillation.

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.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT^∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT^∗ domain. We conclude that interactions with NT^∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT^∗. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.

Antibiotic resistance cassettes are indispensable tools in recombinant DNA technology, synthetic biology, and metabolic engineering. The genetic cassette encoding the TEM-1 β-lactamase (denoted Tn3.1) is one of the most commonly used and can be found in more than 120 commercially available bacterial expression plasmids (e.g., the pET, pUC, pGEM, pQE, pGEX, pBAD, and pSEVA series). A widely acknowledged problem with the cassette is that it produces excessively high titers of β-lactamase that rapidly degrade β-lactam antibiotics in the culture media, leading to loss of selective pressure, and eventually a large percentage of cells that do not have a plasmid. To address these shortcomings, we have engineered a next-generation version that expresses minimal levels of β-lactamase (denoted Tn3.1^MIN). We have also engineered a version that is compatible with the Standard European Vector Architecture (SEVA) (denoted Ap (pSEVA#1^MIN--)). Expression plasmids containing either Tn3.1^MIN or Ap (pSEVA#1^MIN--) can be selected using a 5-fold lower concentration of β-lactam antibiotics and benefit from the increased half-life of the β-lactam antibiotics in the culture medium (3- to 10-fold). Moreover, more cells in the culture retain the plasmid. In summary, we present two antibiotic-efficient genetic cassettes encoding the TEM-1 β-lactamase that reduce antibiotic consumption (an integral part of antibiotic stewardship), reduce production costs, and improve plasmid performance in bacterial cell factories. 

Electrophilic diol epoxide metabolites are involved in the carcinogenicity of benzo[a]pyrene, one of the widely studied polycyclic aromatic hydrocarbons (PAHs). The exposure of humans to this PAH can be assessed by measuring stable blood protein adducts, such as to histidine and lysine in serum albumin, from their reactive metabolites. In this respect, measurement of the adducts originating from the genotoxic (+)-anti-benzo[a]pyrene diol epoxide is of interest. However, these are difficult to measure at such low levels as are expected in humans generally exposed to benzo[a]pyrene from air pollution and the diet. The analytical methods detecting PAH-biomarkers still suffer from low selectivity and/or detectability to enable generation of data for calculation of in vivo doses of specific stereoisomers, for evaluation of risk factors and assessing risk from exposures to PAH. Here, we suggest an analytical methodology based on high-pressure liquid chromatography (HPLC) coupled to high-resolution tandem mass spectrometry (MS) to lower the detection limits as well as to increase the selectivity with improvements in both chromatographic separation and mass determination. Method development was performed using serum albumin alkylated in vitro by benzo[a]pyrene diol epoxide isomers. The (+)-anti-benzo[a]pyrene diol epoxide adducts could be chromatographically resolved by using an HPLC column with a pentafluorophenyl stationary phase. Interferences were further diminished by the high mass accuracy and resolving power of Orbitrap MS. The achieved method detection limit for the (+)-anti-benzo[a]pyrene diol epoxide adduct to histidine was approximately 4 amol/mg serum albumin. This adduct as well as the adducts to histidine from (−)-anti- and (+/−)-syn-benzo[a]pyrene diol epoxide were quantified in the samples from benzo[a]pyrene-exposed mice. Corresponding adducts to lysine were also quantified. In human serum albumin, the anti-benzo[a]pyrene diol epoxide adducts to histidine were detected in only two out of twelve samples and at a level of approximately 0.1 fmol/mg.

Protein homeostasis collapse typically leads to protein aggregation into amyloid fibrils and diffuse amorphous aggregates, which both occur in Alzheimer’s and other neurodegenerative diseases, but their relationship remains to be clarified. Here we examine the interactions between the amorphously aggregated non-chaperone proteins (albumin, β-lactoglobulin, and superoxide dismutase 1) and Alzheimer’s amyloid-β (Aβ) peptides. Amorphous aggregates suppress the primary nucleation and elongation of Aβ fibrillation and modulate Aβ toxicity. The higher inhibitory efficiency of intermediately sized molten globular aggregates (20–300 nm) on Aβ fibrillation is hypothesized to be due to the higher amount of exposed hydrophobic residues and higher free energy. The formed co-aggregates are off-pathway species that favor formation of the amorphous end state instead of fibrillar amyloid structures normally formed by Aβ. Our findings expand our knowledge of how the native and aggregated cellular proteins modulate Aβ aggregation at the molecular and mesoscopic level and point out the major conclusions.

The assembly of proteins and peptides into amyloid fibrils is causally linked to serious disorders such as Alzheimer’s disease. Multiple proteins have been shown to prevent amyloid formation in vitro and in vivo, ranging from highly specific chaperone–client pairs to completely nonspecific binding of aggregation-prone peptides. The underlying interactions remain elusive. Here, we turn to the machine learning–based structure prediction algorithm AlphaFold2 to obtain models for the nonspecific interactions of β-lactoglobulin, transthyretin, or thioredoxin 80 with the model amyloid peptide amyloid β and the highly specific complex between the BRICHOS chaperone domain of C-terminal region of lung surfactant protein C and its polyvaline target. Using a combination of native mass spectrometry (MS) and ion mobility MS, we show that nonspecific chaperoning is driven predominantly by hydrophobic interactions of amyloid β with hydrophobic surfaces in β-lactoglobulin, transthyretin, and thioredoxin 80, and in part regulated by oligomer stability. For C-terminal region of lung surfactant protein C, native MS and hydrogen–deuterium exchange MS reveal that a disordered region recognizes the polyvaline target by forming a complementary β-strand. Hence, we show that AlphaFold2 and MS can yield atomistic models of hard-to-capture protein interactions that reveal different chaperoning mechanisms based on separate ligand properties and may provide possible clues for specific therapeutic intervention.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation. 

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

Although native mass spectrometry is widely applied to monitor chemical or thermal protein denaturation, it is not clear to what extent it can inform about alkali-induced unfolding. Here, we probe the relationship between solution- and gas-phase structures of proteins under alkaline conditions. Native ion mobility-mass spectrometry reveals that globular proteins are destabilized rather than globally unfolded, which is supported by solution studies, providing detailed insights into alkali-induced unfolding events. Our results pave the way for new applications of MS to monitor structures and interactions of proteins at high pH.