Protein crystallisation has gained a new strategic and commercial relevance in the post-genomic era because of its pivotal role in structural genomics. Producing high-quality crystals has always been a bottleneck to structure determination and, with the advent of proteomics, this problem is becoming increasingly acute. The task of producing suitable crystals may be tackled using two approaches. The first relies on empirical techniques that are based mainly on trial and error, and what is perceived to be the 'art' of crystallisation. The second approach is aimed at gaining an understanding of the fundamental principles that govern crystallisation; this knowledge may be applied to design experimental methodology for producing high-quality crystals of medical and industrial interest.Copyright 2004 Elsevier Ltd.

Cryo-electron microscopy, or simply cryo-EM, refers mainly to three very different yet closely related techniques: electron crystallography, single-particle cryo-EM, and electron cryotomography. In the past few years, single-particle cryo-EM in particular has triggered a revolution in structural biology and has become a newly dominant discipline. This Review examines the fascinating story of its start and evolution over the past 40-plus years, delves into how and why the recent technological advances have been so groundbreaking, and briefly considers where the technique may be headed in the future.Copyright © 2018, American Association for the Advancement of Science.

3D cryo-electron microscopy (cryo-EM) is an expanding structural biology technique that has recently undergone a quantum leap progression in its achievable resolution and its applicability to the study of challenging biological systems. Because crystallization is not required, only small amounts of sample are needed, and because images can be classified in a computer, the technique has the potential to deal with compositional and conformational mixtures. Therefore, cryo-EM can be used to investigate complete and fully functional macromolecular complexes in different functional states, providing a richness of biological insight. In this review, we underlie some of the principles behind the cryo-EM methodology of single particle analysis and discuss some recent results of its application to challenging systems of paramount biological importance. We place special emphasis on new methodological developments that are leading to an explosion of new studies, many of which are reaching resolutions that could only be dreamed of just a couple of years ago. Copyright © 2015 Elsevier Inc. All rights reserved.

A deeper understanding of how hydrogen/deuterium exchange mass spectrometry (HDX-MS) reveals allostery is important because HDX-MS can reveal allostery in systems that are not amenable to nuclear magnetic resonance (NMR) spectroscopy. We were able to study thrombin and its complex with thrombomodulin, an allosteric regulator, by both HDX-MS and NMR. In this Perspective, we compare and contrast the results from both experiments and from molecular dynamics simulations. NMR detects changes in the chemical environment around the protein backbone N-H bond vectors, providing residue-level information about the conformational exchange between distinct states. HDX-MS detects changes in amide proton solvent accessibility and H-bonding. Taking advantage of NMR relaxation dispersion measurements of the time scale of motions, we draw conclusions about the motions reflected in HDX-MS experiments. Both experiments detect allostery, but they reveal different components of the allosteric transition. The insights gained from integrating NMR and HDX-MS into thrombin dynamics enable a clearer interpretation of the evidence for allostery revealed by HDX-MS in larger protein complexes and assemblies that are not amenable to NMR.

The study of membrane protein structure and enzymology has traditionally been hampered by the inherent insolubility of membrane proteins in aqueous environments and experimental challenges in emulating an in vivo lipid environment. Phospholipid bilayer nanodiscs have recently been shown to be of great use for the study of membrane proteins since they offer a controllable, stable, and monodisperse model membrane with a nativelike lipid bilayer. Here we report the integration of nanodiscs with hydrogen exchange (HX) mass spectrometry (MS) experiments, thereby allowing for analysis of the native conformation of membrane proteins. gamma-Glutamyl carboxylase (GGCX), an approximately 94 kDa transmembrane protein, was inserted into nanodiscs and labeled with deuterium oxide under native conditions. Analytical parameters including sample-handling and chromatographic separation were optimized to measure the incorporation of deuterium into GGCX. Coupling nanodisc technology with HX MS offers an effective approach for investigating the conformation and dynamics of membrane proteins in their native environment and is therefore capable of providing much needed insight into the function of membrane proteins.

Programmed cell death-1 (PD-1), an antigen co-receptor on cell surfaces, is one of the conspicuous immune checkpoints. Nivolumab, a monoclonal antibody therapeutic approved by the FDA, binds to PD-1 and efficiently blocks its pathways. In this study, an integrated approach was developed to map the epitope/paratope of PD-1/nivolumab. The approach includes hydrogen-deuterium exchange mass spectrometry (HDX-MS) followed by electron-transfer dissociation (ETD), chemical cross-linking, and molecular docking. HDX-ETD offers some binding-site characterization with amino acid resolution. Chemical cross-linking provides complementary information on one additional epitope (i.e., the BC-loop) and a potential paratope at the N-terminus of the heavy chain. Furthermore, cross-linking identifies another loop region (i.e., the C'D-loop) that undergoes a remote conformational change. The distance restraints derived from the cross-links enable building high-confidence models of PD-1/nivolumab, evaluated with respect to a resolved crystal structure. This integrated strategy is an opportunity to characterize comprehensively other antigen-antibody interactions, to enable the understanding of binding mechanisms, and to design future antibody therapeutics.

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a key technique for monitoring structural and dynamic aspects of proteins in solution. This approach relies on the fact that exposure of a protein to D(2)O induces rapid amide H → D exchange in disordered regions that lack stable hydrogen-bonding. Tightly folded elements are much more protected from HDX, resulting in slow isotope exchange that is mediated by the structural dynamics ("breathing motions") of the protein. MS-based peptide mapping is a well established technique for measuring the mass shifts of individual protein segments. This tutorial review briefly discusses basic fundamentals of HDX/MS, before highlighting a number of recent developments and applications. Gas phase fragmentation strategies represent a promising alternative to the traditional proteolysis-based approach, but experimentalists have to be aware of scrambling phenomena that can be encountered under certain conditions. Electron-based dissociation methods provide a solution to this problem. We also discuss recent advances that facilitate the applicability of HDX/MS to membrane proteins, and to the characterization of short-lived protein folding intermediates. It is hoped that this review will provide a starting point for novices, as well as a useful reference for practitioners, who require an overview of some recent trends in HDX/MS.

Understanding as much as possible about proteins in the shortest amount of time has long been a goal of hydrogen exchange (HX) MS. Recent technological advances have led to improvements in the technique, but has this goal yet been achieved? (To listen to a podcast about this Feature, please go to the Analytical Chemistry Web site at pubs.acs.org/journal/ancham.).

Hydrogen-deuterium exchange-mass spectrometry (HDXMS) is a powerful technology to characterize conformations and conformational dynamics of proteins and protein complexes. HDXMS has been widely used in the field of therapeutics for the development of protein drugs. Although sufficient sequence coverage is critical to the success of HDXMS, it is sometimes difficult to achieve. In this study, we developed a HDXMS data analysis strategy that includes parallel post-translational modification (PTM) scanning in HDXMS analysis. Using a membrane-delimited G protein-coupled receptor (vasopressin type 2 receptor; VR) and a cytosolic protein (Na/H exchanger regulatory factor-1; NHERF1) as examples, we demonstrate that this strategy substantially improves protein sequence coverage, especially in key structural regions likely including PTMs themselves that play important roles in protein conformational dynamics and function.

With the development of biomedical technology, epitope mapping of proteins has become critical for developing and evaluating new protein drugs. The application of hydrogen-deuterium exchange for protein epitope mapping holds great potential. Although several reviews addressed the hydrogen-deuterium exchange, to date, only a few systematic reviews have focused on epitope mapping using this technology. Here, we introduce the basic principles, development history, and review research progress in hydrogen-deuterium exchange epitope mapping technology and discuss its advantages. We summarize the main hurdles in applying hydrogen-deuterium exchange epitope mapping technology, combined with relevant examples to provide specific solutions. We describe the epitope mapping of virus assemblies, disease-associated proteins, and polyclonal antibodies as examples of pattern introduction. Finally, we discuss the outlook of hydrogen-deuterium exchange epitope mapping technology. This review will help researchers studying protein epitopes to gain a more comprehensive understanding of this technology.

We demonstrate for different protein samples that 2D 1H-15N correlation NMR spectra can be recorded in a few seconds of acquisition time using a new band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence experiment. This has enabled us to measure fast hydrogen-deuterium exchange rate constants along the backbone of a small globular protein fragment by real-time 2D NMR.

The characterization of residual structures persistent in unfolded proteins is an important issue in studies of protein folding, because the residual structures present, if any, may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the residual structures of the isolated B domain (BDPA) of staphylococcal protein A in 6 M guanidinium chloride. BDPA is a small three-helix-bundle protein, and until recently its folding/unfolding reaction has been treated as a simple two-state process between the native and the fully unfolded states. We employed a dimethylsulfoxide (DMSO)-quenched hydrogen/deuterium (H/D)-exchange 2D NMR techniques with the use of spin desalting columns, which allowed us to investigate the H/D-exchange behavior of individually identified peptide amide (NH) protons. We obtained H/D-exchange protection factors of the 21 NH protons that form an α-helical hydrogen bond in the native structure, and the majority of these NH protons were significantly protected with a protection factor of 2.0-5.2 in 6 M guanidinium chloride, strongly suggesting that these weakly protected NH protons form much stronger hydrogen bonds under native folding conditions. The results can be used to deduce the structure of an early folding intermediate, when such an intermediate is shown by other methods. Among three native helical regions, the third helix in the C-terminal side was highly protected and stabilized by side-chain salt bridges, probably acting as the folding initiation site of BDPA. The present results are discussed in relation to previous experimental and computational findings on the folding mechanisms of BDPA. This article is protected by copyright. All rights reserved.© 2023 The Protein Society.

Natural protein sequences have evolved to fold efficiently to their functional forms, traversing a relatively smooth energy surface with minimal frustration. The optimization of stability in de novo-designed proteins, without regard to dynamical processes required for function, can have unintended consequences on energy surfaces. Electrostatic and hydrophobic networks of side chains for Di-III_14, a small βα protein with a natural topology, create a rough energy surface whose high-energy states reflect the progressive unlocking of a tightly packed interior. Unbiased explorations of sequence space can reveal complex properties not seen in natural proteins, expanding our understanding of the relationships among sequence, structure, folding, and function of these ubiquitous biopolymers.

Many protein structural investigations involve the use of H/D exchange (HDX) techniques. It is commonly thought that amide backbone protection arises from intramolecular H-bonding and/or burial of NH sites. Recently, fundamental HDX-related tenets have been called into question. The current work focuses on ubiquitin for exploring the defining features that distinguish amides in "open" (exchange-competent) and "closed" (exchange-incompetent) environments. Instead of relying on static X-ray structures, we employ all-atom molecular dynamics (MD) simulations for obtaining a dynamic view of the protein ground state and its surrounding solvent. The HDX properties for 57 out of 72 NH sites can be readily explained on the basis of backbone and side chain H-bonding, as well as solvent accessibility considerations. Unexpectedly, the same criteria fail for predicting the HDX characteristics of the remaining 15 amides. Significant protection is seen for numerous exposed NH sites that are not engaged in intramolecular H-bonds, whereas other amides that seemingly share the same features are unprotected. We scrutinize the proposal that H-bonding to crystallographically defined water can cause the protection of surface amides. For ubiquitin, the positioning of crystal water is not compatible with this idea. To further explore possible solvation effects, we tested for the presence of partially immobilized water networks. Our MD data reveal no difference in the solvation properties of protected vs unprotected surface amides, making it unlikely that restricted water dynamics can cause anomalous amide protection. The findings reported here suggest that efforts to deduce protein structural features on the basis of HDX protection factors may yield misleading results. This conclusion is relevant for initiatives that rely on sparse structural data as constraints for elucidating protein conformations. It may be necessary to pursue detailed quantum mechanical studies of the protein, the solvent, and the hydroxide catalyst for obtaining a comprehensive understanding of the factors that govern HDX rates. The considerable size of the systems involved makes such endeavors a daunting task.

Protein hydrogen/deuterium exchange (HDX) followed by protease digestion and mass spectrometric (MS) analysis is accepted as a standard method for studying protein conformation and conformational dynamics. In this article, an improved HDX MS platform with fully automated data processing is described. The platform significantly reduces systematic and random errors in the measurement by introducing two types of corrections in HDX data analysis. First, a mixture of short peptides with fast HDX rates is introduced as internal standards to adjust the variations in the extent of back exchange from run to run. Second, a designed unique peptide (PPPI) with slow intrinsic HDX rate is employed as another internal standard to reflect the possible differences in protein intrinsic HDX rates when protein conformations at different solution conditions are compared. HDX data processing is achieved with a comprehensive HDX model to simulate the deuterium labeling and back exchange process. The HDX model is implemented into the in-house developed software MassAnalyzer and enables fully unattended analysis of the entire protein HDX MS data set starting from ion detection and peptide identification to final processed HDX output, typically within 1 day. The final output of the automated data processing is a set (or the average) of the most possible protection factors for each backbone amide hydrogen. The utility of the HDX MS platform is demonstrated by exploring the conformational transition of a monoclonal antibody by increasing concentrations of guanidine.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is now a routinely used technique to inform on protein structure, dynamics, and interactions. Localizing the incorporated deuterium content on a single residue basis increases the spatial resolution of this technique enabling detailed structural analysis. Here, we investigate the use of ultraviolet photodissociation (UVPD) at 213 nm to measure deuterium levels at single residue resolution in HDX-MS experiments. Using a selectively labeled peptide, we show that UVPD occurs without H/D scrambling as the peptide probe accurately retains its solution-phase deuterium labeling pattern. Our results indicate that UVPD provides an attractive alternative to electron mediated dissociation for increasing the spatial resolution of the HDX-MS experiment, capable of yielding high fragmentation efficiency, high fragment ion diversity, and low precursor ion charge-state dependency.

Top-down hydrogen-deuterium exchange (HDX) analysis using electron capture or transfer dissociation Fourier transform mass spectrometry (FTMS) is a powerful method for the analysis of secondary structure of proteins in solution. The resolution of the method is a function of the degree of fragmentation of backbone bonds in the proteins. While fragmentation is usually extensive near the N- and C-termini, electron capture (ECD) or electron transfer dissociation (ETD) fragmentation methods sometimes lack good coverage of certain regions of the protein, most often in the middle of the sequence. Ultraviolet photodissociation (UVPD) is a recently developed fast-fragmentation technique, which provides extensive backbone fragmentation that can be complementary in sequence coverage to the aforementioned electron-based fragmentation techniques. Here, we explore the application of electrospray ionization (ESI)-UVPD FTMS on an Orbitrap Fusion Lumos Tribrid mass spectrometer to top-down HDX analysis of proteins. We have incorporated UVPD-specific fragment-ion types and fragment-ion mixtures into our isotopic envelope fitting software (HDX Match) for the top-down HDX analysis. We have shown that UVPD data is complementary to ETD, thus improving the overall resolution when used as a combined approach.

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 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.

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.

Epitope mapping of antibodies (Abs) is crucial for understanding adaptive immunity, as well as studying the mode of action of therapeutic antibodies and vaccines. Especially insights into the binding of the entire polyclonal antibody population (pAb) raised upon vaccination would be of unique value to vaccine development. However, very few methods for epitope mapping can tolerate the complexity of a pAb sample. Here we show how hydrogen-deuterium exchange mass spectrometry (HDX-MS) can be used to map epitopes recognized by pAb samples. Our approach involves measuring the HDX of the antigen in absence or presence of varied amounts of pAbs, as well as dissociating additives. We apply the HDX-MS workflow to pAbs isolated from rabbit immunized with factor H-binding protein (fHbp), a vaccine antigen. We identify four immunogenic regions located on the N- and C-terminal region of fHbp and provide insights into the relative abundance and avidity of epitope binding Abs present in the sample. Overall, our results show that HDX-MS can provide a unique and relatively fast method for revealing the binding impact of the entire set of pAbs present in blood samples after vaccination. Such information provides a rare view into effective immunity and can guide the design of improved vaccines against viruses or bacteria.

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformations, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron variant emergence.© 2023. The Author(s).

The susceptibility of the cellular prion protein (PrP(C)) to convert to an alternative misfolded conformation (PrP(Sc)), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23-231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to time-resolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.

Hydrogen/deuterium (H/D) exchange combined with two-dimensional (2D) NMR spectroscopy has been widely used for studying the structure, stability, and dynamics of proteins. When we apply the H/D-exchange method to investigate non-native states of proteins such as equilibrium and kinetic folding intermediates, H/D-exchange quenching techniques are indispensable, because the exchange reaction is usually too fast to follow by 2D NMR. In this article, we will describe the dimethylsulfoxide (DMSO)-quenched H/D-exchange method and its applications in protein science. In this method, the H/D-exchange buffer is replaced by an aprotic DMSO solution, which quenches the exchange reaction. We have improved the DMSO-quenched method by using spin desalting columns, which are used for medium exchange from the H/D-exchange buffer to the DMSO solution. This improvement has allowed us to monitor the H/D exchange of proteins at a high concentration of salts or denaturants. We describe methodological details of the improved DMSO-quenched method and present a case study using the improved method on the H/D-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride.

SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

Multiple crystal structures of CYP2B4 have demonstrated the binding of the detergent 5-cyclohexyl-1-pentyl-β-D-maltoside (CYMAL-5) in a peripheral pocket located adjacent to the active site. To explore the consequences of detergent binding, X-ray crystal structures of the peripheral pocket mutant CYP2B4 F202W were solved in the presence of hexaethylene glycol monooctyl ether (C8E6) and CYMAL-5. The structure in the presence of CYMAL-5 illustrated a closed conformation indistinguishable from the previously solved wild-type. In contrast, the F202W structure in the presence of C8E6 revealed a detergent molecule that coordinated the heme-iron and extended to the protein surface through the substrate access channel 2f. Despite the overall structural similarity of these detergent complexes, remarkable differences were observed in the A, A', and H helices, the F-G cassette, the C-D and β4 loop region. Hydrogen-deuterium exchange mass spectrometry (DXMS) was employed to probe these differences and to test the effect of detergents in solution. The presence of either detergent increased the H/D exchange rate across the plastic regions, and the results obtained by DXMS in solution were consistent in general with the relevant structural snapshots. The study provides insight into effect of detergent binding and the interpretation of associated conformational dynamics of CYP2B4.Copyright © 2016 Elsevier B.V. All rights reserved.

Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR.We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified.Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.© 2022. The Author(s).

Based on Ernst-angle-type excitation and Acceleration by Sharing Adjacent Polarization (ASAP), a fast HSQC-TOCSY experiment is introduced. In the approach, the DIPSI-2 isotropic mixing period of the ASAP-HSQC is simply shifted, which provides a TOCSY period without additional application of rf-energy. The ASAP-HSQC-TOCSY allows the acquisition of a conventional 2D in about 30 s. Alternatively, it allows the acquisition of highly carbon-resolved spectra (several Hz digital resolution) on the order of minutes. An ASAP-HSQC-TOCSY-IPAP variant, finally, allows the sign-sensitive extraction of heteronuclear long-range coupling constants from a pair of highly resolved spectra in less than an hour. Pulse sequences, several example spectra, and a discussion of results are given.Copyright © 2019 Elsevier Inc. All rights reserved.