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2007, 111, 13147-13150 Published on Web 11/01/2007
A Direct Comparison of Protein Structure in the Gas and Solution Phase: The Trp-cage Alexandra Patriksson,† Christopher M. Adams,‡ Frank Kjeldsen,‡,§ Roman A. Zubarev,*,‡ and David van der Spoel*,† Department of Cell and Molecular Biology, Uppsala UniVersity, 596, SE-751 24, Uppsala, Sweden, and Laboratory for Biological and Medical Mass Spectrometry, Uppsala UniVersity, Box 583, SE-751 23, Uppsala, Sweden ReceiVed: October 10, 2007; In Final Form: October 12, 2007
Molecular dynamics simulations of zwitterions of the Trp-cage protein in the gas phase show that the most stable ion in vacuo has preserved the charge locations acquired in solution. A direct comparison of the gas and solution-phase structures reveals that, despite the similarity in charge location, there is significant difference in the structures, with a substantial increase in hydrogen bonds and exposure of hydrophobic parts in the gas phase. The structure of the salt bridge in the gas phase is also much more stable than in the (experimental) solution structure.
To understand the effects of solvent on protein structure, a direct comparison of gas-phase structures of proteins and their solution-phase structures would be very helpful. However, proteins in different phases can be charged differently, which makes direct comparison difficult. For instance, the 20-residue miniprotein Trp-cage1 with pI 9.4 has a net charge of +1 in solution at pH 7, whereas gas-phase Trp-cage ions produced from the same solution by electrospray ionization (ESI) have an average charge of +2.2, that is, a mixture of +2 and +3 ions, wherein the +2 ion is more abundent.2,3 The solutionphase Trp-cage is a zwitterion, with Asp9 and the C-terminus deprotonated and the N-terminus, Lys8, and Arg16, protonated.1 However, the difference in basicity between lysine and other amino acids is much smaller in the gas phase than in solution,4 whereas Coulomb interactions play a much more important role. As a result, the charge in +2 solution-phase ions relocates in the ESI from Lys8 to Gln5, and Asp9 becomes neutral, resulting in collapse of the zwitterionic solution state.2 Not surprisingly, the NMR-derived solution structure of the +1 zwitterion and the minimum-energy gas-phase structure of +2 ions differ quite significantly, with a CR rmsd of 0.39 nm.3 By source condition optimization in ESI, it is possible to obtain +1 ions of Trp-cage, and we have demonstrated that 157 nm photodissociation (PD) can reliably detect zwitterions in the gas phase.5 Briefly, excitation by a 7.9 eV photon leads to facile decarboxylation of anionic carboxylate groups, whereas neutral carboxylic groups remain intact.5 The same study proved that the gas-phase Trp cage +1 is a zwitterion; the +2 ion is not. The site of deprotonation in +1 was shown to be either Asp9 or the C-terminus, with an ∼3:2 ratio between these two * To whom correspondence should be addressed: E-mail (D.v.d.S.):
[email protected]; homepage: http://folding.bmc.uu.se. E-mail (R.Z.):
[email protected]. † Dept. of Cell and Molecular Biology. ‡ Laboratory for Biological and Medical Mass Spectrometry. § Permanent address: Dept. of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
10.1021/jp709901t CCC: $37.00
sites. However, the 157 nm UV PD study on +1 ions could not distinguish whether the second proton was located at Lys8, as in solution, or at Gln5, as in the gas-phase +2 ions. To elucidate the position of the second proton, we have therefore performed molecular dynamics simulations of four isomers of Trp cage +1 ions: two isomers having a deprotonated Asp9 and protonated Gln5 or Lys8, respectively, and two isomers having a deprotonated C-terminus and the same protonated residues. All four isomers also have a protonation located on Arg16. The isomers were all constructed from the structure found by NMR in solution (the first one of the 38 structures in the “native” ensemble),1 and simulations were run in vacuo without any cutoffs. To ensure production of realistic vacuum phase structures, the simulations were run for 1.5 µs using the replica exchange approach6 at 16 different temperatures, ranging from 275 to 419 K. Equilibration of the structures was obtained after 1 µs in all cases. Bond lengths were constrained using the LINCS algorithm7 to allow the use of 2 fs time steps, and the OPLS-AA forcefield8-10 was used. To maintain the temperatures at the predefined levels, the NoseHoover thermostat11,12 was applied with a coupling constant of 0.1 ps. Simulations and analyses were performed using the GROMACS software package.13 In order to find the preferred position of the second proton, the relative stabilities (∆G) of each of the four isomers were calculated from the equilibrated part of the trajectories, that is, the last 500 ns, at 296 K. The conformational entropies were calculated using two different formulas: the quasiharmonic,14,15 recommended by Carlsson and Åqvist, and Schlitter’s.15 Both methods give very similar results, with slightly lower quasiharmonic entropies than obtained from Schlitter’s formula, which is agreement with Andricioaei and Karplus16 (Table 1). The entropy difference of the two methods is, however, constant (0.20 kJ/mol K) and, thus, will not affect the resulting Gibb’s energies; the quasiharmonic values were used for the subsequent calculations. For the enthalpies, the difference in average potential energy was used (Table 1). According to the results, © 2007 American Chemical Society
13148 J. Phys. Chem. B, Vol. 111, No. 46, 2007
Letters
TABLE 1: Gibbs Energies (∆G), Entropies Using the Quasiharmonic Formula (∆SQH) and Schlitter’s Formula (∆SSchlitter), Relative Enthalpies (∆H), PM3 Energies, NMR Distance Violations (), Cr rmsd to the NMR Structure, and the Number of Intramolecular Hydrogen Bonds (no. hb)a ion
∆G (kJ/mol)
∆SQH (kJ/mol K)
∆SSchlitter (kJ/mol K)
∆H (kJ/mol)
PM3 (kJ/mol)
(nm)
rmsd (nm)
no. hb
RDK RCK RDQ RCQ NMR
-1388 ( 6 -1361 ( 3 -1279 ( 5 -1265 ( 3
4.69 ( 0.02 4.57 ( 0.01 4.84 ( 0.01 4.92 ( 0.01
4.90 ( 0.07 4.78 ( 0.05 5.05 ( 0.04 5.13 ( 0.04
0(3 -7.9 ( 0.1 153.3 ( 3 191.0 ( 0.1
0 150.4 168.2 12.1
0.014 0.021 0.030 0.040 0
0.29 0.34 0.29 0.41 0.09
17 18 18 16 9
a
All calculations are performed using the last 500 ns of the simulations run at 296 K.
Figure 1. Violation matrices showing the pairwise sum of distance violations between the residues in (A) the RDK ion and (B) the RCK ion.
Figure 2. Comparison of the NMR structure (A and C) and the most common structure of the RDK isomer (B and D), shown from two directions. Backbone atoms are colored blue, and side chains, cyan. Tyr3 is colored in yellow. The distance vectors between Arg16-Asp9 and Asp9-Lys8, the residues making up the ion bridge in RDK, are shown as a dark blue dotted line.
both of the two most stable isomers have Arg16 and Lys8 protonated, and of these, the one with a deprotonated Asp9 (isomer RDK) has a lower Gibbs energy (by 27 kJ/mol) than the ion with a deprotonated C-terminus (RCK). Thus, the charge configuration in the minimum-energy, gas-phase ions is very similar to that in solution, and the structures can be compared directly for the first time. To quantify the difference between the simulated conformations in vacuo and experimental solution data, time-averaged NOE distance violations were computed17 on the basis of 168
distance restraints from NMR.1 We find that the two most stable ions (RDK and RCK) also have the largest structural similarity to the native NMR structure, with an average violation () of 0.014 and 0.021 nm, respectively (Table 1). For both RDK and RCK, the majority of the violated distances are found on atoms belonging to the residues making up the hydrophobic core of the NMR structure:1 Trp6, Pro12, and Pro18 (Figures 1 and 2). For RCK, a significant number of violations are found on Arg16, which in RDK shows no violations at all (Figure 1). To better understand the relation between NOE violations and structure, the most common structure of each of the four ions was extracted using a clustering procedure18 based on a principal component analysis of the coordinates.19 The RDK ion has a CR-rmsd to the native NMR structure of 0.29 nm, whereas the rmsd between the RCK ion and the NMR structure is 0.34 nm. The rmsd between these two ions (RDK, RCK) is 0.44 nm, and hence, they contain more similarity to the native structure than to each other. The structure that deviates most from the NMR solution conformation, both in terms of violations and rmsd, is the RCQ ion. The average internal rmsd between the 38 solution structures in the NMR ensemble is 0.09 nm, and the average number of hydrogen bonds is only 9, as compared to the zwitterions in vacuo, which have between 16 and 18 intramolecular hydrogen bonds, on average. Optimization of the most common structure of each species was performed using the semiempirical PM3 method,20,21 as implemented within the Gaussian 03 package.22 In these optimizations, the structures changed only slightly. The RDK ion was found to be the most stable in calculations (Table 1) by 12 kJ/mol. This strengthens the results from the MD calculations, even though it is based on a single conformation only. In Figure 2, the NMR solution structure is compared to the most common structure of RDK in vacuo. The backbone configuration of RDK is slightly more compact than the solution backbone (a radius of gyration of 0.68 nm versus 0.73 nm) and Tyr3 has no contact at all with the hydrophobic core in RDK and extends into the vacuum. Of the nine hydrogen bonds found in the NMR structure, only four are present among the 17 hydrogen bonds identified in the RDK ion: Gln5-NH-Leu2O, Leu7-NH-Tyr3-O, Gly10-NH-Leu7-O, andGly11-NHTrp6-O. A close-up of the salt bridge in the four isomers (Figure 3) shows almost perfect interactions among the three charged residues, and the donor-acceptor distances are much shorter in the gas phase than in the NMR structure (Arg16-Asp9 ) 0.37 nm and Lys8-Asp9 ) 0.35 nm for RDK, as compared to 0.58 and 0.86 nm, respectively, for the NMR ensemble average). The structures obtained from a water simulation of one of the NMR structures of Trp-cage,23 however, show an Arg16-Asp9 distance that is even closer than in the gas structure (0.34 nm) but no interaction at all between Lys8 and Asp9 (1.03 nm). In the gas-phase simulations, the interaction between the negative charge (on Asp9 or the C-terminal) and Arg16 is found to fluctuate among the three amine groups of the arginine. These
Letters
J. Phys. Chem. B, Vol. 111, No. 46, 2007 13149 The RDK ion has lower Gibbs energy than the RCK ion (Table 1) and, hence, is the dominant species at low and moderate temperatures, a result that is in good qualitative agreement with 157 nm UV PD data that showed similar abundances for both deprotonated Asp9 and C-terminus species, with a small preference for the Asp9 isomer.5 The large energy barrier between Lys8 and Gln5 protonated isomers, 82 kJ/mol at 296 K, indicates that the location of the second proton is the most important factor for structure stability and that interconversion is unlikely. In this study, we have demonstrated that gas-phase ions can preserve the charge locations acquired in solution, and hence, determination of the charge location in the gas-phase can be meaningful for understanding native structures. The study also shows that, despite the similarity in charge location in Trpcage +1, the solution-phase and gas-phase structures differ significantly, with a substantial increase in hydrogen bonds and exposure of hydrophobic parts to the vacuum. In the simulations, all four charge isomers made a transition from the native structure to a unique, steady state within 1.0 µs. Therefore, the reverse transition could be just as efficient, and gas-phase structures can be good starting approximations for obtaining native conformations, provided solvent effects are well understood. By running molecular dynamics folding simulations in solution using information obtained from experiments such as mass spectrometry as a start, the native state of the proteins could, in principle, be determined by locating the (Gibbs) energy minimum.26,27
Figure 3. Close-ups of the salt bridge of the most common structure of (A) RDK, (B) RCK, (C) RDQ, and (D) RCQ. Only the side-chains or the C-terminal bearing the charges are shown.
Acknowledgment. This work was supported by the Swedish research council (Grant 621-2004-4897 to R.Z.). Jens Carlsson is acknowledged for valuable help with entropy calculations.
salt bridges, therefore, have a wide distribution of donorhydrogen-acceptor angles but at an almost constant donoracceptor distance that varies between 0.33 and 0.38 nm for the four ions. As noted above, the most stable isomers in vacuo are the ones that are closest to the native structure, not only in charge distribution, but also in terms of rmsd and average NMR violations (Table 1). The conformational differences observed between the vacuum structures and the solution-phase structure are completely due to the increased number of hydrogen bonds (Table 1): since there is no hydrophobic effect in vacuo, the Van der Waals interactions between the nonpolar side chains are not strong enough to maintain the solution structure. This effect is, however, dependent on protein size, and it has been shown that larger proteins, such as ubiquitin and lysozyme, have a more nativelike secondary structure in vacuo than the +2 ion of Trp-cage because native hydrogen bonds in R-helices and β-sheets may stabilize the solution conformation in vacuo.23 Nevertheless, these larger proteins also show a considerable increase in accessible hydrophobic surface area in vacuo.23 The pronouncedly increased stability of salt bridges observed in the vacuum structures, as compared to both NMR1 and structures simulated in water,23 is also due to the lack of interacting waters. The strong interaction between Arg16 and Asp9, both in gas and solution, is in complete agreement with fluorescence probe measurements of the conformational stability of a D9N mutated Trp-cage.24 Therefore, strong interactions between charged residues should be expected in gas-phase experiments of other proteins as well. The flexibility of Tyr3 observed in the Trp-cage +1 ions in vacuo has, however, been reported earlier in a simulation study in solution.25
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13150 J. Phys. Chem. B, Vol. 111, No. 46, 2007 Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson,
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