Hydrogen/deuterium Exchange Coupled With Mass Spectrometry Research Articles

Allosteric Regulation and Inhibition of Coronavirus 3CLpro Revealed by HDX-MS.

Coronavirus (CoV) infections have caused contagious and fatal respiratory diseases in humans worldwide. CoV 3-chymotrypsin-like proteases (3CLpro or Mpro) play an important role in viral maturation, and maintenance of their dimeric conformation is crucial for viral activity. Therefore, allosterically regulated dimerization of 3CLpro can be employed as a drug development target. Here, we investigated the allosteric regulatory mechanism of 3CLpro dimerization by using hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) technology. We found that the FLAG tag directly coupled to the N-finger of 3CLpro significantly increased HDX kinetics at the dimer interface, and 3CLpro transformed from a dimer to a monomer. The 3CLpro mutants of SARS-CoV-2, which are monomeric, also exhibited increased deuterium exchange. Binding of the allosteric inhibitor Gastrodenol to most betacoronavirus 3CLpros led to increased allosteric deuterium exchange, resulting in the monomeric conformation of the CoV 3CLpro upon binding. Molecular dynamics (MD) simulation analysis further indicated the molecular mechanism of action of Gastrodenol on CoV 3CLpro: binding of Gastrodenol to SARS-CoV-2 3CLpro destroyed the hydrogen bond in the dimer interface. These results suggest that Gastrodenol may be a potential broad-spectrum anti-betacoronavirus drug.

Hydrogen-Deuterium Exchange Epitope Mapping of Glycosylated Epitopes Enabled by Online Immobilized Glycosidase.

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is widely used for monoclonal antibody (mAb) epitope mapping, which aids in the development of therapeutic mAbs and vaccines, as well as enables the understanding of viral immune evasion. Numerous mAbs are known to recognize N-glycosylated epitopes and to bind in close proximity to an N-glycan site; however, glycosylated protein sites are typically obscured from HDX detection as a result of the inherent heterogeneity of glycans. To overcome this limitation, we covalently immobilized the glycosidase PNGase Dj on a solid resin and incorporated it into an online HDX-MS workflow for post-HDX deglycosylation. The resin-immobilized PNGase Dj exhibited robust tolerance to various buffer conditions and was employed in a column format that can be readily adapted into a typical HDX-MS platform. Using this system, we were able to obtain full sequence coverage of the SARS-CoV-2 receptor-binding domain (RBD) and map the glycosylated epitope of the glycan-binding mAb S309 to the RBD.

The Conformational Changes of Bovine Serum Albumin at the Air/Water Interface: HDX-MS and Interfacial Rheology Analysis.

The characterization and dynamics of protein structures upon adsorption at the air/water interface are important for understanding the mechanism of the foamability of proteins. Hydrogen-deuterium exchange, coupled with mass spectrometry (HDX-MS), is an advantageous technique for providing conformational information for proteins. In this work, an air/water interface, HDX-MS, for the adsorbed proteins at the interface was developed. The model protein bovine serum albumin (BSA) was deuterium-labeled at the air/water interface in situ for different predetermined times (10 min and 4 h), and then the resulting mass shifts were analyzed by MS. The results indicated that peptides 54-63, 227-236, and 355-366 of BSA might be involved in the adsorption to the air/water interface. Moreover, the residues L55, H63, R232, A233, L234, K235, A236, R359, and V366 of these peptides might interact with the air/water interface through hydrophobic and electrostatic interactions. Meanwhile, the results showed that conformational changes of peptides 54-63, 227-236, and 355-366 could lead to structural changes in their surrounding peptides, 204-208 and 349-354, which could cause the reduction of the content of helical structures in the rearrangement process of interfacial proteins. Therefore, our air/water interface HDX-MS method could provide new and meaningful insights into the spatial conformational changes of proteins at the air/water interface, which could help us to further understand the mechanism of protein foaming properties.

Elucidating Protein-Ligand Interactions in Cell Lysates Using High-Throughput Hydrogen-Deuterium Exchange Mass Spectrometry with Integrated Protein Thermal Depletion.

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein-ligand interactions. Currently, there is a growing need for breakthroughs in the application of HDX-MS analysis to protein-ligand interactions in highly complex biological samples such as cell lysates. However, HDX-MS analysis in such systems suffers from extreme spectral complexity as a result of high sample complexity and limited LC separation power due to the traditional use of short LC gradients. Here, we introduced protein thermal depletion (PTD) to reduce protein complexity in E. coli cell lysate for our subzero-temperature long gradient UPLC-HDX-MS platform (PTD-HDX-MS) to facilitate high-throughput analysis of protein-ligand interactions in cell lysates. We spiked bovine carbonic anhydrase II (CaII) and its inhibitor acetazolamide (AZM) into E. coli cell lysate as a model system in our study. We demonstrated that PTD at 60 °C greatly reduces protein complexity in cell lysates, while the AZM-targeted CaII complex remains in solution due to improved thermal stability upon binding. Using both PTD to reduce sample complexity and subzero-temperature long gradient UPLC to boost LC separation power, we successfully elucidated the interaction sites between AZM and CaII in E. coli cell lysate from the high-throughput HDX-MS analysis of thousands of deuterated peptides from hundreds of proteins. Our results highlight the great promise of the PTD-HDX-MS platform for the identification of ligand targets and characterization of protein-ligand interactions in highly complex biological samples such as cell lysates.

Modeling native state ensembles and macromolecular complexes using hydrogen-deuterium exchange ensemble reweighting

Gaining a high-resolution understanding of the conformational landscape of proteins and protein complexes in solution remains a challenge. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is an insightful approach in probing a protein's structural dynamics in solution. It reports on the structural environment of the backbone amide hydrogens of a protein in a time resolved manner. As such, HDX-MS has now been widely applied to study proteins within various biophysical perspectives including the characterization of native state ensembles of proteins, macromolecular interactions, protein-small molecule interactions or protein folding and unfolding studies.

Dynamics of protein kinases and pseudokinases by HDX-MS.

Dynamics of the protein kinase fold are deeply intertwined with its structure. The past three decades of kinase biophysical studies revealed key dynamic features of the kinase domain and, more recently, how these features may endow catalytically impaired kinases-or pseudokinases-with signaling properties. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is proving to be a valuable approach for studies of kinase and pseudokinase domain dynamics. Here, we briefly discuss the methods that have provided insights into protein kinase dynamics, describe how HDX-MS is being used to answer questions in the kinase/pseudokinase field, and provide a detailed protocol for collecting an HDX-MS dataset to study the impacts of small molecule binding to a pseudokinase domain. As more small molecules are discovered that can disrupt pseudokinase conformations, HDX-MS is likely to be a powerful approach for exploring drug-induced changes in pseudokinase dynamics and structure.

Unraveling cardiolipin-induced conformational change of cytochrome c through H/D exchange mass spectrometry and quartz crystal microbalance

Cardiolipin (CL), a crucial component in inner mitochondrial membranes, interacts with cytochrome c (cyt c) to form a peroxidase complex for the catalysis of CL oxidation. Such interaction is pivotal to the mitochondrial regulation of apoptosis and is affected by the redox state of cyt c. In the present study, the redox-dependent interaction of cyt c with CL was investigated through amide hydrogen/deuterium exchange coupled with mass spectrometry (HDXMS) and quartz crystal microbalance with dissipation monitoring (QCM-D). Ferrous cyt c exhibited a more compact conformation compared with its ferric form, which was supported by the lower number of deuterons accumulated and the greater amplitude reduction on dissipation. Upon association with CL, ferrous cyt c resulted in a moderate increase in deuteration, whereas the ferric form caused a drastic increase of deuteration, which indicated that CL-bound ferric cyt c formed an extended conformation. These results were consistent with those of the frequency (f) − dissipation (D) experiments, which revealed that ferric cyt c yielded greater values of |ΔD/Δf| within the first minute. Further fragmentation analysis based on HDXMS indicated that the effect of CL binding was considerably different on ferric and ferrous cyt c in the C-helix and the Loop 9–24. In ferric cyt c, CL binding affected Met80 and destabilized His18 interaction with heme, which was not observed with ferrous cyt c. An interaction model was proposed to explain the aforementioned results.

Interaction interface in the C-terminal parts of centriole proteins Sas6 and Ana2.

The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2's loading to the site of procentriole formation. Four conserved serines in Ana2's STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled–coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6 in vitro and in vivo. Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2–Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously.

Comparative Analysis of Cleavage Specificities of Immobilized Porcine Pepsin and Nepenthesin II under Hydrogen/Deuterium Exchange Conditions.

Hydrogen/Deuterium Exchange (HDX) coupled with Mass Spectrometry (HDX-MS) is a sensitive and robust method to probe protein conformational changes and protein-ligand interactions. HDX-MS relies on successful proteolytic digestion of target proteins under acidic conditions to localize perturbations in exchange behavior to protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. While the acid protease pepsin has been the enzyme of choice for HDX-MS studies, recently, it was shown that aspartic proteases from carnivorous pitcher plants of the genus Nepenthes are active under low-pH conditions and cleave at basic residues that are "forbidden" in peptic digests. In this report, we describe the utility of one of these enzymes, Nepenthesin II (NepII), in a HDX-MS workflow. A systematic and statistical analysis of data from 11 proteins (6391 amino acid residues) digested with immobilized porcine pepsin or NepII under conditions compatible with HDX-MS was performed to examine protease cleavage specificities. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe, Leu, and Met are favored residues, each with a cleavage probability of greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. In contrast, NepII offers advantageous cleavage to all basic residues and produces shortened peptides that could improve the spatial resolution in HDX-MS studies.

Discovery of Nuclear Receptor Ligands and Elucidation of Their Mechanisms of Action.

To develop potent ligands for the vitamin D receptor (VDR), we designed and synthesized a series of vitamin D analogues with and without 22-alkyl substituents. These analogues exhibited agonistic, partial agonistic, or antagonistic activity. To elucidate the mechanism of action of the analogues, we conducted crystal structure analyses of the ligand-binding domain (LBD) of VDR complexed with the analogues. The VDR-LBD/agonist complex exhibited precise interactions, which clearly explained VDR agonism. The VDR-LBD/partial agonist complex showed two conformers (agonist and antagonist binding conformers) in a single crystal, demonstrating that partial agonism could be explained by the sum of the agonistic and antagonistic activities. Antagonist binding to the VDR-LBD structure was elucidated using both crystal structure analysis and in-solution structural analyses with the small-angle X-ray scattering (SAXS)-molecular dynamics (MD) and hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) methods. Several antagonist-binding structures were detected. We found that the antagonist binding structures differed depending on the structure of the antagonist itself, and those structures clearly explained the VDR antagonism. Furthermore, the apo VDR-LBD structure without the ligand in the ligand-binding pocket was revealed and found to have an entrance to accommodate the ligand. Thus we elucidated the mechanisms of action of agonists, partial agonists, and antagonists based on structural changes (differences) in the receptor protein induced by ligand binding.

Molecular regulation of lysine trimethylation by the P. aeruginosa methyltransferase EftM

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of severe life‐threatening lung and other infections in individuals with cystic fibrosis, compromised immune systems or severe burns. P. aeruginosa is also intrinsically resistant to many antibiotics making infections difficult to treat once established. We previously showed that the S‐adenosyl‐L‐methionine‐dependent methyltransferase EftM is responsible for trimethylation of lysine 5 of the translation factor EF‐Tu and that this modification enhances P. aeruginosa adhesion to host epithelial cells. Additionally, methyltransferase activity, and thus EF‐Tu modification, is temperature regulated via the intrinsic thermolability of EftM. Understanding EftM's complete mechanism of action may reveal new strategies for developing effective therapeutics targeting this pathogen. As the next step in this process, we now aim to define the substrate recognition and catalytic mechanisms of EftM. A phylogenetic reconstruction of the larger EftM family was generated to identify conservation patterns of putative key residues involved in interaction with EF‐Tu and facilitating catalysis. This analysis was supported by generation of an EftM homology model and protein‐protein docking studies to propose a EftM:EF‐Tu binding model. This model and phylogentic insights were used to guide site‐directed mutagenesis of residues in EftM and EF‐Tu to test their role in protein‐protein recognition using methyltransferase activity and binding assays. Finally, to begin investigating the role of protein dynamics and potential allosteric effects of SAM cosubstrate binding, other computational methods such as normal mode analysis were used to propose a coarse‐grain model of dynamics of EftM in parallel with experimental hydrogen‐deuterium exchange coupled with mass spectrometry (HDX‐MS) analysis of EftM and its complexes with SAM and EF‐Tu. In conclusion, our results provide the first structural insight into the EftM:EF‐Tu recognition, which paves the way for further understanding EftM's substrate recognition and catalytic mechanisms.Support or Funding InformationCystic Fibrosis Foundation Postdoctoral Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Binding Interface of Gapdh to the Au Rich Elements from TNF-Alpha mRNA Revealed by Hydrogen Deuterium Exchange Coupled with Mass Spectrometry

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous protein primarily known for its glycolytic function. More recent studies have revealed GAPDH as a multifaceted, multifunctional protein involved in various cellular processes including iron homeostasis, heme transport, apoptosis, tumerogenesis, oxidative stress response, chromatin structure among many others. As part of its multifunctional purpose, GAPDH has been shown to bind to various cellular RNAs and has been classified as a non-canonical RNA binding protein. In particular, it has been shown to bind AU-rich element of mRNA transcripts and consequently affecting translation. However, the detailed structural characterization of the RNA bound complex has remained elusive. The RNA binding site remains unknown thereby restricting potential therapeutic strategies. Our previous studies established that GAPDH binds the AU rich element of TNF-α mRNA transcript via a two-step model. In addition, we have shown that a T229K mutant of GAPDH displayed altered RNA and NAD+ binding properties, potentially signaling to a linkage between RNA binding and the cofactor. Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) revealed that the T229K mutant induced conformational changes into distal grooves along the P-axis of the tetrameric protein. In this study, we have mapped the binding interfaces of three GAPDH constructs displaying varying NAD+ binding properties with the AU rich element from TNF-α mRNA using HDX-MS. A largely common pattern of localized protection from exchange pointed to the grooves along the P-axis as the RNA binding interfaces, as hypothesized. Additionally, some variations in HDX protection point to potential conformational basis for the linkage with NAD+ binding.

Histone H3 binding to the PHD1 domain of histone demethylase KDM5A enables active site remodeling

Histone demethylase KDM5A removes methyl marks from lysine 4 of histone H3 and is often overexpressed in cancer. The in vitro demethylase activity of KDM5A is allosterically enhanced by binding of its product, unmodified H3 peptides, to its PHD1 reader domain. However, the molecular basis of this allosteric enhancement is unclear. Here we show that saturation of the PHD1 domain by the H3 N-terminal tail peptides stabilizes binding of the substrate to the catalytic domain and improves the catalytic efficiency of demethylation. When present in saturating concentrations, differently modified H3 N-terminal tail peptides have a similar effect on demethylation. However, they vary greatly in their affinity towards the PHD1 domain, suggesting that H3 modifications can tune KDM5A activity. Furthermore, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) experiments reveal conformational changes in the allosterically enhanced state. Our findings may enable future development of anti-cancer therapies targeting regions involved in allosteric regulation.

Differential Scanning Fluorimetry and Hydrogen Deuterium Exchange Mass Spectrometry to Monitor the Conformational Dynamics of NBD1 in Cystic Fibrosis.

Cystic fibrosis (CF) is one of the most common, lethal autosomal recessive diseases in Caucasians with a life expectancy of 37-47years. The CF transmembrane conductance regulator (CFTR) is a plasma membrane ion channel, confined to apical membrane of epithelia, and ensures transepithelial water and solute movement across secretory epithelia in several organs. Numerous CF mutations, including the most prevalent deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) leads to CFTR global misfolding and premature intracellular degradation at the endoplasmic reticulum (ER). To better understand the misfolding mechanism caused by CF-causing point mutations in the NBD1, which is poorly understood, differential scanning fluorimetry (DSF) and hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) are the choice of techniques. These established methods can measure the conformational dynamics of the NBD1 globally and at peptide resolution level by monitoring backbone amide HDX, respectively, and will be instrumental to evaluate the mechanism of action of CF mutations and folding correctors that rescue CFTR folding defects via stabilizing the mutant NBD1.

Disease‐associated mutation in PTH reveals molecular mechanisms in endosomal GPCR signaling

Parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only PTH sustains GS–mediated cAMP production after PTHR internalization into early endosomes. The physiological relevance of this unexpected behavior for PTHR is unclear. Here we present evidence that endosomal PTHR signaling through cAMP also has disease relevance. We demonstrated that R25C mutation recently identified in PTH as a new cause of hypocalcemia in humans affects receptor binding at endosomal pH resulting in rapid ligand dissociation and recycling of PTHR preventing sustained endosomal cAMP signaling. Using hydrogen–deuterium exchange coupled with mass spectrometry (HDXMS) to map structural changes of PTHR upon PTH binding, we identified acidic clusters in the receptor's first extracellular loop as key determinants for Arg25 binding and endosomal cAMP signaling. Determination of the dynamic of protein‐protein interaction using FRET approaches revealed that PTH R25C has a lower potency to form and stabilize the functional complex between β‐Arrestin, Gβγ and PTHR required for endosomal cAMP generation. Overall, these new findings indicate that PTH‐induced endosomal cAMP signaling is likely a primary signaling component regulating calcium homeostasis.Support or Funding InformationNIH R01‐DK102495 and R01‐DK087688 National Research Foundation of Korea NRF‐2013R1A1A1A05005629This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Cooperative Folding Unit as the Structural Link for Energetic Coupling within a Protein.

Previously, we demonstrated that binding of a ligand to Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), a homodimeric protein, is energetically coupled with dimerization. The equilibrium unfolding of dPGM occurs with a stable, monomeric intermediate. Binding of several nonsubstrate metabolites stabilizes the dimeric native form over the monomeric intermediate, reducing the population of the intermediate. Both the active site and the dimer interface appear to be unfolded in the intermediate. We hypothesized that a loop containing residues 118-152 was responsible for the energetic coupling between the dimer interface and the distal active site and was unfolded in the intermediate. Here, we investigated the structure of the dPGM intermediate by probing side-chain interactions and solvent accessibility of the peptide backbone. By comparing the effect of a mutation on the global stability and the stability of the intermediate, we determine an equilibrium φ value (φeq value), which provides information about whether side-chain interactions are retained or lost in the intermediate. Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to investigate differences in the solvent accessibility of the peptide backbone in the intermediate and native forms of dPGM. The results of φeq value analysis and HDX-MS reveal the least stable folding unit of dPGM, which is unfolded in the intermediate and links the active site to the dimer interface. The structure of the intermediate reveals how the cooperative network of residues in dPGM gives rise to the observed energetic coupling between dimerization and ligand binding.

Computational method allowing Hydrogen-Deuterium Exchange Mass Spectrometry at single amide Resolution

Hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (HDXMS) is a rapid and effective method for localizing and determining protein stability and dynamics. Localization is routinely limited to a peptide resolution of 5 to 20 amino acid residues. HDXMS data can contain information beyond that needed for defining protein stability at single amide resolution. Here we present a method for extracting this information from an HDX dataset to generate a HDXMS protein stability fingerprint. High resolution (HR)-HDXMS was applied to the analysis of a model protein of a spectrin tandem repeat that exemplified an intuitive stability profile based on the linkage of two triple helical repeats connected by a helical linker. The fingerprint recapitulated expected stability maximums and minimums with interesting structural features that corroborate proposed mechanisms of spectrin flexibility and elasticity. HR-HDXMS provides the unprecedented ability to accurately assess protein stability at the resolution of a single amino acid. The determination of HDX stability fingerprints may be broadly applicable in many applications for understanding protein structure and function as well as protein ligand interactions.

Post-translational modifications in PrP expand the conformational diversity of prions in vivo

Misfolded prion protein aggregates (PrPSc) show remarkable structural diversity and are associated with highly variable disease phenotypes. Similarly, other proteins, including amyloid-β, tau, α-synuclein, and serum amyloid A, misfold into distinct conformers linked to different clinical diseases through poorly understood mechanisms. Here we use mice expressing glycophosphatidylinositol (GPI)-anchorless prion protein, PrPC, together with hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and a battery of biochemical and biophysical tools to investigate how post-translational modifications impact the aggregated prion protein properties and disease phenotype. Four GPI-anchorless prion strains caused a nearly identical clinical and pathological disease phenotype, yet maintained their structural diversity in the anchorless state. HXMS studies revealed that GPI-anchorless PrPSc is characterized by substantially higher protection against hydrogen/deuterium exchange in the C-terminal region near the N-glycan sites, suggesting this region had become more ordered in the anchorless state. For one strain, passage of GPI-anchorless prions into wild type mice led to the emergence of a novel strain with a unique biochemical and phenotypic signature. For the new strain, histidine hydrogen-deuterium mass spectrometry revealed altered packing arrangements of β-sheets that encompass residues 139 and 186 of PrPSc. These findings show how variation in post-translational modifications may explain the emergence of new protein conformations in vivo and also provide a basis for understanding how the misfolded protein structure impacts the disease.

New insights into interactions between the nucleotide-binding domain of CFTR and keratin 8.

The intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding domain 1 (NBD1) of the cystic fibrosis (CF) transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508-CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for CF patients bearing the ΔF508 mutation. Here, we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on recombinant wild-type (wt) NBD1 and ΔF508-NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt-NBD1 and ΔF508-NBD1. Finally, we performed HDX-MS analysis of the NBD1 molecules and full-length K8, revealing hydrogen-bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508-NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1.

Analysis of Translocation-Competent Secretory Proteins by HDX-MS.

Protein folding is an intricate and precise process in living cells. Most exported proteins evade cytoplasmic folding, become targeted to the membrane, and then trafficked into/across membranes. Their targeting and translocation-competent states are nonnatively folded. However, once they reach the appropriate cellular compartment, they can fold to their native states. The nonnative states of preproteins remain structurally poorly characterized since increased disorder, protein sizes, aggregation propensity, and the observation timescale are often limiting factors for typical structural approaches such as X-ray crystallography and NMR. Here, we present an alternative approach for the in vitro analysis of nonfolded translocation-competent protein states and their comparison with their native states. We make use of hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS), a method based on differentiated isotope exchange rates in structured vs unstructured protein states/regions, and highly dynamic vs more rigid regions. We present a complete structural characterization pipeline, starting from the preparation of the polypeptides to data analysis and interpretation. Proteolysis and mass spectrometric conditions for the analysis of the labeled proteins are discussed, followed by the analysis and interpretation of HDX-MS data. We highlight the suitability of HDX-MS for identifying short structured regions within otherwise highly flexible protein states, as illustrated by an exported protein example, experimentally tested in our lab. Finally, we discuss statistical analysis in comparative HDX-MS. The protocol is applicable to any protein and protein size, exhibiting slow or fast loss of translocation competence. It could be easily adapted to more complex assemblies, such as the interaction of chaperones with nonnative protein states.