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 (

The β-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA–E), which is essential for viability in E. coli. BAM promotes the folding and insertion of β-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a ‘lateral gating' motion of the β-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex and interactions between BamA, B, D and E, and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM.

Gram-negative pathogens commonly use the chaperone-usher pathway to assemble adhesive multisubunit fibers on their surface. In the periplasm, subunits are stabilized by a chaperone that donates a beta strand to complement the subunits' truncated immunoglobulin-like fold. Pilus assembly proceeds through a "donor-strand exchange" (DSE) mechanism whereby this complementary beta strand is replaced by the N-terminal extension (Nte) of an incoming pilus subunit. Using X-ray crystallography and real-time electrospray ionization mass spectrometry (ESI-MS), we demonstrate that DSE requires the formation of a transient ternary complex between the chaperone-subunit complex and the Nte of the next subunit to be assembled. The process is crucially dependent on an initiation site (the P5 pocket) needed to recruit the incoming Nte. The data also suggest a capping reaction displacing DSE toward product formation. These results support a zip-in-zip-out mechanism for DSE and a catalytic role for the usher, the molecular platform at which pili are assembled.

The search for therapeutic agents which bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult since many protein precursors of aggregation are partially folded or intrinsically disordered, ruling out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or non-specific 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 (Aβ40) aggregation as either specific, non-specific, 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.

The molecular mechanisms by which different proteins assemble into highly ordered fibrillar deposits and cause disease remain topics of debate. Human amylin (also known as islet amyloid polypeptide/hIAPP) is found in vivo as amyloid deposits in the pancreatic islets of sufferers of type II diabetes mellitus, and its self-aggregation is thought to be a pathogenic factor in disease and to contribute to the failure of islet transplants. Here, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to monitor oligomer formation from IAPP. The detection, identification and characterization of oligomers from both human and rat amylin (rIAPP) are described. Oligomers up to and including hexamers have been detected for both peptides. From ESI-IMS-MS derived collision cross sections (CCS), these species are shown to be elongated in conformation. Collision-induced dissociation (CID-MS/MS) revealed differences in the gas-phase stability of the oligomers formed from hIAPP and rIAPP, which may contribute to their differences in amyloid propensity. Using ESI-IMS-MS, the mode of inhibition of amyloid formation from hIAPP using small molecules or co-incubation with rIAPP was also investigated. We show that the polyphenolic compounds epigallocatechin gallate (EGCG) and silibinin bind to specific conformers within a dynamic ensemble of hIAPP monomers, altering the progress of oligomerization and fibril assembly. Hetero-oligomer formation also occurs with rIAPP but leads only to inefficient inhibition. The results indicate that although different small molecules can be effective inhibitors of hIAPP self-assembly, their modes of action are distinct and can be distinguished using ESI-IMS-MS.

The conformational properties of soluble a-synuclein, the primary protein found in patients with Parkinson's disease, are thought to play a key role in the structural transition to amyloid fibrils. In this work, we report that recombinant 100% N-terminal acetylated a-synuclein purified under mild physiological conditions presents as a primarily monomeric protein, and that the N-terminal acetyl group affects the transient secondary structure and fibril assembly rates of the protein. Residue-specific NMR chemical shift analysis indicates substantial increase in transient helical propensity in the first 9 N-terminal residues, as well as smaller long-range changes in residues 28-31, 43-46, and 50-66: regions in which the three familial mutations currently known to be causative of early onset disease are found. In addition, we show that the N-terminal acetylated protein forms fibrils that are morphologically similar to those formed from nonacetylated a-synuclein, but that their growth rates are slower. Our results highlight that N-terminal acetylation does not form significant numbers of dimers, tetramers, or higher molecular Abbreviations: asyn, alpha synuclein; Ac-asyn, acetylated asyn; BOG, b-octyl glucoside; EM, fluorescence electron microscopy; ESI-IMS-MS, ion mobility spectrometry combined with ESI-MS; ESI-MS, noncovalent electrospray ionization mass spectrometry; IDP, intrinsically disordered protein; NatB, N-acetyltransferase B; SEC, analytical size-exclusion chromatography; ThT, Thioflavin T.Additional Supporting Information may be found in the online version of this article. † Lijuan Kang and Gina M. Moriarty contributed equally to this work. weight species, but does alter the conformational distributions of monomeric a-synuclein species in regions known to be important in metal binding, in association with membranes, and in regions known to affect fibril formation rates.

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Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.