Effects of Multidentate Metal Interactions on the Structure of

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Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations Claire E. Bartman, Haidy Metwally, and Lars Konermann Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01627 • Publication Date (Web): 13 Jun 2016 Downloaded from http://pubs.acs.org on June 13, 2016

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Analytical Chemistry

Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations

Claire E. Bartman, Haidy Metwally, and Lars Konermann*

Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada

Running Title: Metal-Protein Interactions in the Gas Phase

* To whom correspondence should be addressed. Telephone: (519) 661-2111 ext. 86313. Email: [email protected]

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ABSTRACT: Much remains to be learned about the way in which bound metal ions modulate the response of electrosprayed proteins and protein complexes to collisional excitation. Nonspecific metal adducts can affect the extent of collision-induced unfolding (CIU) and collision-induced dissociation (CID). Here we examine how Na+ and Ca2+ adducts alter the CIU response of monomeric proteins under native electrospray conditions. Both of these metals are commonly encountered in biological samples. Measured collision cross sections are largely independent of metal adduction as long as in-source excitation is minimized. In contrast, under CIU conditions the metal-adducted proteins are markedly more compact than their metal-free counterparts. This phenomenon is particularly pronounced for Ca2+ binding, but Na+ adducts have significant effects as well. Molecular dynamics simulations reproduce the experimentally observed trends. The simulations show that structural expansion of the collisionally unfolded proteins is limited by multidentate metal contacts that restrict the conformational freedom of the polypeptide chains. Multidentate interactions with carboxylates and other electron-rich moieties are to be anticipated for divalent metals such as Ca2+. It is surprising that Na+ also engages in multidentate ligation. Electrostatic mapping reveals that the propensity of both Na+ and Ca2+ to interact with multiple electron-rich groups is caused by ineffective charge shielding during ion pairing. Despite their compactness, the CIU structures of metalated proteins do not retain native-like elements. Instead, CIU generates inside-out conformations where previously surface-exposed hydrophilic side chains get buried along with most of the metal ions. Our findings caution that the observation of compact conformers after collisional excitation does not imply the survival of solution-like structural features. We also discuss possible implications of adductmediated effects for CIU fingerprinting studies.


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Numerous proteins

in solution contain metal ions that are required for specific biological

functions such as catalysis, signaling, transport, or detoxification.1-5 Typically these metals undergo multidentate ligation by backbone carbonyl oxygens and electron-rich side chains (including Asp-, Glu-, Cys-, His, Asn, and Gln).6-8 One can distinguish catalytic and structural metal binding sites. The latter are characterized by saturated coordination spheres, and their main function is stabilization of the protein conformation in solution.6 Electrospray ionization (ESI) allows the transfer of proteins and multi-protein complexes into the gas phase.9-12 The resulting ions can be analyzed by mass spectrometry (MS), ion mobility spectrometry (IMS), and other gas phase techniques.13-20 The question whether electrosprayed macromolecules retain their solution structures has attracted considerable attention.15,

21-23

Evidence suggests that the use of neutral aqueous solutions

promotes the preservation of native-like conformations, ligand-protein contacts, and proteinprotein interactions in the gas phase.9,

10, 18, 24

Source conditions have to be carefully

optimized for such “native” ESI studies. Moderately energetic collisions with background gas are required for proper desolvation,25 whereas higher activation levels cause collisioninduced unfolding (CIU) as well as collision-induced dissociation (CID) of noncovalent complexes.19, 26-29 Experimentalists often strive to avoid CIU and CID. On the other hand, it is also possible to deliberately expose gaseous proteins to collisional excitation for probing structural and dynamic features.19,

26-29

For example, it has been proposed that CIU

“fingerprints” can help distinguish between different modes of inhibitor binding.28 In the case of protein complexes, CIU and CID go hand in hand. Multi-subunit systems usually eject a highly charged single subunit. This process involves unfolding of one monomer (CIU) in conjunction with migration of mobile protons, and subsequent ejection of the unraveled chain (CID).9, 30-32



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Factors that influence the structural integrity of electrosprayed proteins are of great interest.33-39 Protein binding to low molecular weight ions is of particular importance in this context.35, 37, 40, 41 These bound ions may originate from specific solution phase interactions.4, 20, 42-44

In addition, native ESI is prone to forming nonspecific adducts.45-51 Small cations and

anions tend to associate with biomolecular analytes during the final stages of nanodroplet evaporation. For example, ESI of NaCl-containing protein solutions generates heterogeneous [M + zH + n(Na-H) + m(Cl+H)]z+ assemblies.52-54 It has been reported that non-specifically bound small ions can stabilize collisionally activated multi-protein complexes, i.e., they shift the onset of CID to higher collision energies.37 Anions that dissociate from the protein as protonated neutrals can lower the internal energy of the analyte by evaporative cooling.52, 55 In contrast, metal adducts remain associated with the protein during collisional excitation.52 Divalent species such as Ca2+ affect CIU and CID processes via mechanisms that remain to be fully elucidated.35, 37 One possibility is that metal ions have a lower mobility than protons, thereby suppressing charge migration which is an integral part of the CID mechanism for multi-subunit systems.30-32, 56-58 Alternatively, metal adducts may form multidentate contacts with the protein35, 37 analogous to structural metal ions in solution.6 In the remainder of this work we focus on the role of metal ions, rather than discussing anion effects. Bound metals cause changes in protein behavior that are more pronounced than those of anionic adducts.37 Also, metal adduction is more prevalent in the commonly used positive ESI mode.52, 54 Metal-mediated effects during ESI are not limited to multi-subunit systems, but have also been reported for monomeric proteins. For example, collision cross sections (Ω) measured by IMS are lower for calcium-saturated calmodulin than for the apo-protein.59 Similarly, increased cadmium binding to metallothionein produces more compact gas phase structures in CIU experiments.60 Both of these examples59,

60


refer to metal adducts that

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originate from specific solution interactions. In addition, compaction effects were reported for proteins carrying nonspecific metal adducts.41 Investigations on small monomeric proteins, rather than multi-subunit systems, should be well suited for exploring fundamental aspects of metal-mediated effects during collisional excitation. Monomeric proteins exhibit distinct metalation signals, whereas multi-subunit systems often show broad peaks that obscure the nature of adduct species.61 Also, the absence of subunit ejection simplifies CIU studies on monomeric proteins. Finally, monomeric proteins are readily amenable to molecular dynamics (MD) simulations of the CIU process. MD techniques have been applied to various gaseous proteins,13, 18, 20, 62-66 but there have been only few attempts to use this approach for CIU simulations in the presence of metal ions.60 The present study examines how nonspecific metal adducts influence the CIU behavior of proteins electrosprayed from non-denaturing solutions. We focus on Na+ and Ca2+ binding to ubiquitin (Ubq), cytochrome c (Cyt), and holo-myoglobin (hMb). Under native solution conditions all three proteins adopt a globular fold that comprises a hydrophilic exterior and a hydrophobic core.67-69 None of them have any specific Na+ or Ca2+ binding affinity in solution. The choice of Na+ and Ca2+ reflects the fact that these two cations are commonly encountered in biological samples.70 Experiments reveal that Ca2+ and Na+ binding leads to CIU structures that are more compact than for metal-free proteins. Using MD simulations and electrostatic mapping we demonstrate that this effect is rooted in multidentate protein-metal contacts that dramatically alter the conformational preferences of the collisionally excited analytes. The complementarity of IMS and MD provides detailed insights into the nature of protein-metal interactions, paving the way towards future studies on other ligand-mediated effects in the gas phase.



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Experimental Reagents. Bovine Ubq, horse heart Cyt, equine hMb, ammonium, sodium, and calcium acetate were from Sigma (St. Louis, MO). Neutral aqueous solutions were prepared at a protein concentration of 10 µM with 10 mM ammonium acetate. For metal-binding experiments the solutions were supplemented with 0.1 mM sodium acetate or calcium acetate.

Mass Spectrometry and Ion Mobility Spectrometry. Mass spectra were recorded on a Synapt G1 HDMS time-of-flight mass spectrometer (Waters, Milford, MA). Ω distributions were measured using the instrument’s traveling wave IMS cell, following procedures described previously.71 Instrument parameters and procedures are detailed in the SI.

Molecular Dynamics Simulations. MD simulations on Ubq in the gas phase were conducted using GROMACS 5.1.72 To match the experimental conditions, all runs were conducted with a 6+ net protein charge. Unless noted otherwise, titratable sites were used with their default charges, i.e., N-terminus+, Arg+, Lys+, His0, Glu-, Asp-, and C-terminus-. The canonical charge of Ubq in neutral solution is zero, resulting from equal numbers of protonated groups (Nterminus+, 4 Arg+, 7 Lys+) and deprotonated moieties (6 Glu-, 5Asp-, C-terminus-). In metal-free solution some of the carboxylates will undergo protonation during the final stages of ESI, thereby imparting the protein with its net 6+ charge.53 For modeling [Ubq + 6H]6+ we chose to protonate all six Glu residues, keeping in mind that Glu- is more basic than Asp-.70 Production runs were conducted over a 100 ns period, during which the temperature was ramped linearly from 350 K to 1000 K. This temperature range is comparable to that used for previous CIU simulations.32,

60, 73

All MD runs were repeated five times with different initial metalation

patterns and atom starting velocities. Additional details of the MD procedures used are provided 6 ACS Paragon Plus Environment

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in the SI. Ω values of MD structures were calculated using the EHSS method in MOBCAL74 which provides adequate Ω estimates for Ubq and other small proteins.18,

41, 62, 75, 76

Minor

modifications of the code were required to allow for the presence of Ca2+. An in-house Fortran program was used for electrostatic mapping.

Results and Discussion Nonspecific Metal Adduction. Mass spectra obtained under native ESI conditions exhibit narrow charge state distributions, with [M + 6H]6+, [M + 8H]8+, and [M + 9H]9+ as the most intense signals for Ubq, Cyt, and hMb, respectively (Figure 1A-C, D-F). Supplementing the analyte solutions with salts causes significant adduction without affecting charge state distributions.45-52 Spectra acquired in the presence of sodium acetate are dominated by species of the type [M + zH + n(Na-H)]z+ (Figure 1G-I), while addition of calcium acetate generates [M + zH + n(Ca -2H)]z+ with n = 0, 1, 2, ... (Figure 1J-L). Anion adduction is minimal, reflecting the propensity of acetate to leave as protonated neutral in the ESI source.52

Collision Cross Sections Under Gentle Source Conditions. Traveling-wave IMS data were acquired for [M + zH]z+ ions and for various metal-adducted forms of the three proteins. We initially focus on experiments conducted with minimum collisional activation, where voltages along the ion path were set just above the transmission threshold (this includes a cone voltage of 5 V). [M + zH]z+ ions of Ubq, Cyt, and hMb generated under these conditions exhibit Ω distributions with maxima at 985 Å2, 1324Å2, and 1884 Å2, respectively (Figure 2A-C). These results are consistent with previous reports18, 29, 62, 77 and they are close to Ω values calculated for the corresponding X-ray structures.76 These findings are consistent with the view that gentle ESI conditions allow the preservation of solution-like structures in the gas phase.9, 10, 18, 24, 78, 79 7 ACS Paragon Plus Environment


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It is not clear from the literature41, 53 to what extent nonspecific metal adducts affect the structures of globular proteins under gentle ESI conditions. Figure 2A shows that Ω values of [Ubq + 6H]6+, [Ubq + 6Na]6+, and [Ubq + 3Ca]6+ differ by no more than 2%. For Cyt the protonated, sodiated, and calciated forms share virtually the same IMS characteristics (Figure 2B). Na+ and Ca2+-bound hMb are slightly (~4%) more compact than in the absence of metal adducts (Figure 2C). Overall, metal-induced conformational differences of the three electrosprayed proteins are quite minimal under the gentle conditions of Figure 2A-C. Much larger differences were reported in ref.41, where binding to 6 Na+ lowered Ω of Ubq and Cyt by ~10%. This apparent contradiction is resolved when considering that ref.41 used harsher conditions (including a cone voltage of 40 V), thereby inducing partial CIU with multimodal Ω distributions. This is in contrast to the gentle settings used for the data of Figures 2A-C that generate more compact structures (cone 5 V).

Effects of Metal Adducts on Collision-Induced Unfolding. CIU can be triggered by activating protein ions in the ESI source.26 For the mass spectrometer used here the cone (“declustering”)25 voltage is the most pertinent parameter in this context. On other instruments raising the IMS injection energy has analogous effects.80 CIU can also be implemented further downstream along the ion path, for example in the trapping region.28 However, changes in cone voltage produced data with the highest S/N in our experiments, keeping in mind the low intensities for some of the adduct signals. IMS measurements on Ubq and Cyt were acquired at cone voltages between 5 V and 135 V. In the case of hMb measurements beyond 85 V were precluded by heme loss.26 The occurrence of CIU is readily apparent from shifts of the IMS distributions to larger Ω for all three proteins (Figure 2). Several of the Ω distributions are multimodal, reflecting the presence of co-existing gas phase conformers.19, 8 ACS Paragon Plus Environment

20, 41

The data of Figure 2

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reveal marked differences in the CIU behavior of metal-free and adducted proteins. For example, at 45 V compact structures of [Ubq + 6Na]6+ and [Ubq + 3Ca]6+ are more abundant than for [Ubq + 6H]6+ (Figure 2D). Ubq at 125 V shows large-scale unfolding; protonated Ubq is most expanded under these conditions (Ω =1450 Å2), while the Ω values of the sodiated and calciated forms are smaller by 4% and 6%, respectively (Figure 2J). Similarly, with the cone set to 85 V [Cyt + 8H]8+ is extensively unfolded, whereas more compact conformers remain detectable for the Na+ and Ca2+-bound forms (Figure 2H). In analogous fashion, Ca2+ binding to hMb favors structures that are more compact. At 85 V [hMb + 3Ca]6+ has a Ω value that is 5% below that of [hMb + 9H]9+ (Figure 2L). The charts shown along the bottom of Figure 2 compare CIU profiles of [M + zH]z+ generated in pure ammonium acetate solution with those of proteins carrying six Na+ or three Ca2+. Adductdependent differences are particularly prevalent between 50 V and 85 V. In this transition region the metalated species of all three proteins are more compact than the protonated forms for any given cone voltage (Figure 2M-O). The number of bound metal ions has a striking effect on the protein structure. This is illustrated in Supporting Figures S1 and S2, which show the CIU behavior of [M + zH + n(Na-H)]z+ and [M + zH + n(Ca -2H)]z+ as a function of n. Protein ions generally become more compact with increasing n under CIU conditions. Our IMS data show that during collisional activation the presence of metal adducts tends to produce structures that are more compact than those of metal-free proteins. Previous CIU/CID work on multi-subunit complexes reported comparable effects, but primarily for divalent metal ions.37 The current study shows that for monomeric proteins Na+ can have significant effects as well, although Ca2+ is a more effective “compactor ion” than Na+.

Molecular Dynamics Simulations of the CIU Process. For elucidating the physical basis of the experimentally observed metal adduction effects we resorted to MD simulations. The focus 9 ACS Paragon Plus Environment


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of these simulations will be on Ubq6+ because adducts caused the largest CIU differences for this protein. Also, complicating factors regarding the MD treatment of heme-protein interactions do not have to be considered for Ubq. Starting structures for simulation runs on [Ubq + 6H]6+, [Ubq + 6Na]6+, and [Ubq + 3Ca]6+ are summarized in Supporting Figure S3. Collisional heating was simulated by ramping the temperature from 350 K to 1000 K over a period of 100 ns. Protein structural changes were very minor during the initial stage of the simulations, i.e., for the temperature range of roughly 350 K to 480 K. Figure 3A-C exemplifies typical T = 480 K conformations. Comparison with X-ray coordinates67 (Figure 3A-C, green) confirms that the protonated and metalated species retain native-like conformations at 480 K. Closer examination reveals that protonated Ubq bears the closest resemblance to the X-ray structure, while minor distortions take place for the Na+ and Ca2+ bound forms. The corresponding RMSD values relative to the crystal Cα coordinates are around 0.3 nm, 0.5 nm, and 0.6 nm, respectively. Just like the X-ray structure67 the simulated 480 K conformations in Figure 3A-C exhibit a hydrophobic core, while charged and polar residues (as well as bound H+, Na+, and Ca2+) are on the surface. The calculated Ω values at 480 K (protonated: 1036 Å2, sodiated: 1037 Å2, calciated: 1055 Å2) are close to the Ωav data measured at a cone voltage of 5 V (1020 Å2, 1036 Å2, and 1029 Å2, respectively). Gradual ramping of the simulation temperature to 1000 K caused complete breakdown of the native structure in the all MD runs. Three representative examples of CIU structures produced in this way are shown in Figure 3D-F (see Supporting Figure S3 for a comprehensive summary). Interestingly, the CIU structures of sodiated and calciated Ubq6+ have most of their adduct ions and charged side chains clustered together in buried locations. Most hydrophobic residues point toward the protein exterior (illustrated in Figure 4). The possible existence of such inside-out structures in vacuo has been suggested previously.22,

66

Protonated Ubq at 1000 K

does not show pronounced inside-out character, and its CIU structures are more expanded than 10 ACS Paragon Plus Environment

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those of the metalated species (Figure 3D). None of the MD runs involved charge carrier ejection, consistent with the fact that electrostatic ligand-protein contacts are extremely stable in the vacuum.81 Figure 5A provides a closer look at the simulated CIU behavior of protonated, sodiated, and calciated Ubq6+ by plotting the radius of gyration (Rg) vs. temperature. Unfolding at high temperature is most extensive for [Ubq + 6H]6+, while [Ubq + 3Ca]6+ remains most compact. [Ubq + 6Na]6+ displays intermediate behavior. Figure 5B shows the MD data in a different format, by plotting the temperature dependence of the calculated Ω values. It is gratifying that these MD data qualitatively reproduce the key experimental trends, i.e., (i) bound metal ions lead to CIU structures that are more compact than for protonated Ubq, and (ii) collisionally unfolded Ubq carrying Ca2+ is more compact than the Na+ adducted protein. We do not claim that the T = 1000 K conditions at the endpoint of the MD trajectories exactly mimic the experimental environment at a cone voltage = 135 V. Nonetheless, it is interesting that for [Ubq + 3Ca]6+ the endpoints of the simulated and experimental Ω profiles agree within less than 1%. The simulated protonated and sodiated CIU structures exhibit Ω values that are somewhat larger than the experimental numbers (compare Figures 5B and 2M).

Mechanism of Metal-Mediated CIU Effects. Differences in the CIU behavior of protonated and metalated Ubq are caused by the specific types of protein-charge carrier interactions. (i) [Ubq + 6H]6+ possesses six protonated Glu. The R-COOH moieties generated in this way have low propensity to interact with other parts of the protein. Instead, each of them remains isolated on the surface, both at the onset of the MD runs (Figure 6A) and after CIU (Figure 6B). The effects of excess H+ on the conformational freedom of the protein are minimal. (ii) In the case of [Ubq + 6Na]6+ each of the Na+ forms multidentate interactions with various groups of the protein. Many of these interactions involve metal solvation by one or two 11 ACS Paragon Plus Environment


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side chain carboxylates, often with participation of side chain or backbone carbonyl groups. These metal binding modes are consistent with previous studies.52,

54, 82

Figures 3B, 6C

demonstrate that such multidentate contacts can form without necessitating large-scale distortion of the native fold. The same types of multidentate contacts persist for the inside-out structures generated for [Ubq + 6Na]6+ under CIU conditions (Figure 6D). Multidentate solvation of the partially buried metals by the protein restricts the conformational freedom of the chain and produces CIU structures that are less expanded than for [Ubq + 6H]6+. (iii) Multidentate protein-metal contacts are most prevalent for [Ubq + 3Ca]6+, both at the onset of collisional activation and after heating to 1000 K (Figure 6E,F). The three buried Ca2+ in the inside-out CIU structures of [Ubq + 3Ca]6+ are surrounded by a solvation shell that includes eleven of the protein's thirteen carboxylates (Figure 6F). This extreme side chain sequestration causes highly compact CIU structures, with Rg and Ω values significantly below those of [Ubq + 6H]6+ and [Ubq + 6Na]6+ (Figure 5A,B).

Electrostatic Aspects of Protein-Cation Interactions. Why do Na+ and Ca2+ form multidentate contacts, while such bridging interactions are not observed in protonated Ubq? We will examine this question by focusing on the behavior of carboxylates which represent the main attachment sites for all three charge carrier types. It is straightforward to explain why Ca2+ will interact with more than one R-COO- group; binding of a single Ca2+ to a carboxylate generates (R-COOCa)+. This moiety has one remaining positive charge, allowing for ion pairing with a second R-COO-. However, such simple ion pairing arguments do not fully capture the nature of protein-metal interactions. Our MD data reveal that one Ca2+ can interact with more than two negative side chains; Figure 6F shows three Ca2+ surrounded by eleven carboxylates (corresponding to 3.66 carboxylates per calcium). More importantly, sodiation and protonation both produce moieties that are formally neutral (R-COONa and R-COOH). It has to be examined why R-COOH is 12 ACS Paragon Plus Environment

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incapable of forming multidentate contacts, whereas arrangements such as Glu16...Na...Glu18 (Figure 6C) are quite common. A mapping analysis provides insights into the propensity of the different charge carriers to engage in multidentate contacts. Figure 7 shows the electrostatic potential experienced by a negative charge in the vicinity of a R-COOH, R-COONa, and a (R-COOCa)+ group. Blue/black represents regions that attract negative charges, while orange/red marks regions of repulsion. In other words, the black/blue areas in Figure 7 are where additional electron-rich ligands such as R-COO- will preferentially attach. In the case of R-COOH the bound proton is closely coupled to a carboxylate oxygen, with an O-H bond distance of 1.0 Å. The positive charge of the proton is effectively neutralized, such that the H+ cannot participate in ion pairing with an additional RCOO- (Figure 7A). A very different situation is encountered for R-COONa (Figure 7B). Closerange (van der Waals)83, 84 repulsion between O and Na+ results in O-Na distances of 2-3 Å. This large spacing renders shielding of the positive charge by R-COO- ineffective (black/blue areas in Figure 7B). As a result, ion pairing of Na+ with a single carboxylate produces an unsaturated site that retains high affinity for interaction with additional electron-rich protein moieties. Figure 7C shows an electrostatic map of (R-COOCa)+. The extremely high affinity of bound calcium ion for additional electron-rich ligands partly originates from the net positive charge of the calciated carboxylate. An additional factor is the significant distance between the Ca2+ and the oxygen atoms (2-3 Å, similar to Na+) which prevents effective charge shielding. This explains why Ca2+ maintains the capacity to bind additional electron-rich protein moieties, even after chelation by two R-COO- groups. One example of such a multi-dentate arrangement is the interaction of the Thr22 and Gln25 side chain oxygens with Asp21...Ca...Glu24 (Figure 6E).



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Conclusions The current work reveals that multidentate metal contacts are a key determinant of protein behavior during collisional excitation. Under CIU conditions these metal contacts lead to unfolded structures that are more compact than those of metal-free proteins. Ligation of each metal ion by two or more protein moieties limits the conformational expansion of unfolded polypeptide chains, akin to the effects of disulfide bridges70 or other covalent crosslinks.30 Under gentle source conditions metal-adducted as well as metal-free proteins retain conformations that are close to the native solution structure (Figure 3A-C). In contrast, the observation of relatively compact CIU structures for metalated proteins does not imply the retention of native-like elements after collisional heating. Instead, CIU of metalated proteins triggers the formation of inside-out conformations, where previously solvent-exposed side chains and metals become buried in the interior (Figure 3E,F). For the cases examined here it would thus be misleading to conclude that metal adducts “stabilize” collisionally activated proteins. Instead, metals induce smaller Rg and Ω values for collisionally unfolded chains by tethering parts of the protein together.37 This metal-induced compaction does not promote the survival of native-like structures under CIU conditions. The onset of the CIU transitions occurs at very similar excitation energies for the protonated and metalated proteins, both in experiments (Figure 2M,N,O) and in the MD simulations (Figure 5). On the basis of simple ion pairing arguments one would expect that only divalent cations give rise to multidentate contacts. Surprisingly, we find that even singly charged ions such as Na+ engage in interactions with multiple electron-rich protein moieties. The electrostatic mapping data of Figure 7 provide a simple explanation for this behavior. Both Na+ and Ca2+ therefore induce a significant compaction of CIU structures, although Ca2+ mediated effects are more pronounced.


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The current work has implications for studies aimed at using CIU fingerprints for deciphering structural features of biomolecular analytes.28 Our findings suggest that comparative measurements will yield reliable results only when electrospraying different samples in solutions that generate exactly the same adduction patterns. For small proteins the adduction status is usually apparent in the mass spectra. However, this is not necessarily the case for larger systems, where native ESI tends to generate broad peaks that make it difficult to pinpoint the chemical nature of adducted species.61 In CIU fingerprinting experiments it is therefore imperative to carefully control the concentrations of trace contaminants that might contribute to protein adduction. For multi-protein complexes it has been found that divalent metals shift the onset of CID to higher collision energies.35, 37 Although the current work did not examine the behavior of such complexes, our findings are consistent with the view35, 37 that multidentate contacts within individual subunits and across different chains are a key contributor to this effect. Realistic simulations of CIU/CID processes for multi-protein systems require the inclusion of mobile proton algorithms32 that are difficult to implement with standard MD protocols. Our aim is to address this problem by developing MD strategies that consider both charge migration and metal binding for multi-subunit proteins. Overall, it appears that combined MD/IMS investigations will become a powerful tool for deciphering the role of ligand binding effects on the structure and dynamics of biomolecular analytes in vacuo.

Acknowledgments. Funding for this work was provided by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant 217080-2013).

Supporting Information Available. Additional figures as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. 15 ACS Paragon Plus Environment


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Figure Captions Figure 1. ESI mass spectra of Ubq, Cyt, and hMb. (A-C) Overall spectra acquired in ammonium acetate. (D-F) Close-ups of the base peaks in panels A-C. (G-I) Base peak region of the three proteins acquired in the presence of sodium acetate. (J-L) Base peak region acquired in the presence of calcium acetate. Data in panels D-L are displayed on a deconvoluted mass axis. Numbers indicate the number of bound metal ions (Na+ or Ca2+).

Figure 2. IMS distributions of Ubq6+ (column 1) Cyt8+ (column 2) and hMb9+ (column 3) acquired at different sample cone voltages. Each panel contains data for three types of ions: protonated only (black dashed lines, acquired in ammonium acetate without salt supplements), bound to six Na+ (blue solid lines, acquired with added sodium acetate), and bound to three Ca2+ (red solid lines, acquired with added calcium acetate). Panels shown along the bottom (M, N, O) display the average collision cross section Ωav as function of cone voltage for the different proteins and salt supplements. Typical error bars for triplicate measurements are indicated for some of the data points.

Figure 3. (A) Comparison of the X-ray coordinates 1UBQ (green) with a simulated [Ubq + 6H]6+ structure (gray) at T = 480 K. (B) Same as in panel A, but for [Ubq + 6Na]6+ (blue). (C) Same as in panel A, but for [Ubq + 3Ca]6+ (red). Simulated CIU structures at T = 1000 K are depicted in the bottom row for (D) [Ubq + 6H]6+, (E) [Ubq + 6Na]6+, and (F) [Ubq + 3Ca]6+. Spheres represent excess H+ (gray) Na+ (blue), or Ca2+ (red).

Figure 4. (A) Native Ubq X-ray structure. (B) Simulated CIU structure of [Ubq + 3Ca]6+, taken from Figure 3F. Charged and hydrophilic residues are shown in cyan (termini, Arg, Lys, His, Asp, Glu, Gln, Asn, Thr, Ser, His, Tyr). Hydrophobic residues are highlighted in 19 ACS Paragon Plus Environment


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yellow (Leu, Ile, Val, Ala, Phe).85 All others are shown in gray. Note how for the native conformation the protein surface is dominated by charged/hydrophilic residues. In contrast, Panel B represents an inside-out conformation, where numerous hydrophobic side chains are exposed to the vacuum environment.

Figure 5. Simulated CIU profiles generated by exposing gaseous Ubq to a linear temperature gradient. (A) Radius of gyration Rg for [Ubq + 6H]6+ (black); [Ubq + 6Na]6+ (blue), and [Ubq + 3Ca]6+ (red). (B) Collision cross sections Ω calculated at selected points during the simulations. MD data were averaged over five runs, error bars indicated typical standard deviations.

Figure 6. Charge carrier interactions with the protein for [Ubq + 6H]6+, [Ubq + 6Na]6+, and [Ubq + 3Ca]6+. Top row: Examples of MD starting structures at the onset of collisional heating, bottom row: CIU structures after 100 ns (T = 1000 K). White spheres represent excess protons (A, B); blue spheres denote Na+ (C, D); red spheres indicate Ca2+ (E, F). Panels D-F highlight protein moieties that interact with metal ions, all others are omitted. Regular font denotes side chain contacts, bold letters represent main chain contacts.

Figure 7. Color map of the electrostatic potential V(x,y) experienced by a -e point charge in the x/y plane of a (A) protonated carboxylate, (B) sodiated carboxylate, and (C) calciated carboxylate. Colors represent the potential in MD units (e nm-1). Negatively charged ligands such as R-COO- will be attracted to regions with strongly negative V(x,y), indicated in dark blue and black. Atomic charges (indicated by numbers) and bond distances reflect CHARMM parameters; white areas indicate van der Waals-disallowed regions.


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Figure 1 Ubq

Cyt

6+

ESI-MS Intensity 1000

8+

A

1500

2000

hMb

1000

9+

B

1500 m/z

2000

C

1000 1500 2000 2500

E

D

F

NH4+

0

G

1

4

0

Ca2+

5

3

4

6

J

1

0

5

4

I

5

6

6

K

1

1

2

0

3 2

3

0 2

3

Na

2

1

1

2 +

H

0

L

2 3

Y Data

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3

8560 8600 8640 8680

12360 12410 12460

17570 17610 17650 17690

Mass (Da)



Figure 2 6+

Cyt

Ubq Cone 5V

A

8+

9+

hMb

Cone 5V

B

Cone 5V

C

protonated + with 6 Na 2+ with 3 Ca

45 V

D

45 V

E

45 V

F

65 V

G

85 V

H

65 V

I

125 V

J

125 V

K

85 V

L

900

1200

1500

1800

1200

1500

1800

2100

1500

2000

2500

3000

Collision Cross Section Ω (Å2)

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1850

1200

1600

N

O

2400

protonated + with 6 Na with 3 Ca2+

2200 2000

1350

1000 5

35

65

95

125

1800 5

35

65

95

125

Cone Voltage (V)


0

30

60

90

Ωav (Å2)

M Ωav (Å2)

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Figure 3



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Figure 4


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Figure 5

Time (ns) 0

Radius of Gyration Rg (nm)

2.5

30

60

90

A

2.0 [Ubq + 6H]6+ [Ubq + 6Na]6+ [Ubq + 3Ca]6+ 1.5

1.0

Collision Cross Section Ω (Å2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


B 1600

1400

1200

1000 400

500

600

700

800

900 1000

Temperature (K)



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Figure 6


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Figure 7

1.0

1.0

C

B

V(x,y) -10 -8 -6 -4 -2 0 2 4

0.8

0.6

0.4

0.8

0.6

0.44 -0.61

0.2

H

-0.55

O

O

1.0

2.0

Na

Ca

-0.76

O

C 0.75

-0.76

-0.76

O

O

C0.62

0.4 -0.76

O

0.2

C0.62

0.0

0.0 -0.4

-0.2

0.0

0.2

0.4

-0.4

-0.2

0.0

0.2

0.4

x (nm)


-0.4

-0.2

0.0

0.2

0.4

y (nm)

A

y (nm)

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For TOC Only


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Effects of Multidentate Metal Interactions on the Structure of

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