11189
2008, 112, 11189–11193 Published on Web 08/14/2008
Conformational Preferences and pKa Value of Cysteine Residue Joo Yun Lee,†,‡ Byung Jin Byun,† and Young Kee Kang*,† Department of Chemistry, Chungbuk National UniVersity, Cheongju, Chungbuk 361-763, Republic of Korea ReceiVed: June 14, 2008; ReVised Manuscript ReceiVed: July 17, 2008
The conformational preferences of the Cys dipeptides with thiol and thiolate groups (Ac-Cys-NHMe and Ac-Cys--NHMe, respectively) and the apparent (i.e., macroscopic) pKa value of the Cys dipeptide have been studied at the hybrid density functional B3LYP/6-311++G(d,p)//B3LYP/6-31+G(d) level with the conductorlike polarizable continuum model in the gas phase and in water. The hydrogen bonds and/or favorable interactions between the backbone and the thiol group of the side chain resulted in the different conformational preferences of the Cys and Cys- dipeptides from those of the Ala dipeptide in the gas phase and in water, although the preferred conformations of the Cys dipeptide are in part similar to those of the Ala dipeptide. In particular, the interactions between the thiolate group and the backbone amide groups appear to play a role in stabilizing the R- or 310-helical conformations for the Cys- dipeptide in the gas phase and in water. The pKa value of the Cys residue is estimated to be 8.58 at 25 °C using the statistically weighted free energies of all feasible conformations for the Cys and Cys- dipeptides in the gas phase and solvation free energies, which is consistent with the observed values of 8.3 and 8.22 ( 0.16. Introduction The cysteine (Cys) residue can bind metal ions and various prosthetic groups and form disulfide bonds in proteins.1 The Cys residues with free SH groups cannot be easily phosphorylated nor linked to carbohydrates, but they are more reactive to many external reagents.1 The pKa value of the thiol group for the Cys residue has been recently determined to be 8.32a and 8.22 ( 0.162b with a thiolate-specific reagent idoacetamide at various pH values. Thiol-disulfide oxidoreductases with a CysX-X-Cys motif have been known to play a role in maintaining the redox status of thiol groups in the cell and in catalyzing or regulating various cellular functions.3 There are differences in pKa values and redox potentials depending on the sequence XX and neighboring residues to the motif. In particular, the pKa values for the two Cys residues were determined to be about 4-11, although the value at the N-terminus is relatively smaller than that at the C-terminus.3b Recently, it has been reported that redox-sensitive cysteine residues play a role in sensing and transducing changes in cellular redox status caused by the generation of reactive oxygen species and the presence of oxidized thiols.4 Although many experiments and computations have been carried out to investigate the conformational preferences of L-cysteine in the gas phase and in water,5 there are a limited number of works that quantum-mechanically studied the conformational preferences of the Cys residue blocked with N- and C-terminal groups, i.e., N-acetyl-L-cysteine-N′-methylamide (AcCys-NHMe, the Cys dipeptide) at the HF and B3LYP levels in * To whom correspondence should be addressed. Telephone: +82-43261-2285. Fax: +82-43-273-8328. E-mail:
[email protected]. † Chungbuk National University. ‡ Present address: Center for Drug Discovery Technologies, Korea Research Institute of Chemical Technology, Daejon 305-600, Republic of Korea.
10.1021/jp8052423 CCC: $40.75
the gas phase.6 In particular, 42 local minima were identified at the B3LYP/6-31G(d) level and the potential energy surfaces (PESs) were explored by allowing the conformational interconversions among the local minima at the HF/3-21G level.6c Recently, Canle L. et al. have estimated the microscopic pKa values for L-cysteine using discrete, continuum, and mixed discrete-continuum solvation models at the B3LYP/ 6-31++G(d,p) level.7 They employed only a single conformation for each ionized species and the discrete-continuum model resulted in better pKa values for the amino group than that for the thiol group. Carvalho et al. have determined the pKa values of two Cys residues of the active sites for oxidoreductases thioredoxin and DsbA using QM/MM methods embedded in a dielectric continuum.8 It has been reported that the proton is not shared between the two cysteines but remains attached to the buried cysteine8a and that the difference in pKa values of the nucleophilic cysteines of both enzymes is ascribed to the different ability of the R2-helix in stabilizing their thiolates and the charged residues in the vicinity of the active site are responsible for stabilizing these thiolates.8b However, the conformational preferences of the terminally blocked Cys dipeptides with thiol and thiolate groups in water have never been studied experimentally and theoretically until now. In addition, no theoretical works on the calculation of the pKa value for the thiol group of the Cys dipeptide in water are available to date. Here, we report the conformational preferences of Ac-Cys-NHMe and Ac-Cys--NHMe dipeptides carried out at the hybrid density functional levels with the self-consistent reaction field (SCRF) method in the gas phase and in water. In particular, the apparent (i.e., macroscopic) pKa value of the thiol group for the Cys dipeptide is estimated by considering all feasible conformations in the gas phase and in water. 2008 American Chemical Society
11190 J. Phys. Chem. B, Vol. 112, No. 36, 2008
Letters
Figure 1. Chemical structures and torsional parameters for the Cys (or Cys-) dipeptide.
pairings of oppositely charged ions are folded into the determination.16 In particular, there are several feasible conformations for the Cys and Cys- dipeptides in the gas phase and in water and some conformations are preferred only in the gas phase or in water. Thus, the apparent (i.e., macroscopic) pKa value for the Cys dipeptide can be estimated using the ensemble-averaged values of G°g(AH), G°g(A-), ∆G°s(AH), and ∆G°s(A-) in eq 2, determined by summing over all the feasible conformations weighted according to their Boltzmann factors.10 Lopez et al. applied the similar method to obtain the macroscopic pKa values for phosphoranes.17
Computational Methods Chemical structures and torsional parameters for the Cys (or Cys-) dipeptide are defined in Figure 1. The conformational energies and free energies were computed at the B3LYP/ 6-311++G(d,p)//B3LYP/6-31+G(d) level using the Gaussian 03 package.9,10 In the gas phase, the 39 local minima were obtained at the HF/6-31G(d) level starting from the 56 local minima for the Cys dipeptide identified using the ECEPP force field10,11 and they were followed by the further optimizations at the HF/6-31+G(d) and B3LYP/6-31+G(d) levels. Finally, the 33 local minima were located for the Cys dipeptide at the B3LYP/6-31+G(d) level, which were used as starting points for optimizations of the Cys- dipeptide at the B3LYP/ 6-31+G(d) level, and the seven local minima were identified. The local minima obtained for the Cys and Cys- dipeptides in the gas phase were used as starting points for their optimizations in water. The solvation free energies were calculated using the conductor-like polarizable continuum model (CPCM)12 at the B3LYP/6-31+G(d) level, which recently provided the pKa values for several organic molecules in agreement with available experimental data.13 Vibrational frequencies were calculated for all feasible local minima for the Cys and Cys- dipeptides at the B3LYP/ 6-31+G(d) level in the gas phase and the CPCM B3LYP/ 6-31+G(d) level in water, which were used to estimate relative free energies at 25 °C and 1.0 atm.10 Single-point energies were calculated at the B3LYP/6-311++G(d,p) level for all feasible local minima located in the gas phase and in water. On the basis of a thermodynamic cycle for the dissociation of an acid AH in the gas phase and in water (see Figure S1 of the Supporting Information),14 the pKa value at a temperature T can be calculated using the following equations
pKa ) (∆G°g + ∆∆G°s)/2.303RT
(1)
or
pKa ) [{G°g(H+) + G°g(A-) - G°g(AH)} + {∆G°s(H+) + ∆G°s(A-) - ∆G°s(AH)}]/2.303RT (2) where R is a gas constant and ∆G°g and ∆∆G°s are free-energy changes for the acid dissociation in the gas phase and the solvation in water, respectively. We used the values of G°g(H+) ) -4.39 kcal/mol for a 1 M gas15 and ∆G°s(H+) ) -263.98 kcal/mol for a transfer of an ideal 1 M gas to a 1 M solution determined with the cluster-pair-based approximation.16 Although the value of ∆G°s(H+) has been suggested to be -254 to -265 kcal/mol from experimental and theoretical studies,15 we adopted the value of -263.98 kcal/mol determined by Tissandier et al. with the cluster-pair-based approximation.16 This is because it is the latest value estimated from a set of cluster-ion solvation data without the use of extra thermodynamic assumptions and its uncertainty of ( 0.07 kcal/mol is smaller than expected because the cluster data of 20 different
Results and Discussion The torsion angles for backbone and side chain and thermodynamic properties of preferred local minima for the Cys and Cys- dipeptides calculated at the B3LYP/6-311++G(d,p)// B3LYP/6-31+G(d) level in the gas phase and the B3LYP/ 6-311++G(d,p)//CPCM B3LYP/6-31+G(d) level in water are listed in Tables 1 and 2, respectively. All local minima for the Cys and Cys- dipeptides obtained in the gas phase and in water are listed in Tables S1 and S2 of the Supporting Information, respectively. The most preferred conformations for the Cys and Cys- dipeptides in the gas phase and in water are shown in Figure 2 and the next preferred conformations are drawn in Figures S2 and S3 of the Supporting Information. In the gas phase, 33 and 7 local minima were identified for the Cys and Cys- dipeptides, respectively. The most preferred conformation for the Cys dipeptide is Cg+g+ and is followed by the conformations Etg+ and Dg+g+, whose relative free energies are 0.71 and 0.74 kcal/mol, respectively. Relative free energies of the other local minima are 1.22-11.38 kcal/mol. Two hydrogen bonds between the CdO of the acetyl group and the N-H of the NHMe group, with r(CdO · · · H) ) 2.03 Å, and between the CdO of the Cys residue and the S-H of the side chain, with r(CdO · · · H) ) 2.38 Å, appear to play a role in stabilizing the conformation Cg+g+. The C7 hydrogen bond between two terminal groups was also found to be a crucial factor to stabilize the same backbone conformations of the Ala and Pro dipeptides in the gas phase.18 Although second and third preferred conformations Etg+ and Dg+g+ have comparable free energies, their torsion angles ψ and χ1 are quite different, which can be ascribed to the different patterns of hydrogen bonds or favorable interactions, i.e., the former has a C5 hydrogen bond between the N-H and CdO of the Cys residue, with r(N-H · · · O) ) 2.11 Å. However, the conformation Dg+g+ has two favorable interactions between the amide N of the Cys residue and the N-H of the NHMe group, with r(N · · · H-N) ) 2.34 Å, and between the CdO of the Cys residue and the S-H of the side chain, with r(CdO · · · H) ) 2.80 Å. In particular, two conformations Etg+ and Dg+g+ seem to be more flexible than the conformation Cg+g+ because their relative entropies are computed to be 1.91 and 4.53 cal/K · mol, respectively (Table 1). At the B3LYP/6-31G(d) level in the gas phase, Bombasaro et al. also identified the first and second lowest-energy conformations to be Cg+g+ and Etg+ and the relative energy of the second conformation was computed to be 2.39 kcal/mol,6c which is now 1.34 kcal/mol at the B3LYP/ 6-311++G(d,p)//B3LYP/6-31+G(d) level (Table 1). In the case of the Cys- dipeptide in the gas phase, the most preferred conformation is Ag+ and is followed by the conformation Et with the relative free energy of 1.91 kcal/mol. Relative free energies of the other local minima are 4.71-12.60 kcal/ mol. A bifurcated hydrogen bond between the S- of the side chain and two amide N-H’s of the Cys- residue and the NHMe
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J. Phys. Chem. B, Vol. 112, No. 36, 2008 11191
TABLE 1: Backbone Torsion Angles, Side-Chain Torsion Angles, and Thermodynamic Properties of Preferred Local Minima for Ac-Cys-NHMe and Ac-Cys--NHMe at the B3LYP/6-311++G(d,p)//B3LYP/6-31+G(d) Level in the Gas Phasea backbone torsion anglesb conformerc
ψ
side-chain torsion anglesb χ1
ω′
φ
ω
Cg+g+ Etg+ Dg+g+
-176.9 174.7 -169.9
-82.4 -158.9 -121.8
65.5 172.4 23.0
-178.5 177.2 174.7
Ac-Cys-NHMe 51.8 -161.3 58.1
Ag+ Et
-174.4 175.4
-101.7 -153.0
-63.6 -179.7
-173.1 172.4
Ac-Cys--NHMe 47.1 -171.3
thermodynamic properties
χ2
∆Eed
∆He
∆Gf
pg
66.3 71.4 73.4
0.00 1.34 2.22
0.00 1.28 2.09
0.00 0.71 0.74
0.51 0.15 0.14
0.00 1.82
0.00 1.55
0.00 1.91
0.96 0.04
a Only the conformations with relative free energy
