MD Simulations of Peptide-Containing Electrospray Droplets: Effects of Parameter Settings on the Predicted Mechanisms of Gas Phase Ion Formation.

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.

Effects of Hydrogen/Deuterium Exchange on Protein Stability in Solution and in the Gas Phase.

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 D2O 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 D2O are more stable, with 2-4 K higher melting temperatures than unlabeled proteins in H2O. 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 D2O 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 D2O 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 D2O 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 D2O. 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.

Using Density Functional Theory for Testing the Robustness of Mobile-Proton Molecular Dynamics Simulations on Electrosprayed Ions: Structural Implications for Gaseous 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.

Atomistic Details of Peptide Reversed-Phase Liquid Chromatography from Molecular Dynamics Simulations.

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 SiO2 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.

Formation of Gaseous Peptide Ions from Electrospray Droplets: Competition between the Ion Evaporation Mechanism and Charged Residue Mechanism.

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.

Nrf2, the Major Regulator of the Cellular Oxidative Stress Response, is Partially Disordered.

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.

Mobile Protons Limit the Stability of Salt Bridges in the Gas Phase: Implications for the Structures of Electrosprayed Protein Ions.

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 B0/HA0 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 B0/HA0 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 B0/HA0 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.

Probing the Effects of Heterogeneous Oxidative Modifications on the Stability of Cytochrome c in Solution and in the Gas Phase.

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 (LysCH2-NH2 → 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.

Delineating Heme-Mediated versus Direct Protein Oxidation in Peroxidase-Activated Cytochrome c by Top-Down Mass Spectrometry.

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 (apoSS-cyt c). CT caused MetO formation at Met80 for both holo- and apoSS-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 apoSS-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.

Gas Phase Protein Folding Triggered by Proton Stripping Generates Inside-Out Structures: A Molecular Dynamics Simulation Study.

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.

Effects of electrospray mechanisms and structural relaxation on polylactide ion conformations in the gas phase: insights from ion mobility spectrometry and molecular dynamics simulations.

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.

Protein Ions Generated by Native Electrospray Ionization: Comparison of Gas Phase, Solution, and Crystal Structures.

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.

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation.

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.

Evidence for a Partially Stalled γ Rotor in F1-ATPase from Hydrogen-Deuterium Exchange Experiments and Molecular Dynamics Simulations.

F1-ATPase uses ATP hydrolysis to drive rotation of the γ subunit. The γ C-terminal helix constitutes the rotor tip that is seated in an apical bearing formed by α3ß3. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we interrogated E. coli F1-ATPase by hydrogen-deuterium exchange (HDX) mass spectrometry. Rotation of γ caused greatly enhanced deuteration in the γ C-terminal helix. The HDX kinetics implied that most F1 complexes operate with an intact rotor at any given time, but that the rotor tip is prone to occasional unfolding. A molecular dynamics (MD) strategy was developed to model the off-axis forces acting on γ. MD runs showed stalling of the rotor tip and unfolding of the γ C-terminal helix. MD-predicted H-bond opening events coincided with experimental HDX patterns. Our data suggest that in vitro operation of F1-ATPase is associated with significant rotational resistance in the apical bearing. These conditions cause the γ C-terminal helix to get "stuck" (and unfold) sporadically while the remainder of γ continues to rotate. This scenario contrasts the traditional "greasy bearing" model that envisions smooth rotation of the γ C-terminal helix. The fragility of the apical rotor tip in F1-ATPase is attributed to the absence of a c10 ring that stabilizes the rotation axis in intact FoF1. Overall, the MD/HDX strategy introduced here appears well suited for interrogating the inner workings of molecular motors.

Dimerization interface of osteoprotegerin revealed by hydrogen-deuterium exchange mass spectrometry.

Previous structural studies of osteoprotegerin (OPG), a crucial negative regulator of bone remodeling and osteoclastogenesis, were mostly limited to the N-terminal ligand-binding domains. It is now known that the three C-terminal domains of OPG also play essential roles in its function by mediating OPG dimerization, OPG-heparan sulfate (HS) interactions, and formation of the OPG-HS-receptor activator of nuclear factor κB ligand (RANKL) ternary complex. Employing hydrogen-deuterium exchange MS methods, here we investigated the structure of full-length OPG in complex with HS or RANKL in solution. Our data revealed two noteworthy aspects of the OPG structure. First, we found that the interconnection between the N- and C-terminal domains is much more rigid than previously thought, possibly because of hydrophobic interactions between the fourth cysteine-rich domain and the first death domain. Second, we observed that two hydrophobic clusters located in two separate C-terminal domains directly contribute to OPG dimerization, likely by forming a hydrophobic dimerization interface. Aided by site-directed mutagenesis, we further demonstrated that an intact dimerization interface is essential for the biological activity of OPG. Our study represents an important step toward deciphering the structure-function relationship of the full-length OPG protein.

Electrospray Ionization of Polypropylene Glycol: Rayleigh-Charged Droplets, Competing Pathways, and Charge State-Dependent Conformations.

Recent molecular dynamics (MD) simulations from various laboratories have advanced the general understanding of electrospray ionization (ESI)-related processes. Unfortunately, computational cost has limited most of those previous endeavors to ESI droplets with radii of ∼3 nm or less, which represent the low end of the size distribution in the ESI plume. The current work extends this range by conducting simulations on aqueous ESI droplets with radii of 5.5 nm (∼23 000 water molecules). Considering that computational cost increases with r6, this is a significant step forward. We focused on the ESI process for polypropylene glycol (PPG) which is a common ESI-MS calibrant. Different chain lengths (PPG10, 30, and 60) were tested in droplets that were charged with excess Na+. Solvent evaporation and Na+ ejection, with occasional progeny droplet formation, kept the systems at 80-100% of the Rayleigh limit throughout their life cycle. PPG chains migrated to the droplet surface where they captured Na+ via binding to ether oxygens. Various possible pathways for PPG release into the gas phase were encountered. Some PPG10 runs showed ejection from the droplet surface, consistent with the ion evaporation model (IEM). In other instances, PPG was released after near-complete solvent evaporation, as envisioned by the charged residue model (CRM). A third avenue was the partial separation from the droplet to form double or single-tailed structures, with subsequent chain detachment from the droplet. This last pathway is consistent with the chain ejection model (CEM). Immediately after detachment many chains were electrostatically stretched, but they subsequently collapsed into compact conformers. Extended structures were retained only for the most highly charged ions. Our simulations were complemented by ESI-MS and ion mobility measurements. MD-predicted charge states and collision cross sections were in agreement with these experimental data, supporting the mechanistic insights obtained.

How to run molecular dynamics simulations on electrospray droplets and gas phase proteins: Basic guidelines and selected applications.

The ability to transfer intact proteins and protein complexes into the gas phase by electrospray ionization (ESI) has opened up numerous mass spectrometry (MS)-based avenues for exploring biomolecular structure and function. However, many details regarding the ESI process and the properties of gaseous analyte ions are difficult to decipher when relying solely on experimental data. Molecular dynamics (MD) simulations can provide additional insights into the behavior of ESI droplets and protein ions. This review is geared primarily towards experimentalists who wish to adopt MD simulations as a complementary research tool. We touch on basic points such as force fields, the choice of a proper water model, GPU-acceleration, possible artifacts, as well as shortcomings of current MD models. Following this technical overview, we highlight selected applications. Simulations on aqueous droplets confirm that "native" ESI culminates in protein ion release via the charged residue model. MD-generated charge states and collision cross sections match experimental data. Gaseous protein ions produced by native ESI retain much of their solution structure. Moving beyond classical fixed-charge algorithms, we discuss a simple strategy that captures the mobile nature of H+ within gaseous biomolecules. These mobile proton simulations confirm the high propensity of gaseous proteins to form salt bridges, as well as the occurrence of charge migration during collision-induced unfolding and dissociation. It is hoped that this review will promote the use of MD simulations in ESI-related research. We also hope to encourage the development of improved algorithms for charged droplets and gaseous biomolecular ions.

Crown Ether Effects on the Location of Charge Carriers in Electrospray Droplets: Implications for the Mechanism of Protein Charging and Supercharging.

"Native" electrospray ionization (ESI) mass spectrometry (MS) aims to transfer proteins from solution into the gas phase while maintaining solution-like structures and interactions. The ability to control the charge states of protein ions produced in these experiments is of considerable importance. Supercharging agents (SCAs) such as sulfolane greatly elevate charge states without significantly affecting the protein structure in bulk aqueous solution. The origin of native ESI supercharging remains contentious. According to one model, SCAs trigger unfolding within ESI droplets. In contrast, the "charge trapping model" envisions that SCAs impede the ejection of charge carriers (e.g., NH4+ or Na+) from the droplet. We addressed this controversy experimentally and computationally by employing 18C6 crown ether as a mechanistic probe in native ESI-MS experiments on holo-myoglobin. Remarkably, 18C6 suppressed the supercharging capability of sulfolane. Molecular dynamics (MD) simulations reproduced the experimental charge states. The MD data revealed that 18C6 altered the location of charge carriers in the ESI droplets. Without 18C6, sulfolane covered the droplets in an ionophobic layer that impeded charge carrier access to the surface. In contrast, 18C6 complexation caused charge carrier enrichment in this surface layer, thereby promoting charge ejection. For late droplets, all the water had left and the protein was encapsulated in sulfolane; charge ejection at this stage continued only in the presence of 18C6. As a result, evaporation to dryness of charge-depleted water/sulfolane/18C6 droplets produced low protein charge states, whereas charge-abundant water/sulfolane droplets generated high charge states. Our data support the view that native ESI supercharging is caused by charge trapping. Unfolding within the droplet may play an ancillary role under some conditions, but for the cases examined here, protein structural changes are not a causative factor for supercharging. Our conclusions are bolstered by dendrimer supercharging experiments.

Molecular Dynamics Simulations on Gas-Phase Proteins with Mobile Protons: Inclusion of All-Atom Charge Solvation.

Molecular dynamics (MD) simulations have become a key tool for examining the properties of electrosprayed protein ions. Traditional force fields employ static charges on titratable sites, whereas in reality, protons are highly mobile in gas-phase proteins. Earlier studies tackled this problem by adjusting charge patterns during MD runs. Within those algorithms, proton redistribution was subject to energy minimization, taking into account electrostatic and proton affinity contributions. However, those earlier approaches described (de)protonated moieties as point charges, neglecting charge solvation, which is highly prevalent in the gas phase. Here, we describe a mobile proton algorithm that considers the electrostatic contributions from all atoms, such that charge solvation is explicitly included. MD runs were broken down into 50 ps fixed-charge segments. After each segment, the electrostatics was reanalyzed and protons were redistributed. Challenges associated with computational cost were overcome by devising a streamlined method for electrostatic calculations. Avidin (a 504-residue protein complex) maintained a nativelike fold over 200 ns. Proton transfer and side chain rearrangements produced extensive salt bridge networks at the protein surface. The mobile proton technique introduced here should pave the way toward future studies on protein folding, unfolding, collapse, and subunit dissociation in the gas phase.

Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools.

Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue α-helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high-resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. We also investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrate that the native complexes predominantly exist in a (MRJP14 apisimin4) stoichiometry. Hydrogen/deuterium exchange MS reveals that MRJP1 within these complexes is extensively disordered in the range of residues 20-265. Marginally stable secondary structure (likely antiparallel ß-sheet) exists around residues 266-432. These weakly structured regions interchange with conformers that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a "dimer of dimers" quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316VLFFGLV322. Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP12 apisimin1) stoichiometry. The information uncovered in this work will help pave the way toward a better understanding of the unique physiological role played by MRJP1 during queen differentiation.