Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications.

Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localization of dynamic changes. Consequently, the HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase the data quality by providing an orthogonal mode of peptide ion separation in the gas phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multipass IM separations. Using one-pass and multipass cIM routines, we use the recently commercialized SELECT SERIES Cyclic IM spectrometer for HDX-MS analyses of four detergent solubilized IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multipass cIM data. Interestingly, use of one-pass and multipass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM that has the potential to enable the analysis of more complex systems with greater accuracy and speed.

Linking function to global and local dynamics in an elevator-type transporter.

Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency-yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter GltPh We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

Integrating Hydrogen Deuterium Exchange-Mass Spectrometry with Molecular Simulations Enables Quantification of the Conformational Populations of the Sugar Transporter XylE.

A yet unresolved challenge in structural biology is to quantify the conformational states of proteins underpinning function. This challenge is particularly acute for membrane proteins owing to the difficulties in stabilizing them for in vitro studies. To address this challenge, we present an integrative strategy that combines hydrogen deuterium exchange-mass spectrometry (HDX-MS) with ensemble modeling. We benchmark our strategy on wild-type and mutant conformers of XylE, a prototypical member of the ubiquitous Major Facilitator Superfamily (MFS) of transporters. Next, we apply our strategy to quantify conformational ensembles of XylE embedded in different lipid environments. Further application of our integrative strategy to substrate-bound and inhibitor-bound ensembles allowed us to unravel protein-ligand interactions contributing to the alternating access mechanism of secondary transport in atomistic detail. Overall, our study highlights the potential of integrative HDX-MS modeling to capture, accurately quantify, and subsequently visualize co-populated states of membrane proteins in association with mutations and diverse substrates and inhibitors.

Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein-Lipid Assemblies.

Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein-lipid nanodisc─both empty and loaded with the ∼115 kDa transmembrane protein AcrB─proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads and describe how solution conditions can be optimized to maximize not only the performance of our online but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even native cell membranes.

Deuteros 2.0: peptide-level significance testing of data from hydrogen deuterium exchange mass spectrometry.

SUMMARY: Hydrogen deuterium exchange mass spectrometry (HDX-MS) is becoming increasing routine for monitoring changes in the structural dynamics of proteins. Differential HDX-MS allows comparison of protein states, such as in the absence or presence of a ligand. This can be used to attribute changes in conformation to binding events, allowing the mapping of entire conformational networks. As such, the number of necessary cross-state comparisons quickly increases as additional states are introduced to the system of study. There are currently very few software packages available that offer quick and informative comparison of HDX-MS datasets and even fewer which offer statistical analysis and advanced visualization. Following the feedback from our original software Deuteros, we present Deuteros 2.0 which has been redesigned from the ground up to fulfill a greater role in the HDX-MS analysis pipeline. Deuteros 2.0 features a repertoire of facilities for back exchange correction, data summarization, peptide-level statistical analysis and advanced data plotting features. AVAILABILITY AND IMPLEMENTATION: Deuteros 2.0 can be downloaded for both Windows and MacOS from https://github.com/andymlau/Deuteros_2.0 under the Apache 2.0 license.

Cold Denaturation of Proteins in the Absence of Solvent: Implications for Protein Storage.

The effect of temperature on the stability of proteins is well explored above 298 K, but harder to track experimentally below 273 K. Variable-temperature ion mobility mass spectrometry (VT IM-MS) allows us to measure the structure of molecules at sub-ambient temperatures. Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies (mAbs) on cooling by measuring their collision cross sections (CCS) at discrete drift gas temperatures from 295 to 160 K. The CCS at 250 K is larger than predicted from collisional theory and experimental data at 295 K. This restructure is attributed to change in the strength of stabilizing intermolecular interactions. Below 250 K the CCS of the mAbs increases in line with prediction implying no rearrangement. Comparing data from isotypes suggest disulfide bridging influences thermal structural rearrangement. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the native fold and VT IM-MS measurements at sub ambient temperatures provide new insights to the phenomenon of cold denaturation.

Structure of the CRISPR interference complex CSM reveals key similarities with cascade.

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I-III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family.

Specific cardiolipin-SecY interactions are required for proton-motive force stimulation of protein secretion.

The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of preproteins across the plasma membrane, powered by ATP hydrolysis and the transmembrane proton-motive force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly cardiolipin (CL), a specialized phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific CL binding sites on the Thermotoga maritima SecA-SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry, biochemical analysis, and fluorescence spectroscopy of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional CL binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for CL in the conferral of PMF stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery, and thereby stimulate protein transport, by a hitherto unexplored mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, toward investigation of both the nature and functional implications of protein-lipid interactions.

Hybrid Mass Spectrometry: Towards Characterization of Protein Conformational States.

A current challenge in structural biology is to unravel the conformational states of protein complexes. Hybrid mass spectrometry (MS) has emerged as a key tool to study the structural dynamics of large protein complexes unattainable by traditional methods. Here, we discuss recent advances in hybrid MS allowing characterization of challenging biological systems.

An Unusually Rapid Protein Backbone Modification Stabilizes the Essential Bacterial Enzyme MurA.

Proteins are subject to spontaneous rearrangements of their backbones. Most prominently, asparagine and aspartate residues isomerize to their ß-linked isomer, isoaspartate (isoAsp), on time scales ranging from days to centuries. Such modifications are typically considered "molecular wear-and-tear", destroying protein function. However, the observation that some proteins, including the essential bacterial enzyme MurA, harbor stoichiometric amounts of isoAsp suggests that this modification can confer advantageous properties. Here, we demonstrate that nature exploits an isoAsp residue within a hairpin to stabilize MurA. We found that isoAsp formation in MurA is unusually rapid and critically dependent on folding status. Moreover, perturbation of the isoAsp-containing hairpin via site-directed mutagenesis causes aggregation of MurA variants. Structural mass spectrometry revealed that this effect is caused by local protein unfolding in MurA mutants. Our findings demonstrate that MurA evolved to "mature" via a spontaneous post-translational incorporation of a ß-amino acid, which raises the possibility that isoAsp-containing hairpins may serve as a structural motif of biological importance.

Software Requirements for the Analysis and Interpretation of Native Ion Mobility Mass Spectrometry Data.

The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.

Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology.

Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase noncovalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods have improved, there have been accompanying developments in computational approaches for analyzing and exploiting the profusion of MS data in a structural and biophysical context. In this perspective, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed. Our perspective is based on discussions within the European Cooperation in Science and Technology Action on Native Mass Spectrometry and Related Methods for Structural Biology (EU COST Action BM1403), which involved participants from across Europe and North America. It is intended not as an in-depth review but instead to provide an accessible introduction to and overview of the topic-to inform newcomers to the field and stimulate discussions in the community about addressing existing challenges. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05792) focuses on software tools available to help researchers tackle some of the challenges enumerated here.

Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry.

SUMMARY: Hydrogen deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. Currently, analysis of HDX-MS data remains a laborious procedure, mainly due to the lack of streamlined software to process the large datasets. We present Deuteros which is a standalone software designed to be coupled with Waters DynamX HDX data analysis software, allowing the rapid analysis and visualization of data from differential HDX-MS. AVAILABILITY AND IMPLEMENTATION: Deuteros is open-source and can be downloaded from https://github.com/andymlau/Deuteros, under the Apache 2.0 license. Written in MATLAB and supported on both Windows and MacOS. Requires the MATLAB runtime library. According to the Wellcome Trust and UK research councils' Common Principles on Data Policy on data, software and materials management and sharing, all data supporting this study will be openly available from the software repository.

Structural predictions of the functions of membrane proteins from HDX-MS.

HDX-MS has emerged as a powerful tool to interrogate the structure and dynamics of proteins and their complexes. Recent advances in the methodology and instrumentation have enabled the application of HDX-MS to membrane proteins. Such targets are challenging to investigate with conventional strategies. Developing new tools are therefore pertinent for improving our fundamental knowledge of how membrane proteins function in the cell. Importantly, investigating this central class of biomolecules within their native lipid environment remains a challenge but also a key goal ahead. In this short review, we outline recent progresses in dissecting the conformational mechanisms of membrane proteins using HDX-MS. We further describe how the use of computational strategies can aid the interpretation of experimental data and enable visualisation of otherwise intractable membrane protein states. This unique integration of experiments with computations holds significant potential for future applications.

Protein-Lipid Interactions Stabilize the Oligomeric State of BOR1p from Saccharomyces cerevisiae.

The BOR proteins are integral membrane transporters which mediate efflux of boron. Structures of two BOR family members from Arabidopsis thaliana and Saccharomyces mikitiae indicate that the proteins exist as dimers. However, it remains unclear whether dimer formation is dependent on protein-lipid interactions or whether the dimer is the functional form of the protein. Here, we used the BOR1p protein from Saccharomyces cerevisiae (ScBOR1p), recombinantly expressed in its native host, to explore these aspects of BOR transporter structure and function. Native mass spectrometry (MS) revealed that ScBOR1p isolates as a monomer in a range of detergents. Lipidomics analysis showed that ScBOR1p co-isolates with phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Delipidation of ScBOR1p followed by addition of PS or PE causes formation of ScBOR1p dimers. Using a homology model of ScBOR1p, we identified a possible lipid binding site at the dimer interface comprising residues Arg265, Arg267, Arg480, and Arg481. A quadruple 4R/A mutant was expressed and isolated and also shown to be monomeric by native MS, and addition of PS or PE to this mutant did not reform the dimer. Functional complementation analysis revealed that the 4R/A mutant had boron efflux activity, suggesting that the ScBOR1p monomer is responsible for transport function. Taken together, these data strongly indicate that the physiological form of the ScBOR1p is the dimer and that dimer formation is dependent on association with membrane lipids.

Mechanistic insight into the assembly of the HerA-NurA helicase-nuclease DNA end resection complex.

The HerA-NurA helicase-nuclease complex cooperates with Mre11 and Rad50 to coordinate the repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA. By combining hybrid mass spectrometry with cryo-EM, computational and biochemical data, we investigate the oligomeric formation of HerA and detail the mechanism of nucleotide binding to the HerA-NurA complex from thermophilic archaea. We reveal that ATP-free HerA and HerA-DNA complexes predominantly exist in solution as a heptamer and act as a DNA loading intermediate. The binding of either NurA or ATP stabilizes the hexameric HerA, indicating that HerA-NurA is activated by substrates and complex assembly. To examine the role of ATP in DNA translocation and processing, we investigated how nucleotides interact with the HerA-NurA. We show that while the hexameric HerA binds six nucleotides in an 'all-or-none' fashion, HerA-NurA harbors a highly coordinated pairwise binding mechanism and enables the translocation and processing of double-stranded DNA. Using molecular dynamics simulations, we reveal novel inter-residue interactions between the external ATP and the internal DNA binding sites. Overall, here we propose a stepwise assembly mechanism detailing the synergistic activation of HerA-NurA by ATP, which allows efficient processing of double-stranded DNA.

A Mass-Spectrometry-Based Modelling Workflow for Accurate Prediction of IgG Antibody Conformations in the Gas Phase.

Immunoglobulins are biomolecules involved in defence against foreign substances. Flexibility is key to their functional properties in relation to antigen binding and receptor interactions. We have developed an integrative strategy combining ion mobility mass spectrometry (IM-MS) with molecular modelling to study the conformational dynamics of human IgG antibodies. Predictive models of all four human IgG subclasses were assembled and their dynamics sampled in the transition from extended to collapsed state during IM-MS. Our data imply that this collapse of IgG antibodies is related to their intrinsic structural features, including Fab arm flexibility, collapse towards the Fc region, and the length of their hinge regions. The workflow presented here provides an accurate structural representation in good agreement with the observed collision cross section for these flexible IgG molecules. These results have implications for studying other nonglobular flexible proteins.

Uncovering the Early Assembly Mechanism for Amyloidogenic ß2-Microglobulin Using Cross-linking and Native Mass Spectrometry.

ß2-Microglobulin (ß2m), a key component of the major histocompatibility class I complex, can aggregate into fibrils with severe clinical consequences. As such, investigating the structural aspects of the formation of oligomeric intermediates of ß2m and their subsequent progression toward fibrillar aggregates is of great importance. However, ß2m aggregates are challenging targets in structural biology, primarily due to their inherent transient and heterogeneous nature. Here we study the oligomeric distributions and structures of the early intermediates of amyloidogenic ß2m and its truncated variant ΔN6-ß2m. We established compact oligomers for both variants by integrating advanced mass spectrometric techniques with available electron microscopy maps and atomic level structures from NMR spectroscopy and x-ray crystallography. Our results revealed a stepwise assembly mechanism by monomer addition and domain swapping for the oligomeric species of ΔN6-ß2m. The observed structural similarity and common oligomerization pathway between the two variants is likely to enable ΔN6-ß2m to cross-seed ß2m fibrillation and allow the formation of mixed fibrils. We further determined the key subunit interactions in ΔN6-ß2m tetramer, revealing the importance of a domain-swapped hinge region for formation of higher order oligomers. Overall, we deliver new mechanistic insights into ß2m aggregation, paving the way for future studies on the mechanisms and cause of amyloid fibrillation.

Surface Accessibility and Dynamics of Macromolecular Assemblies Probed by Covalent Labeling Mass Spectrometry and Integrative Modeling.

Mass spectrometry (MS) has become an indispensable tool for investigating the architectures and dynamics of macromolecular assemblies. Here we show that covalent labeling of solvent accessible residues followed by their MS-based identification yields modeling restraints that allow mapping the location and orientation of subunits within protein assemblies. Together with complementary restraints derived from cross-linking and native MS, we built native-like models of four heterocomplexes with known subunit structures and compared them with available X-ray crystal structures. The results demonstrated that covalent labeling followed by MS markedly increased the predictive power of the integrative modeling strategy enabling more accurate protein assembly models. We applied this strategy to the F-type ATP synthase from spinach chloroplasts (cATPase) providing a structural basis for its function as a nanomotor. By subjecting the models generated by our restraint-based strategy to molecular dynamics (MD) simulations, we revealed the conformational states of the peripheral stalk and assigned flexible regions in the enzyme. Our strategy can readily incorporate complementary chemical labeling strategies and we anticipate that it will be applicable to many other systems providing new insights into the structure and function of protein complexes.

A mass spectrometry-based hybrid method for structural modeling of protein complexes.

We describe a method that integrates data derived from different mass spectrometry (MS)-based techniques with a modeling strategy for structural characterization of protein assemblies. We encoded structural data derived from native MS, bottom-up proteomics, ion mobility-MS and chemical cross-linking MS into modeling restraints to compute the most likely structure of a protein assembly. We used the method to generate near-native models for three known structures and characterized an assembly intermediate of the proteasomal base.