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.

Collision-Induced Dissociation of Electrosprayed Protein Complexes: An All-Atom Molecular Dynamics Model with Mobile Protons.

Electrospray ionization mass spectrometry (ESI-MS) has become an indispensable technique for examining noncovalent protein complexes. Collision-induced dissociation (CID) of these multiply protonated gaseous ions usually culminates in ejection of a single subunit with a disproportionately large amount of charge. Experiments suggest that this process involves subunit unfolding prior to separation from the residual complex, as well as H(+) migration onto the unravelling chain. Molecular dynamics (MD) simulations are a promising avenue for gaining detailed insights into these CID events. Unfortunately, typical MD algorithms do not allow for mobile protons. Here we address this limitation by implementing a strategy that combines atomistic force fields (such as OPLS/AA and CHARMM36) with a proton hopping algorithm, focusing on the tetrameric complexes transthyretin and streptavidin. Protons are redistributed over all acidic and basic sites in 20 ps intervals, subject to an energy function that reflects electrostatic interactions and proton affinities. Our simulations predict that nativelike conformers at the onset of collisional heating contain multiple salt bridges. Collisional heating initially causes subtle structural changes that lead to a gradual decline of these zwitterionic patterns. Many of the MD runs show gradual unfolding of a single subunit in conjunction with H(+) migration, culminating in subunit separation from the complex. However, there are also instances where two or more chains start to unfold simultaneously, giving rise to charge competition. The scission point where the "winning" subunit separates from the complex can be attained for different degrees of unfolding, giving rise to product ions in various charge states. The simulated product ion distributions are in close agreement with experimental CID data. Proton enrichment in the departing subunit is driven by charge-charge repulsion, but the combination of salt bridge depletion, charge migration, and proton affinity causes surprising compensation effects among the various energy terms. It appears that this work provides the most detailed account to date of the mechanism whereby noncovalent protein complexes disassemble during CID.

Mechanism of Protein Supercharging by Sulfolane and m-Nitrobenzyl Alcohol: Molecular Dynamics Simulations of the Electrospray Process.

Electrospray ionization (ESI) allows the production of intact gas-phase ions from proteins in solution. Nondenaturing solvent conditions usually culminate in low ESI charge states. However, many mass spectrometric applications benefit from protein ions that are more highly charged. One way to boost protein charge is the addition of supercharging agents (SCAs) such as sulfolane or m-nitrobenzyl alcohol (m-NBA) to the aqueous solution. The supercharging mechanism remains controversial. We use molecular dynamics (MD) simulations to examine how SCAs affect the behavior of ESI nanodroplets. Simulations were conducted on myoglobin in water, water/sulfolane, and water/m-NBA. Na(+) ions served as surrogate charge carriers instead of H(+). We focus on conditions where the protein initially adopts its native conformation. MD-generated charge states show remarkable agreement with experimental data. Droplet shrinkage is accompanied by Na(+) ejection, consistent with the ion evaporation model (IEM). The droplets segregate into an outer SCA shell and an aqueous core. This core harbors protein and Na(+). Unfavorable SCA solvation restricts Na(+) access to the droplet surface, thereby impeding IEM ejection. Rapid water loss causes SCA enrichment, ultimately forcing all remaining Na(+) to bind the protein. IEM ejection is no longer feasible after this point, such that the protein becomes supercharged by Na(+) trapping. SCA-free droplets produce lower charge states because the aqueous environment ensures a higher IEM efficiency. For all scenarios examined here, proteins are released via solvent evaporation to dryness, as envisioned by the charged residue model. Our data provide the first atomistic view of the supercharging mechanism.

Release of Native-like Gaseous Proteins from Electrospray Droplets via the Charged Residue Mechanism: Insights from Molecular Dynamics Simulations.

The mechanism whereby gaseous protein ions are released from charged solvent droplets during electrospray ionization (ESI) remains a matter of debate. Also, it is unclear to what extent electrosprayed proteins retain their solution structure. Molecular dynamics (MD) simulations offer insights into the temporal evolution of protein systems. Surprisingly, there have been no all-atom simulations of the protein ESI process to date. The current work closes this gap by investigating the behavior of protein-containing aqueous nanodroplets that carry excess positive charge. We focus on "native ESI", where proteins initially adopt their biologically active solution structures. ESI proceeds while the protein remains entrapped within the droplet. Protein release into the gas phase occurs upon solvent evaporation to dryness. Droplet shrinkage is accompanied by ejection of charge carriers (Na(+) for the conditions chosen here), keeping the droplet at ∼85% of the Rayleigh limit throughout its life cycle. Any remaining charge carriers bind to the protein as the final solvent molecules evaporate. The outcome of these events is largely independent of the initial protein charge and the mode of charge carrier binding. ESI charge states and collision cross sections of the MD structures agree with experimental data. Our results confirm the Rayleigh/charged residue model (CRM). Field emission of excess Na(+) plays an ancillary role by governing the net charge of the shrinking droplet. Models that envision protein ejection from the droplet are not supported. Most nascent CRM ions retain native-like conformations. For unfolded proteins ESI likely proceeds along routes that are different from the native state mechanism explored here.

Challenges in the interpretation of protein h/d exchange data: a molecular dynamics simulation perspective.

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

Exploring the mechanism of salt-induced signal suppression in protein electrospray mass spectrometry using experiments and molecular dynamics simulations.

Protein analyses by electrospray ionization (ESI) mass spectrometry can suffer from interferences caused by nonvolatile salts. The mechanistic basis of this effect remains to be fully investigated. In the current work we explore the behavior of proteins under native and denaturing conditions in the presence of NaCl, CsCl, and tetrabutyl ammonium chloride (NBu4Cl). All three salts interfere with the formation of "clean" [M + zH](z+) protein ions by progressively deteriorating spectral S/N ratios. We propose that salt interferences can be dissected into two independent aspects, i.e., (i) peak splitting by adduct formation and (ii) protein ion suppression. NaCl degrades the spectral quality by forming heterogeneous [M + zH + n(Na - H) + m(Cl + H)](z+) ions, while the integrated protein ion intensity remains surprisingly robust. Conversely, NBu4Cl does not cause any adduction, while dramatically reducing the protein ion yield. These findings demonstrate that adduct formation and protein ion suppression are indeed unrelated effects that may occur independently of one another. Other salts, such as CsCl, can give rise to a combination of the two scenarios. Molecular dynamics simulations of water droplets charged with either Na(+) or NBu4(+) provide insights into the mechanism underlying the observed effects. Na(+) containing droplets evolve relatively close to the Rayleigh limit (z/z(R) ≈ 0.74), whereas the z/z(R) values of NBu4(+) charged droplets are considerably lower (∼0.59). This difference is due to the high surface affinity of NBu4(+), which facilitates charge ejection from the droplet. We propose that the low z/z(R) values encountered in the presence of NBu4(+) suppress the Rayleigh fission of parent droplets in the ESI plume, thereby reducing the yield of progeny droplets that represent the precursors of gaseous protein ions. In addition, the rate of solvent evaporation is reduced in the presence of NBu4(+). Both of these factors lower the protein signal intensity. NaCl does not interfere with droplet fission, such that protein ions continue to form with high yield­albeit in heavily adducted form. Our findings expand on earlier proposals of charge competition as a key factor during the ESI process for salt-contaminated solutions.

Molecular Dynamics simulations of the electrospray process: formation of NaCl clusters via the charged residue mechanism.

Electrospray ionization (ESI) produces desolvated ions from solution phase analytes for mass spectrometric detection. The final steps of gas phase ion formation from nanometer-sized solvent droplets remain a matter of debate. According to the ion evaporation model (IEM), analytes are ejected from the droplet surface via field emission, whereas the charged residue model (CRM) envisions that ions are released upon droplet evaporation to dryness. Exposure of salt solutions to ESI conditions produces a range of cluster ions. Despite the rich literature on these systems, it is still unclear if these salt clusters form via the CRM or the IEM. The current study explores the formation of Na(n)Cl(m)((n-m)+) clusters from aqueous sodium chloride solution under positive and negative polarity conditions. Molecular dynamics (MD) methods are used for simulating the temporal evolution of charged NaCl-containing water droplets. A trajectory stitching approach is developed for continuously removing evaporated moieties from the simulation, thereby dramatically reducing computational cost. In addition, this procedure ensures adequate temperature control and eliminates evaporative cooling that would otherwise slow down the process. Continuous water evaporation leads to progressive droplet shrinkage, while the emission of solvated single ions ensures that the system remains at ca. 90% of the Rayleigh limit. Early during the process all ions in the droplet behave as freely dissolved species, but after a few nanoseconds at 370 K the systems gradually morph into amorphous wet salt aggregates. Ultimately, free Na(n)Cl(m)((n-m)+) clusters form as the last solvent molecules evaporate. Our data therefore provide direct evidence that sodium chloride cluster formation during ESI proceeds via the CRM. The IEM nonetheless plays an ancillary role, as it allows the system to shed charge (mostly in the form of hydrated Na(+) or Cl(-)) during droplet shrinkage. It appears that this study marks the first successful MD simulation of complete CRM processes.

Lentivector integration sites in ependymal cells from a model of metachromatic leukodystrophy: non-B DNA as a new factor influencing integration.

The blood-brain barrier controls the passage of molecules from the blood into the central nervous system (CNS) and is a major challenge for treatment of neurological diseases. Metachromatic leukodystrophy is a neurodegenerative lysosomal storage disease caused by loss of arylsulfatase A (ARSA) activity. Gene therapy via intraventricular injection of a lentiviral vector is a potential approach to rapidly and permanently deliver therapeutic levels of ARSA to the CNS. We present the distribution of integration sites of a lentiviral vector encoding human ARSA (LV-ARSA) in murine brain choroid plexus and ependymal cells, administered via a single intracranial injection into the CNS. LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA. We identified several genomic hotspots for LV-ARSA integration and identified a consensus target site sequence characterized by two G-quadruplex-forming motifs flanking the integration site. In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells. Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells.