In this review, we attempt to give a concise overview of recent progress made in mechanistic understanding of protein aggregation, particulate formation and protein solution rheology. Recent advances in analytical techniques and methods for characterizing protein aggregation and the formed protein particles as well as advancements, technique limitations and controversies in the field of protein solution rheology are discussed. The focus of the review is primarily on biotherapeutics and proteins/antibodies that are relevant to that area. As per the remit of Current Opinion in Colloid and Interface Science, here we attempt to stimulate interest in areas of debate. While the field is certainly not mature enough that all problems may be considered resolved and accepted by consensus, we wish to highlight some areas of controversy and debate that need further attention from the scientific community.

https://cyberleninka.org/article/n/118092.pdf

Protein aggregation, particle formation, characterization & rheology

Intrinsically disordered protein regions (IDRs) - regions that do not fold into a fixed three-dimensional structure but instead exist in a heterogeneous ensemble of conformations - have recently entered mainstream cell biology in the context of liquid-liquid phase separation (LLPS). IDRs are frequently found to be enriched in phase-separated compartments. Due to this observation, the presence of an IDR in a protein is frequently assumed to be diagnostic of its ability to phase separate. In this review, we clarify the role of IDRs in biological assembly and explore the physical principles through which amino acids can confer the attractive molecular interactions that underlie phase separation. While some disordered regions will robustly drive phase separation, many others will not. We emphasize that rather than 'disorder' driving phase separation, multivalency drives phase separation. As such, whether or not a disordered region is capable of driving phase separation will depend on the physical chemistry encoded within its amino acid sequence. Consequently, an in-depth understanding of that physical chemistry is a prerequisite to make informed inferences on how and why an IDR may be involved in phase separation or, more generally, in protein-mediated intermolecular interactions.

Intrinsically disordered protein regions and phase separation: sequence determinants of assembly or lack thereof.

Sequences rich in glutamine (Q) and asparagine (N) residues often fail to fold at the monomer level. This, coupled to their unusual hydrogen-bonding abilities, provides the driving force to switch between disordered monomers and amyloids. Such transitions govern processes as diverse as human protein-folding diseases, bacterial biofilm assembly, and the inheritance of yeast prions (protein-based genetic elements). A systematic survey of prion-forming domains suggested that Q and N residues have distinct effects on amyloid formation. Here, we use cell biological, biochemical, and computational techniques to compare Q/N-rich protein variants, replacing Ns with Qs and Qs with Ns. We find that the two residues have strong and opposing effects: N richness promotes assembly of benign self-templating amyloids; Q richness promotes formation of toxic nonamyloid conformers. Molecular simulations focusing on intrinsic folding differences between Qs and Ns suggest that their different behaviors are due to the enhanced turn-forming propensity of Ns over Qs.

Opposing Effects of Glutamine and Asparagine Govern Prion Formation by Intrinsically Disordered Proteins

Protein misfolding into amyloid fibrils is linked to more than 40 as yet incurable cell- and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes. So far, however, only one of the numerous anti-amyloid molecules has reached patients. This Minireview gives an overview of molecular strategies and peptide chemistry "tools" to design, develop, and discover peptide-based molecules as anti-amyloid drug candidates. We focus on two major inhibitor rational design strategies: 1) the oldest and most common strategy, based on molecular recognition elements of amyloid self-assembly, and 2) a more recent approach, based on cross-amyloid interactions. We discuss why peptide-based amyloid inhibitors, in particular their advanced generations, can be promising leads or candidates for anti-amyloid drugs as well as valuable tools for deciphering amyloid-mediated cell damage and its link to disease pathogenesis.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/anie.201906908

Peptide‐based molecular strategies to interfere with protein misfolding, aggregation, and cell degeneration

In this article, we review the relevant results obtained during almost 60 years of research on a specific aspect of stereochemistry, namely handedness preference and switches between right-handed and left-handed helical peptide structures generated by protein amino acids or appropriately designed, side-chain modified analogs. In particular, we present and discuss here experimental and theoretical data on three categories of those screw-sense issues: (i) right-handed/left-handed α-helix transitions underwent by peptides rich in Asp, specific Asp β-esters, and Asn; (ii) comparison of the preferred conformations adopted by helical host-guest peptide series, each characterized by an amino acid residue (e.g. Ile or its diastereomer aIle) endowed with two chiral centers in its chemical structure; and (iii) right-handed (type I)/left-handed (type II) poly-(Pro)n helix transitions monitored for peptides rich in Pro itself or its analogs with a pyrrolidine ring substitution, particularly at the biologically important position 4. The unique modular and chiral properties of peptides, combined with their relatively easy synthesis, the chance to shape them into the desired conformation, and the enormous chemical diversity of their coded and non-coded α-amino acid building blocks, offer a huge opportunity to structural chemists for applications to bioscience and nanoscience problems.

Handedness preference and switching of peptide helices. Part II: Helices based on noncoded α-amino acids

β-Amyloid peptide (Aβ) self-associates into oligomers and fibrils, in a process that is believed to directly lead to neuronal death in Alzheimer’s disease. Compounds that bind to Aβ, and inhibit fibrillogenesis and neurotoxicity, are of interest as an anti-Alzheimer therapeutic strategy. Peptides are particularly attractive for this purpose, because they have advantages over small molecules in their ability to disrupt protein–protein interactions, yet they are amenable to tuning of their properties through chemical means, unlike antibodies. Self-complementation and peptide library screening are two strategies that have been employed in the search for peptides that bind to Aβ. We have taken a different approach, by designing Aβ-binding peptides using transthyretin (TTR) as a template. Previously, we demonstrated that a cyclic peptide, with sequence derived from the known Aβ-binding site on TTR, suppressed Aβ aggregation into fibrils and protected neurons against Aβ toxicity. Here, we searched for cyclic pe...

/pdf/tango-inspired-design-of-anti-amyloid-cyclic-peptides-1pzx7ph2pl.pdf

TANGO-Inspired Design of Anti-Amyloid Cyclic Peptides

Of all amino acid repeats in eukaryotes, polyglutamine (polyQ) is the most frequent, followed by polyasparagine (polyN). Glutamine repeats are expanded in proteins associated with several neurodegenerative disorders. The expanded polyQ domain is known to induce aggregation, and it is hypothesized that aggregation is directly causative of pathology. Despite the widespread presence of asparagine repeats in invertebrate eukaryotes, polyN is curiously quite rare in vertebrates. Several investigators have characterized the conformational and aggregation properties of polyQ-containing peptides and proteins, and to a lesser extent, peptides containing mixed glutamine and asparagine, but to our knowledge, there is no detailed characterization of polyN-containing peptides. Such a comparison could elucidate reasons for the paucity of asparagine repeats in humans. In this study, we synthesized a peptide containing a 24-asparagine repeat (N24). For aggregation studies, it is critical to start with monomeric unaggregated peptide. A protocol involving dissolution in mixed trifluoroacetic acid and hexafluoroisopropanol (TFA + HFIP) solvents is widely used for disaggregation of polyQ peptides. We used the same protocol for N24 but discovered that there was both oxidative damage and insufficient disaggregation. Oxidation of tryptophan, used as a flanking residue, was common. Moreover, we found evidence of Forster resonance energy transfer between Trp and its oxidation product N-formylkynurenine, even in chemical denaturants. This suggested that N24 was insufficiently disaggregated, a conclusion that was further supported by gel electrophoresis analysis. Oxidation was reduced, but not eliminated, by addition of methionine to the buffer. Formic acid proved to be a better disaggregator and caused no oxidative damage. The glutamine repeat peptide Q24 also underwent some oxidation after extended incubation in TFA + HFIP, but there was no evidence of Forster resonance energy transfer, and samples appeared monomeric by gel electrophoresis. This result indicates that polyN-containing peptides self-associate more strongly than polyQ-containing peptides. Circular dichroism spectra reveal a greater propensity for β-turn formation in polyN than polyQ, providing an explanation for the increased stability of polyN aggregates relative to polyQ. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis and disaggregation of asparagine repeat-containing peptides.

Protein aggregates are the pathological agents in several neurodegenerative disorders such as Alzheimer’s and Huntington’s disease. In the pharmaceutical industry, protein aggregation poses significant challenges to the manufacture of biologics. Nanoparticle tracking is an emerging technology that allows particle-by-particle measurement of aggregate size and concentration. The technique is solution based, and requires no labeling. Here we describe protocols for using nanoparticle tracking in protein aggregation research, and provide a few examples for illustrative purposes.

Nanoparticle Tracking for Protein Aggregation Research.

Amino acid repeat runs are common occurrences in eukaryotic proteins, with glutamine (Q) and asparagine (N) as particularly frequent repeats. Abnormal expansion of Q-repeat domains causes at least nine neurodegenerative disorders, most likely because expansion leads to protein misfolding, aggregation, and toxicity. The linkage between Q-repeats and disease has motivated several investigations into the mechanism of aggregation and the role of Q-repeat length in aggregation. Curiously, glutamine repeats are common in vertebrates, whereas N-repeats are virtually absent in vertebrates, but common in invertebrates. One hypothesis for the lack of N-repeats in vertebrates is biophysical; that is, there is strong selective pressure in higher organisms against aggregation-prone proteins. If true, then asparagine and glutamine repeats must differ substantially in their aggregation properties despite their chemical similarities. In this work, aggregation of peptides with asparagine repeats of variable length (12–24)...

Asparagine Repeat Peptides: Aggregation Kinetics and Comparison with Glutamine Repeats.

Amyloidogenesis is the process of formation of protein aggregates with fibrillar morphology. Because amyloidogenesis is linked to neurodegenerative disease, there is interest in understanding the mechanism of fibril growth. Kinetic models of amyloidogenesis require data on the number concentration and size distribution of aggregates, but this information is difficult to obtain using conventional methods. Nanoparticle tracking analysis (NTA) is a relatively new technique that may be uniquely suited for obtaining these data. In NTA, the two-dimensional (2-D) trajectory of individual particles is tracked, from which the diffusion coefficient, and, hence, hydrodynamic radius is obtained. Here we examine the validity of NTA in tracking number concentration and size of DNA, as a model of a fibrillar macromolecule. We use NTA to examine three amyloidogenic materials: beta-amyloid, transthyretin, and polyglutamine-containing peptides. Our results are instructive in demonstrating the advantages and some limitations of single-particle diffusion measurements for investigating aggregation in protein systems.

Evaluation of nanoparticle tracking for characterization of fibrillar protein aggregates

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