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As newly synthesized polypeptides emerge from the ribosome, it is crucial that they fold correctly. To prevent premature aggregation, nascent chains interact with chaperones that facilitate folding or prevent misfolding until protein synthesis is complete. Nascent polypeptide-associated complex (NAC) is a ribosome-associated chaperone that is important for protein homeostasis. However, how NAC binds its substrates remains unclear. Using native electrospray ionization MS (ESI-MS), limited proteolysis, NMR, and cross-linking, we analyzed the conformational properties of NAC from Caenorhabditis elegans and studied its ability to bind proteins in different conformational states. Our results revealed that NAC adopts an array of compact and expanded conformations and binds weakly to client proteins that are unfolded, folded, or intrinsically disordered, suggestive of broad substrate compatibility. Of note, we found that this weak binding retards aggregation of the intrinsically disordered protein α-synuclein both in vitro and in vivo These findings provide critical insights into the structure and function of NAC. Specifically, they reveal the ability of NAC to exploit its conformational plasticity to bind a repertoire of substrates with unrelated sequences and structures, independently of actively translating ribosomes., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Amyloid diseases represent a growing social and economic burden in the developed world. Understanding the assembly pathway and the inhibition of amyloid formation is key to developing therapies to treat these diseases. The neurodegenerative condition Machado-Joseph disease is characterised by the self-aggregation of the protein ataxin-3. Ataxin-3 consists of a globular N-terminal Josephin domain, which can aggregate into curvilinear protofibrils, and an unstructured, dynamically disordered C-terminal domain containing three ubiquitin interacting motifs separated by a polyglutamine stretch. Upon expansion of the polyglutamine region above 50 residues, ataxin-3 undergoes a second stage of aggregation in which long, straight amyloid fibrils form. A peptide inhibitor of polyglutamine aggregation, known as polyQ binding peptide 1, has been shown previously to prevent the maturation of ataxin-3 fibrils. However, the mechanism of this inhibition remains unclear. Using nanoelectrospray ionisation-mass spectrometry, we demonstrate that polyQ binding peptide 1 binds to monomeric ataxin-3. By investigating the ability of polyQ binding peptide 1 to bind to truncated ataxin-3 constructs lacking one or more domains, we localise the site of this interaction to a 39-residue sequence immediately C-terminal to the Josephin domain. The results suggest a new mechanism for the inhibition of polyglutamine aggregation by polyQ binding peptide 1 in which binding to a region outside of the polyglutamine tract can prevent fibril formation, highlighting the importance of polyglutamine flanking regions in controlling aggregation and disease.

Soluble oligomers formed during the self-assembly of amyloidogenic peptide and protein species are generally thought to be highly toxic. Consequently, thorough characterization of these species is of much interest in the quest for effective therapeutics and for an enhanced understanding of amyloid fibrillation pathways. The structural characterization of oligomeric species, however, is challenging as they are often transiently and lowly populated, and highly heterogeneous. Electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is a powerful technique which is able to detect individual ion species populated within a complex heterogeneous mixture and characterize them in terms of shape, stoichiometry, ligand binding capability, and relative stability. Herein, we describe the use of ESI-IMS-MS to characterize the size and shape of oligomers of beta-2-microglobulin through use of data calibration and the derivation of models. This enables information about the range of oligomeric species populated en route to amyloid formation and the mode of oligomer growth to be obtained.

The kinetics and thermodynamics of protein folding are commonly studied in vitro by denaturing/renaturing intact protein sequences. How these folding mechanisms relate to de novo folding that occurs as the nascent polypeptide emerges from the ribosome is much less well understood. Here, we have employed limited proteolysis followed by mass spectrometry analyses to compare directly free and ribosome-tethered polypeptide chains of the Src-homology 3 (SH3) domain and its unfolded variant, SH3-m10. The disordered variant was found to undergo faster proteolysis than SH3. Furthermore, the trypsin cleavage patterns observed show minor, but significant, differences for the free and ribosome-bound nascent chains, with significantly fewer tryptic peptides detected in the presence of ribosome. The results highlight the utility of limited proteolysis coupled with mass spectrometry for the structural analysis of these complex systems, and pave the way for detailed future analyses by combining this technique with chemical labeling methods (for example, hydrogen-deuterium exchange, photochemical oxidation) to analyze protein folding in real time, including in the presence of additional ribosome-associated factors., (© 2015 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

Expansion of polyglutamine stretches leads to the formation of polyglutamine-containing neuronal aggregates and neuronal death in nine diseases for which there currently are no treatments or cures. This is largely due to a lack in understanding of the mechanisms by which expanded polyglutamine regions contribute to aggregation and disease. To complicate matters further, several of the polyglutamine-disease related proteins, including ataxin-3, have a multistage aggregation mechanism in which flanking domain self-assembly precedes polyglutamine aggregation yet is influenced by polyglutamine expansion. How polyglutamine expansion influences flanking domain aggregation is poorly understood. Here, we use a combination of mass spectrometry and biophysical approaches to investigate this issue for ataxin-3. We show that the conformational dynamics of the flanking Josephin domain in ataxin-3 with an expanded polyglutamine tract are altered in comparison to those exhibited by its nonexpanded counterpart, specifically within the aggregation-prone region of the Josephin domain (amino acid residues 73-96). Expansion thus exposes this region more frequently in ataxin-3 containing an expanded polyglutamine tract, providing a molecular explanation of why aggregation is accelerated upon polyglutamine expansion. Here, harnessing the power of ion mobility spectrometry-mass spectrometry, oligomeric species formed during aggregation are characterized and a model for oligomer growth proposed. The results suggest that a conformational change occurs at the dimer level that initiates self-assembly. New insights into ataxin-3 fibril architecture are also described, revealing the region of the Josephin domain involved in protofibril formation and demonstrating that polyglutamine aggregation proceeds as a distinct second step after protofibril formation without requiring structural rearrangement of the protofibril core. Overall, the results enable the effect of polyglutamine expansion on every stage of ataxin-3 self-assembly, from monomer through to fibril, to be described and a rationale for expedited aggregation upon polyglutamine expansion to be provided., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Detailed knowledge of the tertiary and quaternary structure of proteins and protein complexes is of immense importance in understanding their functionality. Similarly, variations in the conformational states of proteins form the underlying mechanisms behind many biomolecular processes, numerous of which are disease-related. Thus, the availability of reliable and accurate biophysical techniques that can provide detailed information concerning these issues is of paramount importance. Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) offers a unique opportunity to separate multi-component biomolecular entities and to measure the molecular mass and collision cross-section of individual components in a single, rapid (

Proteomics encompasses several disciplines in the quest to unravel the science of life. Mass spectrometry plays a key rôle in proteomics-associated analyses and this review describes that function in terms of the instrumentation used, the results generated, data interpretation and the advances in analytical techniques being made to deal with the production of highly specific, high throughput data. The review has 151 references.

The precise molecular mechanisms by which different peptides and proteins assemble into highly ordered amyloid deposits remain elusive. The fibrillation of human amylin (also known as islet amyloid polypeptide, hIAPP) and the amyloid-beta peptide (Aβ-40) are thought to be pathogenic factors in Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD), respectively. Amyloid diseases may involve co-aggregation of different protein species, in addition to the self-assembly of single precursor sequences. Here we investigate the formation of heterogeneous pre-fibrillar, oligomeric species produced by the co-incubation of hIAPP and Aβ-40 using electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)-based methods. Conformational properties and gas-phase stabilities of amyloid oligomers formed from hIAPP or Aβ40 alone, and from a 1 : 1 mixture of hIAPP and Aβ40 monomers, were determined and compared. We show that co-assembly of the two sequences results in hetero-oligomers with distinct properties and aggregation kinetics properties compared with the homo-oligomers present in solution. The observations may be of key significance to unravelling the mechanisms of amyloid formation in vivo and elucidating how different sequences and/or assembly conditions can result in different fibril structures and/or pathogenic outcomes. [ABSTRACT FROM AUTHOR]

Abstract: Non-beta amyloid component of Alzheimer’s disease amyloid or NAC is a highly amyloidogenic peptide consisting of 35 amino acids which was first identified associated with senile plaques in the Alzheimer’s disease brain. It is a fragment of the presynaptic protein α-synuclein and, as such, it is implicated in the aetiologies of both Alzheimer’s (AD) and Parkinson’s (PD) disease. Metals are involved in the aggregation of amyloidogenic peptides such as beta amyloid (Aβ), British amyloid peptide (ABri) and α-synuclein though nothing is yet known about how they might influence the aggregation of NAC. We show herein that NAC will form β-pleated conformers at a peptide concentration of only 2.0μM and that metals, and Zn(II) and Cu(II) in particular, accelerate the formation of these fibrils. Cu(II) and Zn(II) did not influence the diameter or general structure of the fibrils which were formed though many more shorter fibrils were observed in their presence and these shorter fibrils were highly thioflavin T positive and they were efficient catalysts of the redox cycling of added Fe(II). By way of contrast, β-pleated conformers of NAC which were formed in the presence of Al(III) showed much lower levels of thioflavin T fluorescence and were poorer catalysts of the redox cycling of added Fe(II) and these properties were commensurate with an increased abundance of a novel amyloid morphology which consisted of twisted fibrils with a periodicity of about 100nm. These spirals of twisted fibrils were especially abundant in the presence of added Al(III) and it is speculated that NAC binding of Al(III) may be important in their formation and subsequent stability. [Copyright &y& Elsevier]

Abstract: British amyloid (ABri) peptide is precipitated as amyloid fibrils in pathological lesions which are characteristic of familial British dementia. Unlike for other amyloidogenic peptides which have been implicated in neurodegenerative disease, for example, Aβ in Alzheimer’s disease and α synuclein in Parkinson’s disease, nothing is yet known as to whether metals mediate the formation of ABri amyloid fibrils. We show herein that a concentration of ABri, which had not previously been shown to spontaneously form amyloid, formed fibrils when incubated for 12 months at 37 °C. The additional presence of Al(III), in particular, or Fe(III) increased significantly both the number and the size of the fibrillar amyloid deposits which were very similar in appearance to amyloid described in hippocampal plaques in familial British dementia. Co-incubation of ABri with either Zn(II) or Cu(II) precipitated the peptide but did not result in the formation of amyloid fibrils. [Copyright &y& Elsevier]

Peptide YY and neuropeptide Y are common peptides with a high degree of primary and tertiary structural homology. They are multifunctional and participate in a diverse array of distinct activities including regulation of gastrointestinal function and neural regulation of satiety. Recently both have been implicated in aluminium chemistry in vivo although their modus opperandi have not been determined. We have used molecular fluorescence, RP-HPLC, ESMS and equilibrium dialysis to identify if either peptide YY or neuropeptide Y will bind aluminium in vitro under near-physiological conditions. We were unable to demonstrate any direct interaction between either peptide and aluminium although we have speculated upon an in vivo mechanism whereby PYY, in particular, might form a stable complex with aluminium. [Copyright &y& Elsevier]

The catabolism of amyloid beta peptides (Abeta) may be important in their accumulation in the brain in both early and late-onset Alzheimer's disease (AD). The serine protease plasmin is one of a suite of proteases implicated in AD. It is a promoter of alpha-cleavage of the amyloid beta precursor protein (AbetaPP) and will degrade Abeta in vitro. Herein we have demonstrated cleavage of the amyloidogenic Abeta(25-35) by plasmin to produce the non-amyloidogenic fragment Abeta(29-35). The activity of plasmin was halved by pre-mixing it with aluminium (Al) prior to its addition to the peptide. An interaction between Al and proteases involved in the catabolism of Abeta might define the putative link between Al and AD. [ABSTRACT FROM AUTHOR]

Abstract: Travelling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS) has the capability to separate ions based on their mobility through a gas-filled travelling wave (T-wave) device in the presence of a train of transient voltage pulses. By calibration of this device using analytes of previously determined cross-sectional area (from conventional IMS experiments), collision cross-sections of ions can be determined based on their drift time through the T-wave device. Comparison of experimentally determined cross-sections with theoretical calculations from structural models has the potential to provide methodology which can be applied to analytes of previously uncharacterised structure; however, this comparison relies on a high degree of confidence in both experimental and theoretical methods. Focussing on small (≤200Da) molecules, collision cross-sections have been measured by TWIMS-MS employing a calibration procedure that uses both oligo-glycine peptides and human haemoglobin-derived tryptic peptides in order to extend the calibration range for the measurement of high mobility ions. The effect of TWIMS wave height parameters on the calibration is addressed. Theoretical TWIMS cross-section calculations have been performed using a rapid, Windows-based, in-house developed projection approximation algorithm. These estimates were optimised by comparison with experimental values for a series of small molecules with rigid core structures by systematic variation of the interaction radii of the atoms comprising these species until theoretical measurements were in agreement with experimentally derived TWIMS cross-sections. The effect of varying the interaction radius for the buffer gas was subsequently studied by comparison of theoretical collision cross-sections calculated using helium and nitrogen radii with TWIMS-MS cross-section measurements determined in either helium or nitrogen buffer gases. It was found that the buffer gas used in theoretical calculations should ideally match the buffer gas in which the cross-sections of the calibrants were originally determined by conventional IMS. The resolving potential of the experimental methodology was demonstrated by separation of two isomeric amino acids, leucine and isoleucine, which showed <3% difference in cross-sectional areas. Furthermore, the application of both experimental and theoretical methods to compare the gas phase and solution conformations of seven amino acids was demonstrated. This study describes important considerations for the comparison of TWIMS-MS collision cross-sections with those obtained by theoretical methods, and also demonstrates that TWIMS-MS could be applied specifically to study a range of small molecules including those of pharmaceutical interest, in particular structural isomers and diastereoisomers that may not be distinguishable by mass spectrometry alone. [Copyright &y& Elsevier]