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Structural Preferences of Gas-Phase M2TMP Monomers upon Sequence Variations

Florian Albrieux,,, Hisham Ben Hamidane,|| Florent Calvo,, Fabien Chirot,*,, Yury O. Tsybin,|| Rodolphe Antoine,, Jr^me Lemoine,, and Philippe Dugourd, eo

Universit de Lyon, CNRS, UMR 5579, LASIM, and CNRS, UMR 5180, LSA, Universit Lyon 1, F-69622 Villeurbanne, France; e e Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fdrale de Lausanne, 1015 Lausanne, Switzerland. e e

S b Supporting Information

ABSTRACT: The conformations of a number of M2TMP(2246) sequence variants have been investigated using ion mobility spectrometry (IMS). Substantial conformational changes were evidenced by IMS upon the variation of a single amino acid in the peptide sequence, with two main drift time signatures. Replica-exchange molecular dynamics simulations were used to help assign the structures of the dierent identied conformers. Even though one-on-one agreement with experiment was found for only two variants, the simulations generally conrmed the existence of two structural families. Based on these results, most of the triply protonated variants, including the wild-type peptide, were found to display collision cross sections in agreement with compact conformations in the gas phase, whereas they tend to form extended R-helices in the condensed phase, as conrmed by circular dichroism and previously reported NMR measurements. The destabilization of R-helices in vacuo upon amino acid substitution is interpreted as being driven by the solvation pattern of the charges.

I. INTRODUCTION The function of biomolecules largely depends on their conformation, and it is crucial to determine which factors inuence this conformation. In this respect, a key issue is the role of the sequence in the way a peptide or protein folds. One way to gain insight into this problem is to question how the structure is stabilized or destabilized upon modications of the properties of the molecule, in relation to the possible enhancement or inhibition of its biological activity. As a complement to other methods, experiments in the gas phase are particularly well-suited to address some of these questions because they allow molecules to be studied in the absence of solvent, thereby providing access to their intrinsic properties.14 Several important factors that aect peptide structure have already been identied. First, the nature of the dierent amino acids present in the sequence has a signicant inuence on the conformation, especially their propensity to form secondary structures, including helices.58 The amount of charges, their location, and their pattern of solvation by the surrounding residues have also been identied as important factors for secondary structure stabilization,812 as well as more distant interactions, for example, the formation of hydrogen bonds. In addition, the roles of temperature and complexation have been examined.1315 To date, the most advanced tool for studying peptide conformation in vacuo experimentally is probably infrared actionspectroscopy (IR/UV or IR multiphoton dissociation). This
r 2011 American Chemical Society

method has proven to be an ecient probe for the local structural arrangement of relatively small peptides16,17 and, more recently, was even able to unravel their entire structure.18,19 Up to now, none of the other fragmentation techniques available for peptides could clearly exhibit a comparable sensitivity to secondary structure, even though some recent results suggest that fragmentation yields by other methods could be correlated with conformation.2023 In addition to fragmentation, neutral molecule beam deection experiments have also provided useful structural data on peptides in the absence of charges through the measurement of electric dipole moments.5,13 General information on the conformation of isolated peptides can also be obtained using hydrogen/deuterium exchange experiments.24,25 Finally, since the pioneering works by the groups of Bowers26 and Jarrold,27 ion mobility spectrometry (IMS) has served as a powerful tool for secondary to quaternary28 structure determination through the measurement of the diusion cross sections of the investigated ions through a buer gas. Most of our current knowledge about helical stability in the gas phase has indeed been obtained through IMS measurements.815 Whereas the wealth of experimental methods oers complementary probes, none of them, alone, can characterize the entire conformation of a gaseous biomolecule. Structural determination requires a
Received: November 10, 2010 Revised: February 22, 2011 Published: April 13, 2011
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The Journal of Physical Chemistry A Table 1. List of the Investigated Variants of M2TMP(2246) with the Codes Used to Name Them in the Texta
sequence name wild H16G D3R S10N V6A V7G G13L
a

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7 Val Val Val Val Val Val Val

10 Ser Ser Ser Ser Ser Ser Ser

11 12 Ile Ile Ile Ile Ile Ile Ile Ile Ile Ile Ile Ile

13 Gly Gly Gly Gly Gly Gly Gly

14

15

16

17

18

19

20

21

22

23

24

25

Ser Ser Asp Pro Leu Val Ser Ser Asp Pro Leu Val Ser Ser Arg Pro Leu Val Ser Ser Asp Pro Leu Val Ser Ser Asp Pro Leu Ala Ser Ser Asp Pro Leu Val

Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu Ile Leu Gly Leu Ile Leu Trp Ile Leu Asp Arg Leu Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu Ile Leu Gly Leu Ile Leu Trp Ile Leu Asp Arg Leu Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu

D3R H16G Ser Ser Arg Pro Leu Val

Ala Ala Asn Ile Ile

Ser Ser Asp Pro Leu Val Gly Ala Ala

Ile Ile Leu Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu

Amino acids diering from the wild sequence are underlined and in bold.

comparison of the measured data with calculations. However, the appropriate theoretical methods largely depend on the system and on the type of measurements, and they generally range from molecular mechanics to explicit descriptions of the electronic structure. One of the main diculties of these calculations aimed at predicting structures is to achieve a broad sampling of the energy landscape accessible for biomolecules. In most cases, this landscape is expected to be highly rugged, and even with force elds, ergodicity in the simulations is dramatically hampered, thus limiting the scope of most successful investigations to relatively small systems. Structural trends can nevertheless be inferred for reasonably large biomolecules by comparing calculated diusion cross sections for trial structures with IMS data. For instance, we recently used replica-exchange molecular dynamics (REMD) coupled with local minimizations to generate realistic structure samples that could be successfully compared with measured IMS cross sections for series of model peptides containing 11 amino acids.9 In the present work, the same technique used in ref 9 was applied to several variants of a subunit of the M2 transmembrane protein from inuenza virus A (M2TMP, residues 2246). When bound as a tetramer bundle in physiological solution, this protein forms a proton channel that is involved in virus replication.29,30 It has been intensively studied to understand the action mechanism of antibiotics, such as amantadine and rimantadine, used to inhibit the function of the proton channel.3134 Recently, the eect of particular mutations on the eciency of the drugs was carefully addressed.35 From previous works, wild-type M2TMP monomers have been shown to adopt a helical conformation in solution,36 as well as to crystallize in either a tetramer37 or isolated forms.38 Moreover, electronic circular dichroism (ECD) experiments suggest the presence of M2TMP helical conformations in the gas phase as well.20 Here, we used IMS and REMD/minimization simulations to investigate the gas-phase conformations of a series of variants of M2TMP(2246). Some of the variants were designed to probe the inuence of factors that have already been identied as strongly inuencing secondary structure, such as charge location. Other variants correspond to variations known to alter the activity of the proton channel formed by the M2TMP tetramer. The IMS data show signicant conformational changes upon the variation of a single amino acid, primarily forming two distinct structural groups by the similarity in the cross sections. The simulations turned out to be much more challenging and generated a very large number of stable conformations, even on relatively limited sampling times. One particular diculty for such a force-eld modeling was the assignment of protonation sites,

which appeared to aect the stable structures to some extent. However, even with these limitations, the simulations conrm the existence of two structural families compatible with experimental measurements, namely, some elongated helices and some more compact conformations.

II. MATERIALS AND METHODS

II.A. Peptide Synthesis. M2TMP wild-type peptide and variants were synthesized by Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis using an Applied Biosystems 433A synthesizer and used without further purification (Protein and Peptide Synthesis Facility, Biochemistry Department, University of Lausanne, Lausanne, Switzerland). The peptides were dissolved in water to approximately 1 mM concentration and further diluted in a standard spraying solution (H2O/CH3OH 50:50 volume ratio with 0.1% acetic acid) to a final peptide concentration of about 100 M. The different mutants studied here differ from the wild-type sequence (SerSer-Asp-Pro-Leu-Val-Val-Ala-Ala-Ser-Ile-Ile-Gly-Ile-Leu-His-LeuIle-Leu-Trp-Ile-Leu-Asp-Arg-Leu) by one or two amino acids. They are listed in Table 1, together with their sequence and the name codes used herein. For instance, H16G stands for the substitution of a histidine by a glycine at position 16 in the wildtype peptide sequence. The rst three variants, H16G, D3R, and H16G D3R, were designed to change the position of the charges. The eect of such modications on helix formation has already been reported in previous works.12,39,40 Other variants correspond to mutations known to alter the eciency of the M2TMP tetramer as a proton channel (G13L or H16G)35 or to cause a resistance to the action of amantadine (S10N or V6A).31,35 II.B. Circular Dichroism Measurements. Far-UV circular dichroism (CD) spectra were collected on a Jasco J-810 spectropolarimeter (Jasco Corporation, Tokyo, Japan) at 37 C using a cuvette with a path length of 0.1 cm. The spectra were recorded between 190 and 250 nm, with 0.2-nm wavelength steps and a bandwidth of 1 nm. Samples were dissolved to a final concentration of 100 M in water, methanol, or acetonitrile. For every reported spectrum, two scans were averaged to improve signalto-noise ratio. Circular dichroism spectra were fitted using the CD-FIT program (B. Rupp, 1997, revised 2003) to estimate the secondary structure contents for the investigated variants. The fitting procedure is based on a least-squares adjustment and takes into account the superposition of R-helix, -sheet, and randomcoil contributions to the spectra.
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II.C. IM-MS Measurements. The IM-MS measurements were carried out using a custom-built ion mobility spectrometer. The experimental setup is described elsewhere.9 Briefly, the ions are formed in an electrospray ion source and transferred to a cylindrical ion trap41 through an ion funnel. Ion pulses from the trap are periodically injected directly in a 1-m-long drift tube filled with 12 Torr of helium and maintained at 293 K owing to a regulated room temperature. The duration of an ion pulse is 1 ms at the injection. At the end of the tube, the ions enter a vacuum chamber through a 0.7-mm-diameter aperture. The ions are finally conveyed through two ion funnels to a time-of-flight mass spectrometer (microTOFQ, Bruker-Daltonics, Bremen, Germany), where they are detected as a function of their mass-to-charge ratio and drift time. At a given electric eld E, the ions travel across the drift tube at a constant speed vD, and the arrival time, tD, is related to this eld through

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tD

L L vD KE

where K denes the ion mobility and L is the length of the drift tube. Under the experimental conditions, K is inversely proportional to the orientationally averaged diusion cross section 42     3 ze 1 1 1=2 2 1=2 1 2 K 16 N m M kT In eq 2, ze is the charge of the ion; N is the buer gas density; k is the Boltzmann constant; T is the temperature; and m and M are the masses of the helium atom and the ion, respectively. As a direct consequence of this formula, the most compact conformers have the shortest drift times. In practice, the drift time tD is measured for various drift voltages V, and the ion mobility is obtained from the slope of tD as a function of V1. The corresponding cross section can then be derived using eqs 1 and 2. The experimental precision on cross sections is estimated to be no larger than 1%. II.D. Theoretical Methods. The conformational landscapes of [M2TMP 3H]3 and its variants have been explored using REMD conformational sampling based on the Amber99 force field,43 coupled with local minimizations to locate low-energy structures. The numerical protocol is the same as was already used in ref 9. It is divided into three stages: Starting from an elongated conformer (not even locally optimized), a series of 1-ns-long REMD trajectories in the temperature range 100 1000 K with 40 replicas and a geometric progression in temperatures were carried out in which instantaneous configurations were saved every 1 ps. Thermalization was achieved using a Berendsen thermostat with coupling constant of 1 ps1. The time step used for all REMD simulations was 1 fs, and the dielectric constant was set to 1. Every 100 fs, an exchange was attempted between two random adjacent replicas. Configurations, saved for all replicas every 1 ps, were locally optimized, and the lowest-energy structure was subsequently used as the new starting configuration for a new REMD simulation. Up to three such simulations are performed in order to approximately locate the putative global minimum. The second stage was to perform a longer REMD simulation of 10 ns, with the temperatures now being chosen in relation to experimental relevance, namely, in the 100500 K range with an arithmetic progression of 50 K. A set of 1000 configurations extracted from these trajectories was then subjected to further conformational and statistical analysis.
Figure 1. Circular dichroism spectra for dierent variants of the M2TMP peptide recorded in water (black curves) and t of the CD spectra (red curve). See text for details.

The collision cross sections were calculated using the direct trajectory method from Mesleh et al.,44 yielding a distribution of cross sections. Finally, simulated arrival time distributions were generated using eqs 1 and 2 to calculate the drift time, tD, corresponding to each calculated cross section. The arrival time distribution (ATD) for a single isomer was modeled through the convolution of the injected ion pulse with Fick's law for the diffusion in the tube.42,45 The expression of the ion flux at time t at the end of the tube for isomers with average drift time tD and injected at time t0 is then ! Z 2 vD 2 tD t0 t dt0 It0 exp vD st, tD p 4DtD 4DtD 3 where I(t0) represents the initial ion pulse and D = KkBT/q is the diffusion constant at temperature T for ions of charge q and mobility K.42 The global ATD was then calculated as a sum of the contributions of the identified isomers weighted by their abundance, C(tD), in the simulated structure sample St

CtD st, tD t
D

All simulations were carried out on the 3 charge states of the variants. In most cases, the charges were assumed to be located on the N-terminus amide and on the side chains of His16 and Arg23, as no other basic sites were available. However, for two of the investigated variants, no obvious charge location could be inferred. D3R, for instance, has four basic sites available for only three charges. The relatively large size of the present peptides makes conformational sampling challenging already with a
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Figure 2. Histogram of secondary structure content for M2TMP wild type and G13L and H16G variants determined by circular dichroism in pure H2O. Error bars were directly obtained from the correlation factor of the tting algorithm.

simple, nonpolarizable force eld; hence, we did not consider possible extensions able to deal with intramolecular proton transfer.4649 Ideally, such models could be used also to predict the most favorable protonation sites. However, the complex interplay between temperature, conformation, and proton transfer11,46 makes it unlikely that the energy landscape of such relatively large peptides could be sampled eciently with such reactive models. Because of the higher proton anity of arginine, we assumed that both Arg3 and Arg23 were charged, leaving two possibilities corresponding to protonation at the N-terminus with a neutral His16 (further denoted NRR) or to a neutral N-terminus with a charged His16 (further denoted RHR). Simulations were performed for both cases. More problematic was the case of H16G, for which only two naturally basic sites were available for the three extra protons. Here, we assumed protonation at Pro4, because proline appears to have a higher proton anity than the other residues.50

Figure 3. Experimental arrival time distributions recorded for the dierent variants using a 770 V drift voltage and a helium pressure of 12 Torr. The red dotted line shows the width of the arrival time distribution calculated from eq 3 assuming the presence of only one conformer.

III. RESULTS AND DISCUSSION

III.A. Circular Dichroism Results. Circular dichroism spectra recorded for the wild-type peptide and for the G13L and H16G variants in 100% water solutions are displayed in Figure 1. The measurements in methanol and acetonitrile (data not shown) yielded similar spectra. A fit using the procedure described in section II.B was applied to decipher secondary structure contributions in each spectrum. The R-helical and -sheet contents (random-coil contribution of 0% in all cases after the fit) were extracted for each variant and are represented as a histogram where the error bars were determined by the correlation factor of the fit (Figure 2). In each case, the R-helix contribution was found to be around 6075%, which is consistent with condensed-phase structural data from previous works concerning the wild-type peptide.36,51 III.B. IMS Results. The arrival time distributions (ATDs) measured for the 3 charge state of all of the M2TMP variants are plotted in Figure 3. These data were recorded using a 770 V drift field under a helium pressure of 12 Torr. The results

obtained for the 2 charge state are not discussed here because of the weakness of the measured signal. The 4 charge state was also present in the mass spectra, but no significant changes were observed between the corresponding ATDs (data not shown), likely because the higher number of charges confers a higher rigidity to the system. Most distributions in Figure 3 are dominated by one peak. For H16G and D3R, one peak centered at 74 ms is visible; it corresponds to a cross section of 610 2. The distributions recorded for the other variants are more structured. The main spectral features for D3R H16G, S10N, V6A, and V7G are all centered at 60 ms, whereas the main peak for the wild peptide is slightly shifted at 63 ms. In terms of cross section, the values for these peaks range from 500 to 515 2. Additional peaks at higher arrival times with smaller intensities are also observed in these four ATDs. This is particularly clear for the wild type (at 72 ms) and for D3R H16G (at 67 and 73 ms). Finally, the distribution obtained for G13L is clearly bimodal, with a peak at 66 ms and another one at 74 ms (i.e., 540 and 610 2, respectively). Beyond their mere positions, the shapes of the identied peaks can also yield valuable information. The ATD expected from the natural diusion broadening in the drift tube for a single conformer calculated from eq 3 is shown in Figure 3 (in the case of D3R). The peaks visible at 75 ms for H16G, D3R, and G13L have widths in good agreement with that expected for
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Figure 4. Calculated arrival time distributions for dierent M2TMP variants. The corresponding lowest-lying structure is also shown, together with an estimate of its helix content h (see text for details). For D3R, the results for the two envisaged protonation schemes NRR and RHR are denoted a and b, respectively.

a single isomer, based on Ficks law. Thus, they correspond to either a single conformational family or dierent conformers with similar diusion cross sections. This is also the case for the main peak in the ATD recorded for S10N. For the other variants, the main spectral feature is signicantly broader than the simulated distribution. Specically, a shoulder is visible in the case of V6A, and the ATDs for wild type and for the V7G and G13L variants exhibit a tail on the long-time side. These broad peaks are due to the coexistence of dierent conformers in the ion beam. The less intense features between 60 and 74 ms (especially for D3R H16G, S10N, and V6A) also support the conclusion that a large variety of conformations are accessible under the experimental conditions. However, the asymmetric shape of the main peaks observed for the wild peptide, for V7G, and for the left peak in the case of G13L could possibly be related to a conformational transition occurring during the diusion in the tube, as discussed below. To summarize, the above overview already allows the identication of two main conformational families with distinct collision cross sections. The sharp peak at 74 ms is common to H16G, D3R, and G13L and corresponds to a cross section of approximately 610 2. From its shape, it could originate from a single conformer or from several conformers with similar cross sections. The second family encompasses more compact structures with cross sections ranging from 500 to 540 2. Because of this broad range of cross sections, several conformations are probably at the origin of this second family. It appears that the peptide can switch from a compact to a more extended structure upon the modication of a single amino acid in its sequence. Moreover, with dierences in the cross sections being on the order of 20%, the conformational changes between variants are expected to be important. In addition to these two families, the presence of secondary peaks, in particular for D3R H16G, S10N, and V6A, denotes the complexity of the conformational landscape accessible to these variants. In contrast, the ATDs for H16G and D3R display a single peak, which suggests that a single conformational family is favored for these variants. III.C. Simulation Results. As an attempt to determine the conformations corresponding to the two conformational families identified from experiment, the structures of different variants

were investigated through REMD simulations and local minimizations following the protocol described in section II.D. The calculated ATDs are plotted in Figure 4, together with a sketch of the structure of the lowest-lying isomer in each case. An estimate of the helix content h for each structure is also given for comparison with the CD experiments. It corresponds to the proportion of the amino acids whose dihedral angles (, ) are compatible with an R-helical structure. Four families of structures emerge from the calculations. The most extended conformations are obtained for the wild-type peptide and for the D3R variant, provided that the latter is charged at RHR. The corresponding cross sections were found to be on the order of 610 2, which yields drift times of about 75 ms. The associated conformations are elongated R-helices. The difference between the two structures lies in the conformation near the carboxyl end. Whereas D3R is a slightly bent helix, the wild-type peptide is unfolded between Ile18 and Ile21. This also corresponds to different charge solvation schemes for His16 and Arg24. More compact structures with shorter helical domains and folded ends were found for the G13L and V16G variants. Their calculated cross sections are on the order of 565 2, and the corresponding ATDs are centered at 67 and 70 ms, respectively. The simulations for H16G and D3R, if the latter is charged at NRR, also yielded compact structures, but without significant helix content, and average cross sections of 530 and 534 2, respectively (with corresponding drift times of 64 and 65 ms). Finally, the most compact structure was found at 510 2 (average drift time of 62 ms) for the doubly substituted D3R H16G variant. This structure is not globular, but the peptide is folded around residues 6 and 7, yielding two subdomains. The N-terminus part is extended and lies parallel to the C-terminus part, which is R-helical. A direct comparison with experimentally measured distributions reveals some possible agreement for the G13L variant, for which the calculated distribution extends on the high-crosssection side up to values that would be compatible with the experimental peak at 610 2. The structures corresponding to this tail are elongated R-helices similar to those computed for the wild peptide. The agreement between experiment and simulations is quantitative only for the D3R and D3R H16G variants. Remarkably, the structure obtained for the triply protonated D3R, although slightly bent in its C-terminus region, is very close to that obtained by solid-state NMR spectroscopy for the M2TMP in a tetramer (PDB 1NYJ).38 Alignment of the Rcarbons of the two structures yields a root-mean-square deviation of 3.304 (see Supporting Information, Figure S1). Moreover, the helix content estimated for the corresponding lowest-energy structure is close to the value estimated for G13L or for the wildtype peptide in the CD experiments. Unlike in the experiments, no clear bimodal character is visible in the simulated crosssection distributions for G13L. This is not surprising, because the simulation time of 10 ns is probably far too short to sample the full isomerization pathway and reproduce such a structural equilibrium for a system of more than 400 atoms, even with the parallel tempering strategy. For other variants, the experimental and calculated peaks are not in full correspondence with each other. However, as in the measured distributions, two main structural families emerge from the simulations, both having cross sections that are compatible with experiments. Beyond insucient sampling, the disagreement between simulations and experiments might reect a lack of accuracy of the Amber99 force eld, but it is unclear whether other nonreactive force elds would be more successful.
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III.D. Discussion. Comparison between the experimental and calculated distributions (see Figures 3 and 4) does not, in general, show good agreement for each individual variant, except for D3R and D3R H16G. This reflects the difficulty of performing a complete exploration of the conformational landscape of a 25amino-acid peptide with an accurate potential energy surface. In particular, screening effects due to charge solvation might result in changes of the local electric susceptibility, which are not reproduced by the nonpolarizable Amber99 force field. Nevertheless, examination of the calculated distributions can give cross-section benchmarks to get more detailed information on the two structural families identified from experiment. The most extended structures were observed with cross sections of about 610 2, which is in good agreement with the calculated values for the elongated R-helices obtained for the wild-type peptide and the D3R variant protonated at RHR. For D3R, the position of the charges strongly stabilizes the helix. This is illustrated by the calculated structure where the charges on R3, H16, and R24 are solvated by the side chain of Ser2, the backbone carbonyls of Ile12 and Gly13, and the carbonyl of Ile21, respectively. In each case, no important constraint is applied on the helix because the solvation occurs with carbonyls located on the next helix loop. Moreover, the regular repartition of the charges along the sequence minimizes the effect of Coulomb repulsion. The above elements and experimental results strongly support the conclusion that D3R adopts a helical conformation in the gas phase. The experimental ATD recorded for H16G is similar to that of D3R, which would then suggest that the former adopts a helical conformation, as no other candidate structure seems extended enough to account for the experimental cross section. However, unlike for D3R, the calculations predict a compact structure. One difficulty for this variant was to localize three charges when only two clearly identified basic groups were available on the N-terminus and on Arg24. Choosing to protonate Pro4 was apparently not realistic, given the discrepancy with experiment. Because no other basic site is present along the chain, a possible scenario would be the protonation at the backbone amide groups. This could favor the helical conformation by allowing an optimal charge repartition, especially if this charge is mobile.11 Unfortunately, this again lies beyond the capabilities of the present force field, and the calculations necessary to test this hypothesis with a reactive model would be far too time-consuming. Whereas H16G and D3R display experimental ATDs in agreement with collision cross sections calculated for helical structures, when the two variations are carried out simultaneously (D3R H16G variant), the dominant structure is one of the most compact observed by experiment. This is in relatively good agreement with the two-subdomain structure predicted for this variant (see calculated ATDs in Figure 4). This structure is favored by the absence of charge at residue 16, which, according to our simulations, could induce a solvation scheme completely dierent from those found for the other variants. Note that the conformation obtained for D3R H16G would then require the absence of a charge at residue 16 and is therefore not likely to be adopted by other variants. The experimental proles observed for V7G, V6A, S10N, and the wild-type peptide (Figure 3) all show a dominant peak consistent with the collision cross section calculated for relatively compact structures (see Figure 4). The assignment of a dened structure for these experimental peaks is more dicult, as the widths of the peaks indicate the coexistence of dierent isomers in this region. However, the comparison with simulated structures

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discards fully helical conformations. These four variants are expected to bring charges at the same locations, which supports the hypothesis that charge location is the main driving eect in the conformations adopted by these peptides in the gas phase. G13L is peculiar in that it displays a bimodal experimental ATD. Neither Gly nor Leu has particular basic properties, and charge location is not a priori responsible for the dierence in ATD as compared to the four peptides just discussed. The main dierence between Gly and Leu is the size of their side chains; therefore, replacing Gly by Leu mainly modies the exibility of the peptide.52 The impact of such a dierence on conformation is not obvious, but it becomes relevant if structural reorganization occurs between solution and the gas phase. We found that, under the given experimental conditions, most of the systems studied here, including the wild-type peptide, adopt rather compact structures, whose cross sections are not compatible with an extended R-helix. However, the previously reported ECD product ion abundance data on the wild-type peptide suggests the presence of helical conformations in the gas phase.20 It should be recalled that, in ECD experiments, peptide ions are also generated by electrospray but transferred to and manipulated under high-vacuum conditions; thus, they experience signicantly fewer collisions with background gas than in the present experiments. The complete thermalization obtained in the IMS experiments through a large number of collisions might favor conformational changes, in contrast to ECD measurements.53,54 Circular dichroism measurements (see Figures 1 and 2) indeed support the conclusion that the wild-type peptide and the G13L variant form R-helices under the solvent conditions used in this work, as their helix contents were estimated to be on the order of 80%. In the present IMS experiments, the ions travel for about 70 ms in a buer gas at room temperature, where they can undergo structural transitions to adopt more stable gas-phase conformations. Under this hypothesis, a lower exibility would slow down the process. The peak at 74 ms would then likely be due to the helical peptides remaining at the end of the drift. Other arguments favor this scenario. First, small spectral features are visible near 74 ms for the wild-type peptide and for D3R H16G, even if the main peaks lie between 60 and 63 ms. This means that some of the gas-phase ions for these peptides adopt structures whose cross sections are compatible with elongated helices. Then, the peaks corresponding to the most compact family are asymmetric for the wild-type peptide and for the V7G and G13L variants, whereas the peak at 74 ms is symmetric. These dierences in shape would be consistent with the expected prole if a transition between two structural families occurred during diusion, provided that the transition time was comparable to the drift time. Similar features have been observed in the case of structural transitions in sodium chloride clusters.55,56 The intermediate features observed between the two dominant peaks in the ATDs could be assigned to minor conformers but could also correspond to structural intermediates trapped during the transition. A good way to test the validity of the present hypotheses would be to follow the relative intensities of the dierent peaks as a function of temperature, which, unfortunately, was not possible with our setup. To summarize, the collision cross sections corresponding to the two main structural families observed in the gas phase are in agreement with calculated values for (i) elongated R-helices, close to the conformation observed in the condensed phase and to the structure adopted by the peptide in its tetramer form, and (ii) more compact structures. The wild-type peptide and the D3R H16G, S10N, V6A, and V7G variants were found to
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The Journal of Physical Chemistry A predominantly adopt a compact structure, with the rst two also showing some isomers with collision cross sections in agreement with helical structures, the D3R and H16G variants forming helical-type of structures, and the G13L variant displaying both conformations. Even without assuming conformational transitions during ion ight, it can be inferred from our results that most of the investigated variants adopt a compact conformation in the gas phase, that is, these peptides do not intrinsically tend to form helices without surrounding solvent. Even in the cell membrane, where the dielectric constant is close to that in vacuo, the helical conformation might be stabilized by the environment. Moreover, noncovalent interactions in the tetramer may also play a role. One possible driving force for the destabilization of helical structures in the gas phase could be the unfavorable interaction of the charges on the N-terminus side of the peptide with the helix dipole.9,10

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cross section is far more computationally expensive than the energy/gradient estimation. The biasing strategy then needs to be performed on other conformational order parameters, and this will be the object of future investigations.

ASSOCIATED CONTENT
S b

Supporting Information. Alignment of the structure obtained through REMD simulations for the triply protonated D3R variant with the solid-state NMR structure. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION
Corresponding Author

*Phone: 334 724 327 80. E-mail: fabien.chirot@univ-lyon1.fr.

IV. CONCLUSIONS The conformation of the 3 charge state of the M2TMP monomer has been investigated as a function of dierent sequence variations using ion mobility spectrometry. The variation of a single amino acid was found to cause major conformational changes, essentially yielding two conformational families. Comparison with replica-exchange molecular dynamics simulations using the Amber99 force eld and augmented with local optimizations conrms the two types of structures, although, in most cases, assignment specic to each variant could not be achieved. According to these combined experimental and simulation results, the dominant conformation of most variants appears to be rather compact, even if they tend to form helices in solution. In general, the precise location of the charges appears as a crucial factor aecting secondary structures, and even small variations in protonation sites could lead to important dierences in the ATDs. One variant was also shown to exhibit bistability, with comparable weights for both of the identied conformational families. It cannot be inferred from the present work whether these two conformations are in equilibrium or whether they isomerize inside the drift tube, and it would be useful in the future to address this specic issue separately. The limitations shown by the present simulation analysis could largely be due to the employed rather crude nonpolarizable force eld, which might not be accurate enough for the studied large multiply-charged biomolecules. More importantly, it is dicult to account for competing protonation sites. Here, we chose to protonate the most basic amino acids, but backbone protonation could have been envisaged as well. An adequate modeling of intramolecular proton transfer would be much more realistic but would require reactive force elds such as empirical valence-bond models4648 or the recent extension of ReaxFF to peptides.49 Furthermore, for the present molecules, these approaches would probably not be feasible in the context of global optimization or nite-temperature statistics. Another strategy worth pursuing would be the introduction of biases aimed at reproducing the experimental cross sections. There are several diculties inherent to biasing simulations, including the nonphysical artifacts this might introduce in the potential energy surface, as well as the appropriate determination of the forms and parameters for the bias potential. In the present case, the determination of cross sections from additional molecular dynamics trajectories at xed molecular conformation makes it even more troublesome, because the single-point calculation of a

ACKNOWLEDGMENT Y.O.T. and H.B.H. acknowledge nancial support from the Swiss National Science Foundation (SNF Project 200021125147/1) and the Swiss Academy for Engineering Sciences (SATW Project 2009-28). REFERENCES
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