Hydrogen–Deuterium Exchange Mass Spectrometry Reveals Unique Conformational and Chemical Transformations Occurring upon [4Fe-4S] Cluster Binding in the Type 2 l‑Serine Dehydratase from <i>Legionella pneumophila</i>

The type 2 l-serine dehydratase from <i>Legionella pneumophila</i> (<i>lp</i>LSD) contains a [4Fe-4S]²⁺ cluster that acts as a Lewis acid to extract the hydroxyl group of l-serine during the dehydration reaction. Surprisingly, the crystal structure shows that all four of the iron atoms in the cluster are coordinated with protein cysteinyl residues and that the cluster is buried and not exposed to solvent. If the crystal structure of <i>lp</i>LSD accurately reflects the structure in solution, then substantial rearrangement at the active site is necessary for the substrate to enter. Furthermore, repair of the oxidized protein when the cluster has degraded would presumably entail exposure of the buried cysteine ligands. Thus, the conformation required for the substrate to enter may be similar to those required for a new cluster to enter the active site. To address this, hydrogen–deuterium exchange combined with mass spectrometry (HDX MS) was used to probe the conformational changes that occur upon oxidative degradation of the Fe–S cluster. The regions that show the most significant differential HDX are adjacent to the cluster location in the holoenzyme or connect regions that are adjacent to the cluster. The observed decrease in flexibility upon cluster binding provides direct evidence that the “tail-in-mouth” conformation observed in the crystal structure also occurs in solution and that the C-terminal peptide is coordinated to the [4Fe-4S] cluster in a precatalytic conformation. This observation is consistent with the requirement of an activation step prior to catalysis and the unusually high level of resistance to oxygen-induced cluster degradation. Furthermore, peptide mapping of the apo form under nonreducing conditions revealed the formation of disulfide bonds between C396 and C485 and possibly between C343 and C385. These observations provide a picture of how the cluster loci are stabilized and poised to receive the cluster in the apo form and the requirement for a reduction step during cluster formation

Conformational-Sensitive Fast Photochemical Oxidation of Proteins and Mass Spectrometry Characterize Amyloid Beta 1–42 Aggregation

Preventing and treating Alzheimer’s disease require understanding the aggregation of amyloid beta 1–42 (Aβ_1–42) to give oligomers, protofibrils, and fibrils. Here we describe footprinting of Aβ_1–42 by hydroxyl radical-based fast photochemical oxidation of proteins (FPOP) and mass spectrometry (MS) to monitor the time-course of Aβ_1–42 aggregation. We resolved five distinct stages characterized by two sigmoidal behaviors, showing the time-dependent transitions of monomers-paranuclei-protofibrils-fibrillar aggregates. Kinetic modeling allows deciphering the amounts and interconversion of the dominant Aβ_1–42 species. Moreover, the irreversible footprinting probe provides insights into the kinetics of oligomerization and subsequent fibrillar growth by allowing the conformational changes of Aβ_1–42 at subregional and even amino-acid-residue levels to be revealed. The middle domain of Aβ_1–42 plays a major role in aggregation, whereas the N-terminus retains most of its solvent-accessibility during aggregation, and the hydrophobic C-terminus is involved to an intermediate extent. This approach affords an in situ, real-time monitoring of the solvent accessibility of Aβ_1–42 at various stages of oligomerization, and provides new insights on site-specific aggregation of Aβ_1–42 for a sample state beyond the capabilities of most other biophysical methods

Fast Photochemical Oxidation of Proteins and Mass Spectrometry Follow Submillisecond Protein Folding at the Amino-Acid Level

We report a study of submillisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laser-induced temperature jump (T jump) from ∼0 °C to ∼room temperature. Subsequent reactions via fast photochemical oxidation of proteins (FPOP) at various fractional millisecond points after the T jump lead to oxidative modification of solvent-accessible side chains whose “protection” changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix₁ shows a decreasing trend of oxidative modification during the first 0.1–1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these residues as folding occurs. We propose that in the early folding stage, barstar forms a partially solvent-accessible hydrophobic core consisting of several residues that have long-range interaction with other, more remote residues in the protein sequence. Our data not only are consistent with the previous conclusion that barstar fast folding follows the nucleation-condensation mechanism with the nucleus centered on helix₁ formed in a folding intermediate but also show the efficacy of this new approach to following protein folding on the submillisecond time range

Primary and Higher Order Structure of the Reaction Center from the Purple Phototrophic Bacterium Blastochloris viridis: A Test for Native Mass Spectrometry

The reaction center (RC) from the phototrophic bacterium Blastochloris viridis was the first integral membrane protein complex to have its structure determined by X-ray crystallography and has been studied extensively since then. It is composed of four protein subunits, H, M, L, and C, as well as cofactors, including bacteriopheophytin (BPh), bacteriochlorophyll (BCh), menaquinone, ubiquinone, heme, carotenoid, and Fe. In this study, we utilized mass spectrometry-based proteomics to study this protein complex via bottom-up sequencing, intact protein mass analysis, and native MS ligand-binding analysis. Its primary structure shows a series of mutations, including an unusual alteration and extension on the C-terminus of the M-subunit. In terms of quaternary structure, proteins such as this containing many cofactors serve to test the ability to introduce native-state protein assemblies into the gas phase because the cofactors will not be retained if the quaternary structure is seriously perturbed. Furthermore, this specific RC, under native MS, exhibits a strong ability not only to bind the special pair but also to preserve the two peripheral BCh’s

Hydrogen–Deuterium Exchange and Mass Spectrometry Reveal the pH-Dependent Conformational Changes of Diphtheria Toxin T Domain

The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen–deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our “standard condition” (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W⁺-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8–9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8–9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain

Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen−Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3

We describe a platform utilizing two methods based on hydrogen–deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein–ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 μM for the three different regions, giving an overall binding affinity of 7.4 μM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides <i>site-specific</i> binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, Δ<i>C</i>_1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein–ligand interactions

Continuous and Pulsed Hydrogen–Deuterium Exchange and Mass Spectrometry Characterize CsgE Oligomerization

We report the use of hydrogen–deuterium amide exchange coupled to mass spectrometry (HDX-MS) to study the interfaces of and conformational changes accompanying CsgE oligomerization. This protein plays an important role in enteric bacteria biofilm formation. Biofilms provide protection for enteric bacteria from environmental extremes and raise concerns about controlling bacteria and infectious disease. Their proteinaceous components, called curli, are extracellular functional amyloids that initiate surface contact and biofilm formation. The highly regulated curli biogenesis involves a major subunit, CsgA, a minor subunit CsgB, and a series of other accessory proteins. CsgE, possibly functioning as oligomer, is a chaperonin-like protein that delivers CsgA to an outer-membrane bound oligomeric CsgG complex. No higher-order structure, or interfaces and dynamics of its oligomerization, however, are known. In this work, we determined regions involved in CsgE self-association by continuous HDX, and, on the basis of that, prepared a double mutant W48A/F79A, derived from interface alanine scan, and verified that it exists as monomer. Using pulsed HDX and MS, we suggest there is a structural rearrangement occurring during the oligomerization of CsgE

Hydrogen–Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM)

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca²⁺-binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca²⁺ but detaches from DRE under Ca²⁺ stimulation, allowing gene expression. The Ca²⁺ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen–deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca²⁺ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca²⁺. Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca²⁺-binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca²⁺ signal to CREB-mediated gene transcription

Structural Analysis of Diheme Cytochrome <i>c</i> by Hydrogen–Deuterium Exchange Mass Spectrometry and Homology Modeling

A lack of X-ray or nuclear magnetic resonance structures of proteins inhibits their further study and characterization, motivating the development of new ways of analyzing structural information without crystal structures. The combination of hydrogen–deuterium exchange mass spectrometry (HDX-MS) data in conjunction with homology modeling can provide improved structure and mechanistic predictions. Here a unique diheme cytochrome <i>c</i> (DHCC) protein from <i>Heliobacterium modesticaldum</i> is studied with both HDX and homology modeling to bring some definition of the structure of the protein and its role. Specifically, HDX data were used to guide the homology modeling to yield a more functionally relevant structural model of DHCC