Long-Range Conformational Changes in Monoclonal Antibodies Revealed Using FPOP-LC-MS/MS

Differences in conformational dynamics between two full-length monoclonal antibodies have been probed in detail using Fast Photochemical Oxidation of Proteins (FPOP) followed by proteolysis and LC-ESI-MS/MS analyses. FPOP uses hydroxyl radical labelling to probe the surface-accessible regions of proteins and has the advantage that the resulting covalent modifications are irreversible, thus permitting optimal down-stream analysis. Despite the two monoclonal antibodies (mAbs) differing by only three amino acids in the heavy chain complementarity determining regions (CDRs), one mAb, MEDI1912-WFL, has been shown to undergo reversible self-association at high concentrations and exhibited poor pharmacokinetic properties in vivo, properties which are markedly improved in the variant, MEDI1912-STT. Identifying the differences in oxidative labelling between the two antibodies at residue level revealed long-range effects which provide a key insight into their conformational differences. Specifically, the amino acid mutations in the CDR region of the heavy chain resulted in significantly different labelling patterns at the interfaces of the CL–CH1 and CH1–CH2 domains, with the non-aggregating variant undergoing up to four times more labelling in this region than the aggregation prone variant, thus suggesting a change in the structure and orientation of the CL – CH1 interface. The wealth of FPOP and LC-MS data obtained enabled the study of the LC elution properties of FPOP-oxidised peptides. Some oxidised amino acids, specifically histidine and lysine, were noted to have unique effects on the retention time of the peptide, offering the promise of using such an analysis as an aid to MS/MS in assigning oxidation sites

Understanding co-polymerization in amyloid formation by direct observation of mixed oligomers

Although amyloid assembly in vitro is commonly investigated using single protein sequences, fibril formation in vivo can be more heterogeneous, involving co-assembly of proteins of different length, sequence and/or post-translational modifications. Emerging evidence suggests that co-polymerization can alter the rate and/or mechanism of aggregation and can contribute to pathogenicity. Electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is uniquely suited to the study of these heterogeneous ensembles. Here, ESI-IMS-MS combined with analysis of fibrillation rates using thioflavin T (ThT) fluorescence, is used to track the course of aggregation of variants of islet-amyloid polypeptide (IAPP) in isolation and in pairwise mixtures. We identify a sub-population of extended monomers as the key precursors of amyloid assembly, and reveal that the fastest aggregating sequence in peptide mixtures determines the lag time of fibrillation, despite being unable to cross-seed polymerization. The results demonstrate that co-polymerization of IAPP sequences radically alters the rate of amyloid assembly by altering the conformational properties of the mixed oligomers that form

Characterization of Amyloid Oligomers by Electrospray Ionization-Ion Mobility Spectrometry-Mass Spectrometry (ESI-IMS-MS)

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

Molecular dynamics simulations of mechanical failure in polymorphic arrangements of amyloid fibrils containing structural defects

We examine how the different steric packing arrangements found in amyloid fibril polymorphs can modulate their mechanical properties using steered molecular dynamics simulations. Our calculations demonstrate that for fibrils containing structural defects, their ability to resist force in a particular direction can be dominated by both the number and molecular details of the defects that are present. The simulations thereby suggest a hierarchy of factors that govern the mechanical resilience of fibrils, and illustrate the general principles that must be considered when quantifying the mechanical properties of amyloid fibres containing defects

Conformational flexibility within the nascent polypeptide–associated complex enables its interactions with structurally diverse client proteins

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 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 analysed 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 having unrelated sequences and structures independently of actively translating ribosomes

Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry

The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition. Here we report a high-throughput method based on ion mobility spectrometry–mass spectrometry (IMS–MS) that is capable of rapidly detecting small molecules that bind to amyloid precursors, identifying the interacting protein species and defining the mode of inhibition. Using this method we have classified a variety of small molecules that are potential inhibitors of human ​islet amyloid polypeptide (​hIAPP) aggregation or ​amyloid-beta 1-40 aggregation as specific, nonspecific, colloidal or non-interacting. We also demonstrate the ability of IMS–MS to screen for inhibitory small molecules in a 96-well plate format and use this to discover a new inhibitor of ​hIAPP amyloid assembly

Topological dissection of the membrane transport protein Mhp1 derived from cysteine accessibility and mass spectrometry

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallised in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-L-hydantoin (L-BH) does not shift this conformational equilibrium, but, systematic co-addition of the two results in an attenuation of labelling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion-substrate coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilisation of structure. The methodology combines covalent labeling, mass spectrometry and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein’s conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions

Inter-domain dynamics in the chaperone SurA and multi-site binding to its outer membrane protein clients

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release

An in vivo platform to select and evolve aggregation-resistant proteins

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve ‘manufacturable’ biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease