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Time-resolved electrospray ionization mass spectrometry and stopped-flow absorption spectroscopy were used to explain the mechanism of the acid-induced denaturation of holo-myoglobin (hMb). Results identify two lifetimes. The shorter lifetime indicates the formation of a transient intermediate which is characterized as the unfolded variant of hMb with an intact heme-protein complex. The longer lifetime, on the other hand, is associated with the formation of a product that disrupts the native heme-protein interactions and exhibits a high degree of folding.

Time-resolved electrospray ionization mass spectrometry was utilized to investigate the acid-denaturated cytochrome c refolding kinetics. It was demonstrated that the kinetics of the refolding from state II to state III through combined electrospray ionization (ESI) and mass spectrometry (MS) with continuous flow mixing technique. The information obtained through this technique proved to be complementary to the two-dimensional nuclear magnetic resonance studies.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) probes dynamic motions of proteins by monitoring the kinetics of backbone amide deuteration. Dynamic regions exhibit rapid HDX, while rigid segments are more protected. Current data readouts focus on qualitative comparative observations (such as "residues X to Y become more protected after protein exposure to ligand Z"). At present, it is not possible to decode HDX protection patterns in an atomistic fashion. In other words, the exact range of protein motions under a given set of conditions cannot be uncovered, leaving space for speculative interpretations. Amide back exchange is an under-appreciated problem, as the widely used (m-m 0 )/(m 100 -m 0 ) correction method can distort HDX kinetic profiles. Future data analysis strategies require a better fundamental understanding of HDX events, going beyond the classical Linderstrøm-Lang model. Combined with experiments that offer enhanced spatial resolution and suppressed back exchange, it should become possible to uncover the exact range of motions exhibited by a protein under a given set of conditions. Such advances would provide a greatly improved understanding of protein behavior in health and disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Electrospray ionization (ESI) of serine (Ser) solution generates Ser 8 H + as an abundant magic number cluster. ESI clustering of most other solutes yields nonspecific stoichiometries. It is unclear why Ser 8 H + dominates in the case of Ser, and how Ser 8 H + forms during ESI. Even the location of Ser 8 H + formation is contentious (in solution, in ESI droplets, or elsewhere). Here we unravel key aspects of the l-Ser 8 H + formation pathway. Harsh ion sampling conditions promote the collision-induced dissociation (CID) of regular ESI analytes. Unexpectedly, Ser 8 H + was seemingly resistant against CID during ion sampling, despite its extremely low tandem mass spectrometry (MS/MS) stability. This unusual behavior reveals that Ser 8 H + forms during ion sampling. We propose the following pathway: (1) Nonspecific Ser clusters are released when ESI droplets evaporate to dryness. These initial clusters cover a wide size range, from a few Ser to hundreds or thousands of monomers. (2) The clusters undergo dissociation during ion sampling, mostly via successive loss of neutral monomers. For any source activation voltage, there is a subpopulation of clusters for which this CID cascade tends to terminate at the octamer level, culminating in Ser 8 H + -dominated product distributions. Mobile proton molecular dynamics simulations were used to model the entire pathway. Ser 8 H + structures formed in these simulations were consistent with ion mobility experiments. The most compact structures resembled the model of [Scutelnic, V. J. Am. Chem. Soc. 2018, 140, 7554-7560], with numerous intermolecular salt bridges and H-bonds. Our findings illustrate how the interplay of association and dissociation reactions across phase boundaries can culminate in magic number clusters.

Electrospray ionization (ESI) mass spectrometry is widely used for interrogating peptides, proteins, and other biomolecular analytes. A growing number of laboratories use molecular dynamics (MD) simulations for uncovering ESI mechanisms by modeling the behavior of highly charged nanodroplets. The outcome of any MD simulation depends on certain assumptions and parameter settings, and it is desirable to optimize these factors by benchmarking computational data against experiments. Unfortunately, benchmarking of ESI simulations is difficult because experimentally generated gaseous ions do not generally retain any features that would reveal their formation pathway [e.g., the charged residue mechanism (CRM) or the ion evaporation mechanism (IEM)]. Here, we tackle this problem by examining the effects of various MD settings on the ESI behavior of the 9-residue peptide bradykinin in acidic aqueous droplets. Several parameters were found to significantly affect the kinetic competition between peptide IEM and CRM. By systematically probing the droplet behavior, we uncovered problems associated with certain settings, including peptide/solvent temperature imbalances, unexpected peptide deceleration during IEM, and a dependence of the ESI mechanism on the water model. We also noted different simulation outcomes for different force fields. On the basis of comprehensive tests, we propose a set of "best practice" parameter settings for MD simulations of ESI droplets. The strategies used here should be transferable to other types of droplet simulations, paving the way toward a more solid understanding of ESI mechanisms.

Age-associated remodeling processes affect the intramuscular connective tissue (IMCT) network, which may significantly impair muscle function. Thus, we aimed to test whether including exercises shown to efficiently target the IMCT to a conventional resistance exercise intervention (CONV) would result in greater functional gains as compared to CONV alone. Fifty-three men and women (66.2 ± 3.3 years) were assigned to either CONV (n = 15), multimodal training (MULTI; n = 17) or a control (CTRL; n = 21) group. All subjects were tested at baseline, and those assigned to CONV or MULTI underwent a 16-week training intervention. The CONV group followed a progressive resistance training program, in which the number of weekly training sessions gradually increased from 1 to 3. In the MULTI group, one of these sessions was replaced with plyometric training, followed by self-myofascial release. Testing included maximal strength and power, imaging-based muscle volume, architecture, and functional performance. The intervention effects were analyzed using two- or three-way repeated measures ANOVA models (α = 0.05). Briefly, the maximal knee extension isometric contraction, one-repetition maximum, and isokinetic peak torque increased in all groups (p < 0.05), albeit to a lesser extent in CTRL. On the other hand, quadriceps femoris muscle volume (p = 0.019) and vastus lateralis pennation angle (p < 0.001) increased only in the MULTI group. Handgrip strength did not change in response to the intervention (p = 0.312), whereas Sit-to-Stand performance improved in all groups after the first 8-wks, but only in MULTI and CONV after 16-wks (all p < 0.001). In conclusion, we found that a resistance training intervention in which one weekly training session is replaced by plyometric training is feasible and as effective as a program consisting solely of conventional strength training sessions for inducing gains in muscle strength and function in older adults. Muscle size and architecture improved only in the MULTI group. German Clinical Trials: DRKS00015750., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aggregation refers to the assembly of proteins into nonphysiological higher order structures. While amyloid has been studied extensively, much less is known about amorphous aggregation, a process that interferes with protein expression and storage. Free arginine (Arg + ) is a widely used aggregation inhibitor, but its mechanism remains elusive. Focusing on myoglobin (Mb), we recently applied atomistic molecular dynamics (MD) simulations for gaining detailed insights into amorphous aggregation (Ng J. Phys. Chem. B 2021, 125, 13099). Building on that approach, the current work for the first time demonstrates that MD simulations can directly elucidate aggregation inhibition mechanisms. Comparative simulations with and without Arg + reproduced the experimental finding that Arg + significantly decreased the Mb aggregation propensity. Our data reveal that, without Arg + , protein-protein encounter complexes readily form salt bridges and hydrophobic contacts, culminating in firmly linked dimeric aggregation nuclei. Arg + promotes the dissociation of encounter complexes. These "unproductive" encounter complexes are favored because Arg + binding to D - and E - lowers the tendency of these anionic residues to form interprotein salt bridges. Side chain blockage is mediated largely by the guanidinium group of Arg + , which binds carboxylates through H-bond-reinforced ionic contacts. Our MD data revealed Arg + self-association into a dynamic quasi-infinite network, but we found no evidence that this self-association is important for protein aggregation inhibition. Instead, aggregation inhibition by Arg + is similar to that mediated by free guanidinium ions. The computational strategy used here should be suitable for the rational design of aggregation inhibitors with enhanced potency.

Ammonium acetate (NH 4 Ac) is a widely used solvent additive in native electrospray ionization (ESI) mass spectrometry. NH 4 Ac can undergo proton transfer to form ammonia and acetic acid (NH 4 + + Ac - → NH 3 + HAc). The volatility of these products ensures that electrosprayed ions are free of undesired adducts. NH 4 Ac dissolution in water yields pH 7, providing "physiological" conditions. However, NH 4 Ac is not a buffer at pH 7 because NH 4 are not a conjugate acid/base pair (Konermann, L. + and Ac - are not a conjugate acid/base pair (Konermann, L. J. Am. Soc. Mass Spectrom. 2017 , 28 , 1827-1835.). In native ESI, it is desirable that analytes experience physiological conditions not only in bulk solution but also while they reside in ESI droplets. Little is known about the internal milieu of NH 4 Ac-containing ESI droplets. The current work explored the acid/base chemistry of such droplets, starting from a pH 7 analyte solution. We used a two-pronged approach involving evaporation experiments on bulk solutions under ESI-mimicking conditions, as well as molecular dynamics simulations using a newly developed algorithm that allows for proton transfer. Our results reveal that during droplet formation at the tip of the Taylor cone, electrolytically generated protons get neutralized by Ac - , making NH 4 + the net charge carriers in the weakly acidic nascent droplets. During the subsequent evaporation, the droplets lose water as well as NH 3 and HAc that were generated by proton transfer. NH 3 departs more quickly because of its greater volatility, causing the accumulation of HAc. Together with residual Ac - , these HAc molecules form an acetate buffer that stabilizes the average droplet pH at 5.4 ± 0.1, as governed by the Henderson-Hasselbalch equation. The remarkable success of native ESI investigations in the literature implies that this pH drop by ∼1.6 units relative to the initially neutral analyte solution can be tolerated by most biomolecular analytes on the short time scale of the ESI process.

Mass spectrometry (MS)-based techniques are widely used for probing protein structure and dynamics in solution. H/D exchange (HDX)-MS is one of the most common approaches in this context. HDX is often considered to be a "benign" labeling method, in that it does not perturb protein behavior in solution. However, several studies have reported that D 2 O pushes unfolding equilibria toward the native state. The origin, and even the existence of this protein stabilization remain controversial. Here we conducted thermal unfolding assays in solution to confirm that deuterated proteins in D 2 O are more stable, with 2-4 K higher melting temperatures than unlabeled proteins in H 2 O. Previous studies tentatively attributed this phenomenon to strengthened H-bonds after deuteration, an effect that may arise from the lower zero-point vibrational energy of the deuterated species. Specifically, it was proposed that strengthened water-water bonds (W···W) in D 2 O lower the solubility of nonpolar side chains. The current work takes a broader view by noting that protein stability in solution also depends on water-protein (W···P) and protein-protein (P···P) H-bonds. To help unravel these contributions, we performed collision-induced unfolding (CIU) experiments on gaseous proteins generated by native electrospray ionization. CIU profiles of deuterated and unlabeled proteins were indistinguishable, implying that P···P contacts are insensitive to deuteration. Thus, protein stabilization in D 2 O is attributable to solvent effects, rather than alterations of intraprotein H-bonds. Strengthening of W···W contacts represents one possible explanation, but the stabilizing effect of D 2 O can also originate from weakened W···P bonds. Future work will be required to elucidate which of these two scenarios is correct, or if both contribute to protein stabilization in D 2 O. In any case, the often-repeated adage that "D-bonds are more stable than H-bonds" does not apply to intramolecular contacts in native proteins.

Current experiments only provide low-resolution information on gaseous protein ions generated by electrospray ionization (ESI). Molecular dynamics (MD) simulations can yield complementary insights. Unfortunately, conventional MD does not capture the mobile nature of protons in gaseous proteins. Mobile-proton MD (MPMD) overcomes this limitation. Earlier MPMD data at 300 K indicated that protein ions generated by "native" ESI retain solution-like structures with a hydrophobic core and zwitterionic exterior [Bakhtiari, M.; Konermann, L. J. Phys. Chem. B 2019, 123, 1784-1796]. MPMD redistributes protons using electrostatic and proton affinity calculations. The robustness of this approach has never been scrutinized. Here, we close this gap by benchmarking MPMD against density functional theory (DFT) at the B3LYP/6-31G* level, which is well suited for predicting proton affinities. The computational cost of DFT necessitated the use of small peptides. The MPMD energetic ranking of proton configurations was found to be consistent with DFT single-point energies, implying that MPMD can reliably identify favorable protonation sites. Peptide MPMD runs converged to DFT-optimized structures only when applying 300-500 K temperature cycling, which was necessary to prevent trapping in local minima. Temperature cycling MPMD was then applied to gaseous protein ions. Native ubiquitin converted to slightly expanded structures with a zwitterionic core and a nonpolar exterior. Our data suggest that such inside-out protein structures are intrinsically preferred in the gas phase, and that they form in ESI experiments after moderate collisional excitation. This is in contrast to native ESI (with minimal collisional excitation, simulated by MPMD at 300 K), where kinetic trapping promotes the survival of solution-like structures. In summary, this work validates the MPMD approach for simulations on gaseous peptides and proteins.

Peptide separations by reversed-phase liquid chromatography (RPLC) are an integral part of bottom-up proteomics. These separations typically employ C18 columns with water/acetonitrile gradient elution in the presence of formic acid. Despite the widespread use of such workflows, the exact nature of peptide interactions with the stationary and mobile phases is poorly understood. Here, we employ microsecond molecular dynamics (MD) simulations to uncover details of peptide RPLC. We examined two tryptic peptides, a hydrophobic and a hydrophilic species, in a slit pore lined with C18 chains that were grafted onto SiO 2 support. Our simulations explored peptide trapping, followed by desorption and elution. Trapping in an aqueous mobile phase was initiated by C18 contacts with Lys butyl moieties. This was followed by extensive anchoring of nonpolar side chains (Leu/Ile/Val) in the C18 layer. Exposure to water/acetonitrile triggered peptide desorption in a stepwise fashion; charged sites close to the termini were the first to lift off, followed by the other residues. During water/acetonitrile elution, both peptides preferentially resided close to the pore center. The hydrophilic peptide exhibited no contacts with the stationary phase under these conditions. In contrast, the hydrophobic species underwent multiple transient Leu/Ile/Val binding interactions with C18 chains. These nonpolar interactions represent the foundation of differential peptide retention, in agreement with the experimental elution behavior of the two peptides. Extensive peptide/formate ion pairing was observed in water/acetonitrile, particularly at N-terminal sites. Overall, this work uncovers an unprecedented level of RPLC molecular details, paving the way for MD simulations as a future tool for improving retention prediction algorithms and for the design of novel column materials.

Events taking place during electrospray ionization (ESI) can trigger the self-assembly of various nanoclusters. These products are often dominated by magic number clusters (MNCs) that have highly symmetrical structures. The literature rationalizes the dominance of MNCs by noting their high stability. However, this argument is not necessarily adequate because thermodynamics cannot predict the outcome of kinetically controlled reactions. Thus, the mechanisms responsible for MNC dominance remain poorly understood. Molecular dynamics (MD) simulations can provide atomistic insights into self-assembly reactions, but even this approach has thus far failed to provide pertinent answers. The current work overcomes this limitation. We focused on salt clusters formed from aqueous NaCl solutions during ESI. The corresponding mass spectra are dominated by the Na 14 Cl 13 + MNC. Simulations of ESI droplets showed nonspecific association of Na + and Cl - , culminating in gaseous clusters via solvent evaporation to dryness (charged residue mechanism). These nascent clusters did not show any preference for MNCs. In mass spectrometry experiments, analyte ions undergo in-source activation prior to detection. We emulated in-source activation by heating nascent clusters in our MD runs. Heating triggered structural fluctuations and dissociation events, generating MNC-dominated product distributions. Why are MNCs preferred after in-source activation? Thermally excited clusters frequently adopt structures consisting of a preformed MNC and a stringlike protrusion that contains the surplus ions. Facile separation of these protrusions releases the MNC (Cluster hot → MNC-protrusion → MNC + protrusion). This work marks the first time that MD simulations were able to capture cluster self-assembly with subsequent "molecular pruning", generating MNC-dominated product distributions that agree with experiments.

Differential scanning calorimetry (DSC) is a standard tool for probing the resilience of monoclonal antibodies (mAbs) and other protein therapeutics against thermal degradation. Unfortunately, DSC usually only provides insights into global unfolding, although sequential steps are sometimes discernible for multidomain proteins. Temperature-dependent hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has the potential to probe heat-induced events at a much greater level of detail. We recently proposed a strategy to deconvolute temperature-dependent HDX data into contributions from local dynamics, global unfolding/refolding, as well as chemical labeling. However, that strategy was validated only for a small protein (Tajoddin, N. N.; Konermann, L. Anal. Chem. 2020, 92, 10058). The current work explores the applicability of this HDX framework to the NIST reference mAb (NISTmAb), a large multidomain protein that is prone to aggregation and has three melting points. Using global fitting, we were able to model HDX profiles across the NISTmAb sequence between zero and 95 °C, and for time points between 15 s and 20 min. We uncovered the enthalpic and entropic contributions of local fluctuations that govern the conformational dynamics at low temperatures. The CH2 and CH3 domains were found to be increasingly affected by global unfolding/refolding in the vicinity of their melting points, although the transiently unfolded protein displayed significant residual protection. Global dynamics were not involved in the deuteration of the Fab domains (which have the highest melting point). Instead, global Fab unfolding was followed immediately by irreversible aggregation. Our results reveal that the thermodynamic HDX-MS strategy applied in this work is well suited for probing spatially resolved dynamics of thermally stressed large proteins such as mAbs, complementing data obtained by DSC.

Excess protons in water exhibit unique transport properties because they can rapidly hop along H-bonded water wires. Considerable progress has been made in unraveling this Grotthuss diffusion mechanism using quantum mechanical-based computational techniques. Unfortunately, high computational cost tends to restrict those techniques to small systems and short times. Molecular dynamics (MD) simulations can be applied to much larger systems and longer time windows. However, standard MD methods do not permit the dissociation/formation of covalent bonds, such that Grotthuss diffusion cannot be captured. Here, we bridge this gap by combining atomistic MD simulations (using Gromacs and TIP4P/2005 water) with proton hopping. Excess protons are modeled as hydronium ions that undergo H 3 O + + H 2 O → H 2 O + H 3 O + transitions. In accordance with ab initio MD data, these Grotthuss hopping events are executed in "bursts" with quasi-instantaneous hopping across one or more waters. The bursts are separated by regular MD periods during which H 3 O + ions undergo Brownian diffusion. The resulting proton diffusion coefficient agrees with the literature value. We apply this Grotthuss MD technique to highly charged water droplets that are in a size regime encountered during electrospray ionization (5 nm radius, ∼17,000 H 2 O). The droplets undergo rapid solvent evaporation and occasional H 3 O + ejection, keeping them at ca. 81% of the Rayleigh limit. The simulated behavior is consistent with phase Doppler anemometry data. The Grotthuss MD technique developed here should be useful for modeling the behavior of various proton-containing systems that are too large for high-level computational approaches. In particular, we envision future applications related to electrospray processes, where earlier simulations used metal cations while in reality excess protons dominate.

The transfer of peptide ions from solution into the gas phase by electrospray ionization (ESI) is an integral component of mass spectrometry (MS)-based proteomics. The mechanisms whereby gaseous peptide ions are released from charged ESI nanodroplets remain unclear. This is in contrast to intact protein ESI, which has been the focus of detailed investigations using molecular dynamics (MD) simulations and other methods. Under acidic liquid chromatography/MS conditions, many peptides carry a solution charge of 3+ or 2+. Because of this pre-existing charge and their relatively small size, prevailing views suggest that peptides follow the ion evaporation mechanism (IEM). The IEM entails analyte ejection from ESI droplets, driven by electrostatic repulsion between the analyte and droplet. Surprisingly, recent peptide MD investigations reported a different behavior, that is, the release of peptide ions via droplet evaporation to dryness which represents the hallmark of the charged residue mechanism (CRM). Here, we resolved this conundrum by performing MD simulations on a common model peptide (bradykinin) in Rayleigh-charged aqueous droplets. The primary focus was on pH 2 conditions (bradykinin solution charge = 3+), but we also verified that our MD strategy captured pH-dependent charge state shifts seen in ESI-MS experiments. In agreement with earlier simulations, we found that droplets with initial radii of 1.5-3 nm predominantly release peptide ions via the CRM. In contrast, somewhat larger radii (4-5 nm) favor IEM behavior. It appears that these are the first MD data to unequivocally demonstrate the viability of peptide IEM events. Electrostatic arguments can account for the observed droplet size dependence. In summary, both CRM and IEM can be operative in peptide ESI-MS. The prevalence of one over the other mechanism depends on the droplet size distribution in the ESI plume.

Proteins that encounter unfavorable solvent conditions are prone to aggregation, a phenomenon that remains poorly understood. This work focuses on myoglobin (Mb) as a model protein. Upon heating, Mb produces amorphous aggregates. Thermal unfolding experiments at low concentration (where aggregation is negligible), along with centrifugation assays, imply that Mb aggregation proceeds via globally unfolded conformers. This contrasts studies on other proteins that emphasized the role of partially folded structures as aggregate precursors. Molecular dynamics (MD) simulations were performed to gain insights into the mechanism by which heat-unfolded Mb molecules associate with one another. A prerequisite for these simulations was the development of a method for generating monomeric starting structures. Periodic boundary condition artifacts necessitated the implementation of a partially immobilized water layer lining the walls of the simulation box. Aggregation simulations were performed at 370 K to track the assembly of monomeric Mb into pentameric species. Binding events were preceded by multiple unsuccessful encounters. Even after association, protein-protein contacts remained in flux. Binding was mediated by hydrophobic contacts, along with salt bridges that involved hydrophobically embedded Lys residues. Overall, this work illustrates that atomistic MD simulations are well suited for garnering insights into protein aggregation mechanisms.

Many aspects of protein function rely on conformational fluctuations. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) provides a window into these dynamics. Despite the widespread use of HDX-MS, it remains unclear whether this technique provides a truly comprehensive view of protein dynamics. HDX is mediated by H-bond-opening/closing events, implying that HDX methods provide an H-bond-centric view. This raises the question if there could be fluctuations that leave the H-bond network unaffected, thereby rendering them undetectable by HDX-MS. We explore this issue in experiments on cytochrome c (cyt c ). Compared to the Fe(II) protein, Fe(III) cyt c shows enhanced deuteration on both the distal and proximal sides of the heme. Previous studies have attributed the enhanced dynamics of Fe(III) cyt c to the facile and reversible rupture of the distal M80-Fe(III) bond. Using molecular dynamics (MD) simulations, we conducted a detailed analysis of various cyt c conformers. Our MD data confirm that rupture of the M80-Fe(III) contact triggers major reorientation of the distal Ω loop. Surprisingly, this event takes place with only miniscule H-bonding alterations. In other words, the distal loop dynamics are almost "HDX-silent". Moreover, distal loop movements cannot account for enhanced dynamics on the opposite (proximal) side of the heme. Instead, enhanced deuteration of Fe(III) cyt c is attributed to sparsely populated conformers where both the distal (M80) and proximal (H18) coordination bonds have been ruptured, along with opening of numerous H-bonds on both sides of the heme. We conclude that there can be major structural fluctuations that are only weakly coupled to changes in H-bonding, making them virtually impossible to track by HDX-MS. In such cases, HDX-MS may provide an incomplete view of protein dynamics.

Native electrospray ionization (ESI)-mass spectrometry (MS) is widely used for the detection and characterization of multi-protein complexes. A well-known problem with this approach is the possible occurrence of nonspecific protein clustering in the ESI plume. This effect can distort the results of binding affinity measurements, and it can even generate gas-phase complexes from proteins that are strictly monomeric in bulk solution. By combining experiments and molecular dynamics (MD) simulations, the current work for the first time provides detailed insights into the ESI clustering of proteins. Using ubiquitin as a model system, we demonstrate how the entrapment of more than one protein molecule in an ESI droplet can generate nonspecific clusters (e.g., dimers or trimers) via solvent evaporation to dryness. These events are in line with earlier proposals, according to which protein clustering is associated with the charged residue model (CRM). MD simulations on cytochrome c (which carries a large intrinsic positive charge) confirmed the viability of this CRM avenue. In addition, the cytochrome c data uncovered an alternative mechanism where protein-protein contacts were formed early within ESI droplets, followed by cluster ejection from the droplet surface. This second pathway is consistent with the ion evaporation model (IEM). The observation of these IEM events for large protein clusters is unexpected because the IEM has been thought to be associated primarily with low-molecular-weight analytes. In all cases, our MD simulations produced protein clusters that were stabilized by intermolecular salt bridges. The MD-generated charge states agreed with experiments. Overall, this work reveals that ESI-induced protein clustering does not follow a tightly orchestrated pathway but can proceed along different avenues.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription regulator that plays a pivotal role in coordinating the cellular response to oxidative stress. Through interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), CREB-binding protein (CBP), and retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes critical for removing toxicants and preventing DNA damage, thereby playing a significant role in chemoprevention. Dysregulation of Nrf2 is linked to tumorigenesis and chemoresistance, making Nrf2 a promising target for anticancer therapeutics. However, despite the physiological importance of Nrf2, the molecular details of this protein and its interactions with most of its targets remain unknown, hindering the rational design of Nrf2-targeted therapeutics. With this in mind, we used a combined bioinformatics and experimental approach to characterize the structure of full-length Nrf2 and its interaction with Keap1. Our results show that Nrf2 is partially disordered, with transiently structured elements in its Neh2, Neh7, and Neh1 domains. Moreover, interaction with the Kelch domain of Keap1 leads to protection of the binding motifs in the Neh2 domain of Nrf2, while the rest of the protein remains highly dynamic. This work represents the first detailed structural characterization of full-length Nrf2 and provides valuable insights into the molecular basis of Nrf2 activity modulation in oxidative stress response.

Electrosprayed protein ions can retain native-like conformations. The intramolecular contacts that stabilize these compact gas-phase structures remain poorly understood. Recent work has uncovered abundant salt bridges in electrosprayed proteins. Salt bridges are zwitterionic BH + /A - contacts. The low dielectric constant in the vacuum strengthens electrostatic interactions, suggesting that salt bridges could be a key contributor to the retention of compact protein structures. A problem with this assertion is that H + are mobile, such that H + transfer can convert salt bridges into neutral B 0 /HA 0 contacts. This possible salt bridge annihilation puts into question the role of zwitterionic motifs in the gas phase, and it calls for a detailed analysis of BH + /A - versus B 0 /HA 0 interactions. Here, we investigate this issue using molecular dynamics (MD) simulations and electrospray experiments. MD data for short model peptides revealed that salt bridges with static H + have dissociation energies around 700 kJ mol -1 . The corresponding B 0 /HA 0 contacts are 1 order of magnitude weaker. When considering the effects of mobile H + , BH + /A - bond energies were found to be between these two extremes, confirming that H + migration can significantly weaken salt bridges. Next, we examined the protein ubiquitin under collision-induced unfolding (CIU) conditions. CIU simulations were conducted using three different MD models: (i) Positive-only runs with static H + did not allow for salt bridge formation and produced highly expanded CIU structures. (ii) Zwitterionic runs with static H + resulted in abundant salt bridges, culminating in much more compact CIU structures. (iii) Mobile H + simulations allowed for the dynamic formation/annihilation of salt bridges, generating CIU structures intermediate between scenarios (i) and (ii). Our results uncover that mobile H + limit the stabilizing effects of salt bridges in the gas phase. Failure to consider the effects of mobile H + in MD simulations will result in unrealistic outcomes under CIU conditions.

It is well-known that supercharging agents (SCAs) such as sulfolane enhance the electrospray ionization (ESI) charge states of proteins, although the mechanistic origins of this effect remain contentious. Only very few studies have explored SCA effects on analytes other than proteins or peptides. This work examines how sulfolane affects electrosprayed NaI salt clusters. Such alkali metal halide clusters have played a key role for earlier ESI mechanistic studies, making them interesting targets for supercharging investigations. ESI of aqueous NaI solutions predominantly generated singly charged [Na n I ( n -1) ] + clusters. The addition of sulfolane resulted in abundant doubly charged [Na n I ( n -2) Sulfolane s ] 2+ species. These experimental data for the first time demonstrate that electrosprayed salt clusters can undergo supercharging. Molecular dynamics (MD) simulations of aqueous ESI nanodroplets containing Na + /I - with and without sulfolane were conducted to obtain atomistic insights into the supercharging mechanism. The simulations produced [Na n I i ] z + and [Na n I i Sulfolane s ] z + clusters similar to those observed experimentally. The MD trajectories demonstrated that these clusters were released into the gas phase upon droplet evaporation to dryness, in line with the charged residue model. Sulfolane was found to evaporate much more slowly than water. This slow evaporation, in conjunction with the large dipole moment of sulfolane, resulted in electrostatic stabilization of the shrinking ESI droplets and the final clusters. Hence, charge-dipole stabilization causes the sulfolane-containing droplets and clusters to retain more charge, thereby providing the mechanistic foundation of salt cluster supercharging.

Various activation methods are available for the fragmentation of gaseous protein complexes produced by electrospray ionization (ESI). Such experiments can potentially yield insights into quaternary structure. Collision-induced dissociation (CID) is the most widely used fragmentation technique. Unfortunately, CID of protein complexes is dominated by the ejection of highly charged monomers, a process that does not yield any structural insights. Using hemoglobin (Hb) as a model system, this work examines under what conditions CID generates structurally informative subcomplexes. Native ESI mainly produced tetrameric Hb ions. In addition, "noncanonical" hexameric and octameric complexes were observed. CID of all these species [(αβ) 2 , (αβ) 3 , and (αβ) 4 ] predominantly generated highly charged monomers. In addition, we observed hexamer → tetramer + dimer dissociation, implying that hexamers have a tetramer··dimer architecture. Similarly, the observation of octamer → two tetramer dissociation revealed that octamers have a tetramer··tetramer composition. Gas-phase candidate structures of Hb assemblies were produced by molecular dynamics (MD) simulations. Ion mobility spectrometry was used to identify the most likely candidates. Our data reveal that the capability of CID to produce structurally informative subcomplexes depends on the fate of protein-protein interfaces after transfer into the gas phase. Collapse of low affinity interfaces conjoins the corresponding subunits and favors CID via monomer ejection. Structurally informative subcomplexes are formed only if low affinity interfaces do not undergo a major collapse. However, even in these favorable cases CID is still dominated by monomer ejection, requiring careful analysis of the experimental data for the identification of structurally informative subcomplexes.

Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c ) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH 2 -NH 2 → LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a ∼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently.

Oxidation of key residues in cytochrome c (cyt c ) by chloramine T (CT) converts the protein from an electron transporter to a peroxidase. This peroxidase-activated state represents an important model system for exploring the early steps of apoptosis. CT-induced transformations include oxidation of the distal heme ligand Met80 (MetO, +16 Da) and carbonylation (LysCHO, -1 Da) in the range of Lys53/55/72/73. Remarkably, the 15 remaining Lys residues in cyt c are not susceptible to carbonylation. The cause of this unusual selectivity is unknown. Here we applied top-down mass spectrometry (MS) to examine whether CT-induced oxidation is catalyzed by heme. To this end, we compared the behavior of cyt c with (holo-cyt c ) and without heme (apo SS -cyt c ). CT caused MetO formation at Met80 for both holo- and apo SS -cyt c , implying that this transformation can proceed independently of heme. The aldehyde-specific label Girard's reagent T (GRT) reacted with oxidized holo-cyt c , consistent with the presence of several LysCHO. In contrast, oxidized apo-cyt c did not react with GRT, revealing that LysCHO forms only in the presence of heme. The heme dependence of LysCHO formation was further confirmed using microperoxidase-11 (MP11). CT exposure of apo SS -cyt c in the presence of MP11 caused extensive nonselective LysCHO formation. Our results imply that the selectivity of LysCHO formation at Lys53/55/72/73 in holo-cyt c is caused by the spatial proximity of these sites to the reactive (distal) heme face. Overall, this work highlights the utility of top-down MS for unravelling complex oxidative modifications.

The mechanisms whereby protein ions are released into the gas phase from charged droplets during electrospray ionization (ESI) continue to be controversial. Several pathways have been proposed. For native ESI the charged residue model (CRM) is favored; it entails the liberation of proteins via solvent evaporation to dryness. Unfolded proteins likely follow the chain ejection model (CEM), which involves the gradual expulsion of stretched-out chains from the droplet. According to the ion evaporation model (IEM) ions undergo electrostatically driven desorption from the droplet surface. The IEM is well supported for small precharged species such as Na + . However, it is unclear whether proteins can show IEM behavior as well. We examined this question using molecular dynamics (MD) simulations, mass spectrometry (MS), and ion mobility spectrometry (IMS) in positive ion mode. Ubiquitin was chosen as the model protein because of its structural stability which allows the protein charge in solution to be controlled via pH adjustment without changing the protein conformation. MD simulations on small ESI droplets (3 nm radius) showed CRM behavior regardless of the protein charge in solution. Surprisingly, many MD runs on larger droplets (5.5 nm radius) culminated in IEM ejection of ubiquitin, as long as the protein carried a sufficiently large positive solution charge. MD simulations predicted that nonspecific salt adducts are less prevalent for IEM-generated protein ions than for CRM products. This prediction was confirmed experimentally. Also, collision cross sections of MD structures were in good agreement with IMS data. Overall, this work reveals that the CRM, CEM, and IEM all represent viable pathways for generating gaseous protein ions during ESI. The IEM is favored for proteins that are tightly folded and highly charged in solution and for droplets in a suitable size regime.

H/D exchange (HDX) mass spectrometry (MS) is a widely used technique for interrogating protein structure and dynamics. Backbone HDX is mediated by opening/closing (unfolding/refolding) fluctuations. In traditional HDX-MS, proteins are incubated in D 2 O as a function of time at constant temperature ( T ). There is an urgent need to complement this traditional approach with experiments that probe proteins in a T -dependent fashion, e.g., for assessing the stability of therapeutic antibodies. A key problem with such studies is the absence of strategies for interpreting HDX-MS data in the context of T -dependent protein dynamics. Specifically, it has not been possible thus far to separate T -induced changes of the chemical labeling step ( k ch ) from thermally enhanced protein fluctuations. Focusing on myoglobin, the current work solves this problem by dissecting T -dependent HDX-MS profiles into contributions from k ch ( T ), as well as local and global protein dynamics. Experimental profiles started off with surprisingly shallow slopes that seemed to defy the quasi-exponential k ch ( T ) dependence. Just below the melting temperature ( T m ) the profiles showed a sharp increase. Our analysis revealed that local dynamics dominate at low T , while global events become prevalent closer to T m . About half of the backbone NH sites exhibited a canonical scenario, where local opening/closing was associated with positive Δ H and Δ S . Many of the remaining sites had negative Δ H and Δ S , thereby accounting for the shallowness of the experimental HDX-MS profiles at low T . In summary, this work provides practitioners with the tools to analyze proteins over a wide temperature range, paving the way toward T -dependent high-throughput screening applications by HDX-MS.

The properties of electrosprayed protein ions continue to be enigmatic, owing to the absence of high-resolution structure determination methods in the gas phase. There is considerable evidence that under properly optimized conditions these ions preserve solution-like conformations and interactions. However, it is unlikely that these solution-like conformers represent the "intrinsic" structural preferences of gaseous proteins. In an effort to uncover what such intrinsically preferred conformers might look like, we performed molecular dynamics (MD) simulations of gaseous ubiquitin. Our work was inspired by recent gas phase experiments, where highly extended 13+ ubiquitin ions were transformed to compact 3+ species by proton stripping (Laszlo, K. J.; Munger, E. B.; Bush, M. F. J. Am. Chem. Soc. 2016 , 138 , 9581-9588). Our simulations covered several microseconds and used a mobile-proton algorithm to account for the fact that a H + in gaseous proteins can migrate between different titratable sites. Proton stripping caused folding of ubiquitin into heterogeneous "inside-out" structures. The hydrophilic core of these conformers was stabilized by charge-charge and polar interactions, while hydrophobic residues were located on the protein surface. Collision cross sections of these MD structures were in good agreement with experimental results. The inside-out structures generated during gas phase folding are in striking contrast to the solution behavior which is dominated by the hydrophobic effect, i.e., the tendency to bury hydrophobic side chains in the core (instead of exposing them to the surface). We do not dispute that native-like proteins can be transferred into the gas phase as kinetically trapped species. However, those metastable conformers do not represent the intrinsic structural preferences of gaseous proteins. Our work for the first time provides detailed insights into the properties of intrinsically preferred gas phase conformers, and we unequivocally find them to have inside-out architectures.

Recent advances in molecular dynamics (MD) simulations have made it possible to examine the behavior of large charged droplets that contain analytes such as proteins or polymers, thereby providing insights into electrospray ionization (ESI) mechanisms. In the present study, we use this approach to investigate the release of polylactide (PLA) ions from water/acetonitrile ESI droplets. We found that cationized gaseous PLA ions can be formed via various competing pathways. Some MD runs showed extrusion and subsequent separation of polymer chains from the droplet, as envisioned by the chain ejection model (CEM). On other occasions the PLA chains remained inside the droplets and were released after solvent evaporation to dryness, consistent with the charge residue model (CRM). Following their release from ESI droplets, the nascent gaseous PLA ions were subjected to structural relaxation for several μs in vacuo. The MD conformations generated in this way for various PLA charge states compared favorably to experimental results obtained by ion mobility spectrometry-mass spectrometry (IMS-MS). The structures of all PLA ions evolved during relaxation in the gas phase. However, some macroion species retained features that resembled their nascent structures. For this subset of ions, the IMS-MS response appears to be strongly correlated with the ESI release mechanism (CEM vs. CRM). The former favored extended structures, whereas the latter preferentially generated compact conformers.

The structure and reactivity of electrosprayed protein ions is governed by their net charge. Native proteins in non-denaturing aqueous solutions produce low charge states. More highly charged ions are formed when electrospraying proteins that are unfolded and/or exposed to organic supercharging agents. Numerous studies have explored the electrospray process under these various conditions. One phenomenon that has received surprisingly little attention is the charge enhancement caused by multivalent metal ions such as La 3+ when electrospraying proteins out of non-denaturing solutions. Here, we conducted mass spectrometry and ion mobility spectrometry experiments, in combination with molecular dynamics (MD) simulations, to uncover the mechanistic basis of this charge enhancement. MD simulations of aqueous ESI droplets reproduced the experimental observation that La 3+ boosts protein charge states relative to monovalent metals (e.g., Na + ). The simulations showed that gaseous proteins were released by solvent evaporation to dryness, consistent with the charged residue model. Metal ion ejection kept the shrinking droplets close to the Rayleigh limit until ∼99% of the solvent had left. For droplets charged with Na + , metal adduction during the final stage of solvent evaporation produced low protein charge states. Droplets containing La 3+ showed a very different behavior. The trivalent nature of La 3+ favored adduction to the protein at a very early stage, when most of the solvent had not evaporated yet. This irreversible binding via multidentate contacts suppressed La 3+ ejection from the vanishing droplets, such that the resulting gaseous proteins carried significantly more charge. Our results illustrate that MD simulations are suitable for uncovering intricate aspects of electrospray mechanisms, paving the way toward an atomistic understanding of mass spectrometry based analytical workflows.

Electrospray ionization (ESI) is an essential technique for transferring proteins from solution into the gas phase for mass spectrometry and ion mobility spectrometry. The mechanisms whereby [M + zH] z+ protein ions are released from charged nanodroplets during ESI have been controversial for many years. Here we discuss recent computational and experimental studies that have shed light on many of the mysteries in this area. Four types of protein ESI experiments can be distinguished, each of which appears to be associated with a specific mechanism. (i) Native ESI proceeds according to the charged residue model (CRM) that entails droplet evaporation to dryness, generating compact protein ions in low charge states. (ii) Native ESI supercharging is also a CRM process, but the dried-out proteins accumulate additional charge because supercharging agents such as sulfolane interfere with the ejection of small ions (Na + , NH 4 + , etc.) from the shrinking droplets. (iii) Denaturing ESI follows the chain ejection model (CEM), where protein ions are gradually expelled from the droplet surface. H + equilibration between the droplets and the protruding chains culminates in highly charged gaseous proteins, analogous to the collision-induced dissociation of multi-protein complexes. (iv) Denatured ESI supercharging also generates protein ions via the CEM. Supercharging agents stabilize protonated sites on the protein tail via charge-dipole interactions, causing the chain to acquire additional charge. There will likely be scenarios that fall outside of these four models, but it appears that the framework outlined here covers most of the experimentally relevant conditions.

It is believed that electrosprayed proteins and protein complexes can retain solution-like conformations in the gas phase. However, the lack of high-resolution structure determination methods for gaseous protein ions implies that their properties remain poorly understood. Many practitioners tackle this difficulty by complementing mass spectrometry-based experiments with molecular dynamics (MD) simulations. It is a potential problem that the standard MD force fields used for this purpose (such as OPLS-AA/L and CHARMM) were optimized for solution conditions. The question whether these force fields produce meaningful gas-phase data has received surprisingly little attention. Standard force fields are overpolarized to account for an aqueous environment, i.e., atomic charges and intramolecular dipole moments are ∼20% larger than predicted by gas-phase ab initio methods. Here, we examined the implications of this overpolarization by conducting a series of MD simulations on electrosprayed proteins. Force fields were modified via a charge scaling factor (CSF), while ensuring that the net protein charge remained unchanged. CSF = 0.8 should roughly eliminate water-associated overpolarization. Gas-phase CHARMM simulations on myoglobin with CSF = 0.8 and with unmodified parameters (CSF = 1) yielded similar results, preserving a compact structure that was consistent with ion mobility experiments. Major structural changes caused by weakened charge-dipole and dipole-dipole contacts occurred only when lowering CSF to physically unreasonable values (0.5 and 0.1). Similar results were obtained in mobile-proton OPLS-AA/L simulations on the collision-induced dissociation of transthyretin. Our data support the view that gas-phase MD simulations with standard (solution) force fields are suitable for modeling gaseous protein ions in a semiquantitative manner. Although this is welcome news for the mass spectrometry community, it is hoped that dedicated gas-phase MD force fields will become available in the near future.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Proteins that are unfolded in solution produce higher charge states during electrospray ionization (ESI) than their natively folded counterparts. Protein charge states can be further increased by the addition of supercharging agents (SCAs) such as sulfolane. The mechanism whereby these supercharged [M + zH] z+ ions are formed under unfolded conditions remains unclear. Here we employed a combination of mass spectrometry (MS), ion mobility spectrometry (IMS), and molecular dynamics (MD) simulations for probing the ESI mechanism under denatured supercharging conditions. ESI of acid-unfolded apo-myoglobin (aMb) in the presence of sulfolane produced charge states around 27+, all the way to fully protonated (33+) aMb. MD simulations of aMb 27+ to 33+ in Rayleigh-charged water/sulfolane droplets culminated in electrostatically driven protein expulsion, consistent with the chain ejection model (CEM). The electrostatically stretched conformations predicted by these simulations were in agreement with IMS experiments. The CEM involves partitioning of mobile H + between the droplet and the departing protein. Our results imply that supercharging of unfolded proteins is caused by residual sulfolane that stabilizes protonated sites on the protruding chains, thereby promoting H + retention on the protein. The stabilization of charged sites is due to charge-dipole interactions mediated by the large dipole moment and the low volatility of sulfolane. Support for this mechanism comes from the experimental observation of sulfolane adducts on the most highly charged ions, a phenomenon previously noted by Venter ( J. Am. Soc. Mass Spectrom. 2012, 23, 489-497). The "CEM supercharging model" proposed here for unfolded proteins is distinct from the charge trapping mechanism believed to be operative during native ESI supercharging.

The assembly of proteins into dimers and oligomers is a necessary step for the proper function of transcription factors, muscle proteins, and proteases. In uncontrolled states, oligomerization can also contribute to illnesses such as Alzheimer's disease. The S100 protein family is a group of dimeric proteins that have important roles in enzyme regulation, cell membrane repair, and cell growth. Most S100 proteins have been examined in their homodimeric state, yet some of these important proteins are found in similar tissues implying that heterodimeric molecules can also be formed from the combination of two different S100 members. In this work, we have established co-expression methods in order to identify and quantify the distribution of homo- and heterodimers for four specific pairs of S100 proteins in their calcium-free states. The split GFP trap methodology was used in combination with other GFP variants to simultaneously quantify homo- and heterodimeric S100 proteins in vitro and in living cells. For the specific S100 proteins examined, NMR, mass spectrometry, and GFP trap experiments consistently show that S100A1:S100B, S100A1:S100P, and S100A11:S100B heterodimers are the predominant species formed compared to their corresponding homodimers. We expect the tools developed here will help establish the roles of S100 heterodimeric proteins and identify how heterodimerization might alter the specificity for S100 protein action in cells., (© 2019 Federation of European Biochemical Societies.)

Experiments and molecular dynamics (MD) simulations in the literature indicate that gaseous proteins generated by electrospray ionization (ESI) can retain native-like structures. However, the exact properties of these ions remain to be explored. Focusing on ubiquitin and lysozyme, we examined several pertinent questions. (1) We applied solvent MD runs to test whether the X-ray structures of both proteins are affected by crystal packing. Main and side-chain orientations were retained in solution, providing a justification for the hitherto unscrutinized approach of relying on crystal data for "solution" versus gas-phase comparisons. (2) Most earlier gas-phase protein MD investigations employed short (ns) simulation windows. By extending this time frame to 1 μs, we were able to observe rare unfolding/folding transitions in ubiquitin. These predicted fluctuations were consistent with a semi-unfolded subpopulation detected by ion mobility spectrometry (IMS). (3) Most earlier modeling studies did not account for the high H + mobility in gaseous proteins. For the first time, we compared static and mobile H + simulations, focusing on both positively and negatively charged ions. The MD runs revealed a strong preference for retention of a solution-like backbone fold, whereas titratable/polar side chains collapsed onto the protein surface. This side-chain collapse was caused by a multitude of intramolecular salt bridges, H-bonds, and charge-dipole interactions. Our results generalize the findings of Steinberg et al. ( ChemBioChem, 2008, 9, 2417-2423) who had first proposed the occurrence of such side-chain contacts on the basis of short-term simulations with static H + . (4) Calculated collision cross sections of the MD conformers were in close agreement with IMS experiments. Overall, this study supports the view that solution-like protein structures can be retained because of kinetic trapping on the time scale of typical ESI-IMS experiments.

The peroxidase activity of cytochrome c (cyt c ) plays a key role during apoptosis. Peroxidase catalysis requires a vacant Fe coordination site, i.e. , cyt c must undergo an activation process involving structural changes that rupture the native Met80-Fe contact. A common strategy for dissociating this bond is the conversion of Met80 to sulfoxide (MetO). It is widely believed that this MetO formation in itself is sufficient for cyt c activation. This notion originates from studies on chloramine-T-treated cyt c (CT-cyt c ) which represents a standard model for the peroxidase activated state. CT-cyt c is considered to be a "clean" species that has undergone selective MetO formation, without any other modifications. Using optical, chromatographic, and mass spectrometry techniques, the current work demonstrates that CT-induced activation of cyt c is more complicated than previously thought. MetO formation alone results in only marginal peroxidase activity, because dissociation of the Met80-Fe bond triggers alternative ligation scenarios where Lys residues interfere with access to the heme. We found that CT causes not only MetO formation, but also carbonylation of several Lys residues. Carbonylation is associated with -1 Da mass shifts that have gone undetected in the CT-cyt c literature. Proteoforms possessing both MetO and Lys carbonylation exhibit almost fourfold higher peroxidase activity than those with MetO alone. Carbonylation abrogates the capability of Lys to coordinate the heme, thereby freeing up the distal site as required for an active peroxidase. Previous studies on CT-cyt c may have inadvertently examined carbonylated proteoforms, potentially misattributing effects of carbonylation to solely MetO formation.

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho-ubiquitin-loaded C-terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re-modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)