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J. Phys. Chem. B 2008, 112, 16782–16787
Site-selective Intramolecular Hydrogen-Bonding Interactions in Phosphorylated Serine and Threonine Dipeptides Kyung-Koo Lee,†,‡ Eunmyung Kim,‡,§,|,⊥ Cheonik Joo,| Jaewook Song,| Hogyu Han,| and Minhaeng Cho*,†,⊥ Department of Chemistry and Center for Multidimensional Spectroscopy, Korea UniVersity, Seoul 136-701, Korea and Multidimensional Spectroscopy Laboratory, Korea Basic Science Institute, Seoul 136-713, Korea ReceiVed: April 16, 2008; ReVised Manuscript ReceiVed: October 6, 2008
To study the phosphorylation effect on the peptide conformation, we carried out nuclear magnetic resonance (NMR), circular dichroism (CD), Fourier transform (FT)-IR, and vibrational circular dichroism (VCD) experiments with serine and threonine dipeptides (SD and TD) and their phosphorylated ones (pSD and pTD). It is found that both unphosphorylated and phosphorylated serine and threonine dipeptides adopt two conformations, polyproline II (PII) and β-strand. The pH-dependent NMR study shows that the side-chain dianionic phosphoryl group can form direct intramolecular hydrogen bonds with the backbone amide protons at both the acetyl and amide ends of pTD, but only at the acetyl end of pSD. Temperature- and pH-dependent CD studies reveal that, unlike pSD, pTD undergoes conformational transition from PII to β-strand upon double ionization of the phosphoryl group. The subtle but distinct differences between pTD and pSD in site-selective intramolecular hydrogen-bonding interaction and charge-dependent conformational transition may sometimes become significant when choosing between serine and threonine for the conformational control of peptides and proteins by phosphorylation. I. Introduction The phosphorylation of proteins plays a pivotal role in the control of various cellular processes including metabolism, signal transduction, ion channel, and cell cycle.1-5 The phosphorylation of serine, threonine, and tyrosine at their side-chain hydroxyl group alters the electrostatic features and quite often the conformation of proteins. The electrostatic and steric change upon phosphorylation can also have significant consequences for protein-protein interactions. Despite the importance of this ubiquitous post-translational modification, little is still known about exactly how phosphorylation is translated into the alteration of molecular interactions and thereby protein conformations. To gain insights into the phosphorylation effect on the protein conformation, a few experimental and theoretical studies have been carried out within the context of short peptides, R-helix, and proteins. Tholey et al. studied the phosphorylation effect on the backbone conformation of short tetrapeptides, Gly-SerXaa-Ser (Xaa ) Ser, Thr, or Tyr in either phosphorylated or unphosphorylated form), by using one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy.6 The pH-dependence of 3JNH-HR coupling constants and chemical shifts indicates that intramolecular hydrogen bonds form between the dianionic phosphoryl group and the nearby amide protons in the tetrapeptides containing phosphorylated serine and threonine. An additional intramolecular electrostatic interaction of the phosphoryl group with the ammonium cation * To whom correspondence should be addressed. E-mail:
[email protected]. † Department of Chemistry and Center for Multidimensional Spectroscopy, Korea University. ‡ These two authors contributed equally to this work. § Current address: Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA. | Department of Chemistry, Korea University. ⊥ Korea Basic Science Institute.
or the carboxylate anion at the two termini of the phosphorylated tetrapeptides is also indicated when their terminal amino and carboxyl groups are not blocked by acetylation and amidation, respectively. All-atom Brownian dynamics simulation and molecular dynamics (MD) simulation have been performed to obtain detailed information about the backbone conformation and intramolecular hydrogen-bonding interactions in the phosphorylated tetrapeptides.7-9 It was suggested that the backbone conformation of the phosphorylated tetrapeptides is close to the right-handed R-helix, which is inconsistent with our recent experimental findings.10 The discrepancy between these two results seems to be largely due to the inaccuracies of force fields used for predicting their conformation. By using circular dichroism (CD) spectroscopy, Doig and co-workers showed that the phosphorylation of serine near the N terminus of an alaninebased R-helix significantly increases its stability via the electrostatic interaction of the dianionic phosphoryl group with the helix peptide bonds.11,12 Phosphorylation can also modulate the association of four monomeric helices by electrostatically stabilizing the individual helices and their contacts within the tetramer.13 Lemke et al. studied the photolytic control of protein phosphorylation with a genetically encoded photocaged serine.14 When incorporated into the transcription factor Pho4, photocaged serine can block its phosphorylation and subsequent nuclear export, which can be triggered upon photolytic conversion of this caged serine into free one. Sahoo and Nau showed that phosphorylation-induced conformational changes in short peptides can be probed by fluorescence resonance energy transfer.15 Conformational changes upon phosphorylation are made possibly by the electrostatic interaction between the dianionic phosphoryl group of threonine and the positively charged guanidino group of arginine at neutral pH. Recently, we have carried out Fourier transform (FT)-IR, vibrational circular dichroism (VCD), CD, and MD simulation studies of both unphosphorylated and phosphorylated tetrapep-
10.1021/jp803285x CCC: $40.75 2008 American Chemical Society Published on Web 12/02/2008
Site-selective Hydrogen-bonding in SD and TD
J. Phys. Chem. B, Vol. 112, No. 51, 2008 16783
Figure 1. Structures of compounds 1-4. Only the L-form was considered in Figures 2-5 unlesss otherwise stated.
SCHEME 1: Syntheses of Compounds 1-4a
a Reagents and conditions: (a) Ac2O, Et3N, DMAP, CH2Cl2, rt, (6a, 49%; 6b, 62%; 9a, 46%; 9b, 78%); (b) 40% MeNH2, H2O, rt, (1a, 84%; 1b, 86%; 3a, 59%; 3b, 90%); (c) (i) N,N-dibenzyl diisopropylphosphoramidite, 1H-tetrazole, DMF, rt, (ii) m-CPBA, CH2Cl2, -20 °C, (7a, 35%; 7b, 70%; 10a, 55%; 10b, 67%); (d) 10% Pd/C, 1 atm H2, AcOH/MeOH (1:9), rt, (2a, 85%; 2b, 84%; 4a, 99%; 4b, 99%).
tides, GSSS and GSSpS.10 The conformations of both GSSS and GSSpS are found to be close to polyproline II (PII) with nonnegligible population of β-strand, indicating that the phosphorylation of serine does not affect the backbone conformation in such a short tetrapeptide. However, the pD-dependent FTIR study provides critical evidence for direct intramolecular hydrogen-bonding interaction between the dianionic phosphoryl group and the amide proton and thus enables the estimation of the corresponding interaction enthalpy and entropy by van’t Hoff analysis. In addition, it is found that the force fields used for MD simulations are not accurate enough to reproduce the experimental IR and VCD spectra, so a constrained MD simulation was introduced and thenceforth information about intramolecular hydrogen-bonding interaction could be obtained. Here, we present various spectroscopic studies of serine and threonine dipeptides (SD and TD), and their phosphorylated ones (pSD and pTD) (Figure 1). Note that their amino and carboxyl termini are acetylated and amidated, respectively, and thus each dipeptide has two different amide protons available for intramolecular hydrogen-bonding interaction with the phosphoryl group. It is found that both serine and threonine dipeptides adopt two conformations, PII and β-strand, which still exist predominantly even after phosphorylation. Therefore, a propensity for such conformations is not affected sterically and electrostatically by the side-chain β-methyl and phosphoryl groups in the dipeptides. However, there are distinct differences between pTD and pSD in their intramolecular hydrogen-bonding interaction and its
Figure 2. 1H NMR chemical shifts (δ) of the two amide protons in pSD (a) and pTD (b) for varying pH. The 1H NMR spectra were measured for 5 mg/0.6 mL pSD and pTD in D2O/H2O (1:9) at 10 °C over the pH range of 1.79-8.49 and 1.77-10.23, respectively. The chemical shifts of the two amide protons HAc and HAm at the acetyl and amide ends are shown in blue and red, respectively. The insets show the structures of pSD2- and pTD2-, the dianionic forms of pSD and pTD, respectively.
possible effect on their conformational transition. These subtle differences may sometimes allow the phosphorylation-induced conformational control of peptides and proteins in a quite significant and distinctive manner. II. Experimental Section A. NMR Spectroscopy. Compounds 1-4 were synthesized and characterized (see Figure 1, Scheme 1, and the Supporting Information).16-18 For the determination of pKa for pSD and pTD, 1H NMR experiments were carried out on a Bruker Avance 500 NMR spectrometer. pSD and pTD were dissolved in D2O/ H2O (1:9) to a concentration of 5 mg/0.6 mL and pH was adjusted at 10 °C by directly adding 0.1 N NaOH or HCl. 1H NMR spectra were recorded using an indirect probe at 10 °C and referenced to D2O (δ 4.75). B. CD Spectroscopy. CD spectra were measured on a JASCO J-810 CD spectrometer equipped with a Peltier heating block. For electronic CD measurements, the sample concentration was 0.2-0.3 mg/mL, the sample cell was 1.0 mm quartz, the CD spectrometer sensitivity was 100 mdeg, the spectral resolution was 0.5 nm, the scanning mode was continuous, the scanning speed was 100 nm/min with a 2 s response time, the
16784 J. Phys. Chem. B, Vol. 112, No. 51, 2008 frequency range was set to vary from 180 to 260 nm, and all spectra presented here are the ones averaged over 10 scans. All CD spectra were measured at 10 °C, except for the temperaturevariant CD experiments, where T varies from 10 to 70 °C. The pH of dipeptide in H2O for CD measurement was adjusted by directly adding NaOH or HCl, and the resultant solution thus buffered was found to be little dependent on temperatures ranging from 10 to 70 °C. C. IR and VCD Spectroscopy. VCD spectra were measured on a Bomem/BioTools Chiralir FT-VCD spectrometer with a circulating water bath for temperature control. This spectrometer is equipped with a HgCdTe detector having a cutoff at 800 cm-1 and a ZeSe photoelastic modulator (PEM) to create left and right circularly polarized IR radiation. The dual PEM designed to reduce the baseline artifact19 was optimized for maximum quarter-wave response at 1600 cm-1. To substitute the labile hydrogen atoms by deuterium atoms, the dipeptide/D2O solution was lyophilized twice on a SpeedVac (ThermoSavant), and subsequently, it was dissolved in D2O to a concentration of 40 mg/mL. The pD of pSD (pTD) was adjusted to 7.0 (7.5) by directly adding NaOD or DCl. VCD and IR spectra were measured with a frequency resolution of 4 cm-1 using a CaF2 cell with a path length of 25 µm, and the sample cell chamber was purged with dehydrated air. The collection time for a VCD spectrum was about 6-12 h (3000 AC and 50 DC scans collected in one block for 1 h). The baseline-corrected VCD spectra were obtained by taking half of the difference between the normalized VCD spectra of D and L forms, where the two VCD spectra were separately measured. To examine the possible aggregation of dipeptides in water, IR spectra of their D2O solutions at various concentrations were measured on a JASCO FT/IR-4100 spectrometer equipped with a MCT detector. The normalized IR spectra were nearly identical over the concentration range from 0.4 to 40 mg/mL, which indicates that dimers or higher aggregates are not formed in this range. The temperature of the sample cell was controlled with a circulating water bath (Sam Heung Instrument SH-R-WB10), and a temperature sensor was directly attached to the cell surface for an additional monitoring of any temperature variation during data collection. All IR and VCD spectra were measured at 15 °C. III. Results and Discussion A. NMR Spectra. From the NMR spectra of pSD and pTD in D2O/H2O (1:9), it is possible to identify the two amide proton peaks. For notational simplicity, the amide proton at the acetyl (amide) end will be denoted HAc (HAm). The chemical shifts of HAc and HAm in pSD and pTD for varying pH are plotted in Figure 2. One of the most important findings is that the chemical shifts of HAc and HAm in pSD and pTD are strongly pH dependent. We attributed this pH dependence to deprotonations of pSD- and pTD-, the monoanionic forms of pSD and pTD, into pSD2- and pTD2-, their dianionic forms. For its confirmation and comparison, the chemical shifts of HAc and HAm in unphosphorylated dipeptides SD and TD for varying pH are also plotted (data not shown). We found that their chemical shifts remain unchanged over the pH range from 2 to 9, where the HAc and HAm peaks appear around 8.26 (8.12) and 7.97 (7.96) ppm in SD (TD), respectively. This supports the proposition that the chemical shift changes with pH titration shown in Figure 2, panels a and b, are associated with the further ionization of the monoanionic phosphoryl group in pSD- and pTD-. A similar titration curve for a phophopeptide was also reported by Du et al.20
Lee et al. In the case of pSD, the chemical shift of HAc increases, that is, the HAc peak is shifted downfield, when the phosphoryl group becomes dianionic (Figure 2a). The corresponding pK2 value of the phosphoryl group can therefore be estimated to be 6.05 (pH meter reading value). This value is close to that obtained from the 31P NMR spectra of O-phosphoserine in lyophilizates (average pK2 value of 5.9) and in solution (average pK2 value of 5.72);21,22 note that the O-phosphoserine is structurally different from our pSD at the N and C termini. In contrast to HAc, HAm in pSD has the nearly identical chemical shift over the pH range from 2 to 8.5. This is important evidence that only one of the two nearby amide protons in pSD participates in the intramolecular H-bonding interaction with the dianionic phosphoryl group. Unlike pSD, however, pTD shows the pHdependent chemical shifts for both amide protons (Figure 2b). The HAc and HAm peaks in pTD are shifted downfield when the phosphoryl group becomes dianionic. Accordingly, the pK2 value of the phosphoryl group can be estimated to be 6.49; note that the pK2 value for pTD is slightly higher than that for pSD. This clearly indicates that both amide protons in pTD can form intramolecular H bonds with the dianionic phosphoryl group. This is an important piece of information for further elucidating different mechanisms of phosphorylation effects on dipeptide conformation in these two systems. B. CD Spectra. To investigate the backbone conformations of SD, pSD, TD, and pTD, their CD spectra were measured for varying temperature (from 10 to 70 °C near neutral pH) and pH (from 3.00 to 7.20 at 10 °C) in water (Figure 3). Phosphorylated Serine. The CD spectra of SD and pSD at pH 7.00 for varying temperature are shown in Figure 3, panels a and b, respectively. Note that the phosphoryl group of pSD is mainly dianionic at pH 7.00, as shown by NMR study. First of all, the CD spectra of SD and pSD exhibit a strong negative band at 198 nm and a broad negative band at 210-230 nm. This is a characteristic line shape of polypeptide having mainly PII (strong negative band at 198 nm) and some β-strand (broad negative band at 210-230 nm) conformations.10,23-26 With increasing temperature, the absolute CD intensity of the negative band at 198 nm decreases, but that at 210-230 nm increases. This indicates that the relative population of β-strand increases with temperature. Interestingly, there is an isodichroic point at 206 nm in the CD spectra of SD and pSD. This is strong evidence that there exist two distinguished conformers with free energy barrier larger than the thermal energy. By comparing CD spectra of SD and pSD, we can conclude that the backbone conformation of SD is nearly identical to that of pSD and thus seems unaffected by phosphorylation. Another evidence for the little dependence of the SD conformation on phosphorylation can be found in the CD spectra of pSD at 10 °C for varying pH (see Figure 3c). Except for the slight decrease in the absolute CD intensity, pSD does not show any CD spectral line shape change over the pH range from 3.00 to 7.15. More importantly there is no isodichroic point found at all. This indicates that the relative population of PII and β-strand remains unchanged when the phosphoryl group becomes doubly ionized at pH > 6.05. Thus, one can conclude that pSD has a strong PII conformational propensity in water despite the electrostatic perturbation to the backbone peptides by the dianionic phosphoryl group. Furthermore, it should be noted that the intramolecular H-bonding interaction between the dianionic phosphoryl group and HAc in pSD, which is suggested by NMR study, does not induce any conformational transition. Phosphorylated Threonine. The CD spectra of TD and pTD at pH 7.00 and 7.50 for varying temperature are shown in Figure
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Figure 3. CD spectra of SD, pSD, TD, and pTD for varying temperature and pH. The CD spectra were measured for 0.2 mg/mL SD (a) and 0.3 mg/mL pSD (b) in H2O at pH 7.00 over the temperature range of 10-70 °C; (c) 0.3 mg/mL pSD in H2O at 10 °C over the pH range of 3.00-7.15; 0.2 mg/mL TD in H2O at pH 7.00 (d) and 0.25 mg/mL pTD in H2O at pH 7.50 (e) over the temperature range of 10-70 °C; (f) 0.25 mg/mL pTD in H2O at 10 °C over the pH range of 5.30-7.20.
3, panels d and e, respectively. Similar to SD and pSD, both TD and pTD have a strong negative band at 198 nm and a broad negative band at 210-230 nm. Again, with increasing temperature, the absolute CD intensity of the negative band at 198 nm decreases, but that at 210-230 nm increases. Likewise, TD and pTD have an isodichroic point at 206 nm. This indicates that they also have two possible conformations, PII and β-strand. Interestingly, however, TD and pTD have the larger absolute amplitudes of the CD band at 210-230 nm in comparison to SD and pSD. This means that TD and pTD tend to have more population of β-strand than SD and pSD do. In stark contrast to pSD, pTD undergoes notable conformational transition upon double ionization of the phosphoryl group as shown in the CD spectra of pTD at 10 °C for varying pH (Figure 3f). With increasing pH, the absolute CD intensity of the negative band at 198 nm decreases, but that at 210-230 nm increases. One can immediately identify an isodichroic point at 209 nm. Note that there is no isodichroic point in the CD spectra of pSD at 10 °C for varying pH (see Figure 3c). This result indicates that pTD undergoes conformational transition from PII to β-strand upon double ionization of the phosphoryl group. We suggest that the presence of the dianionic phosphoryl group capable of forming an intramolecular H bond with HAm is associated with such an increase of β-strand. As found by NMR study, HAc in both pSD and pTD can form an H bond with the dianionic phosphoryl group, whereas HAm only in pTD can make an additional H-bonding interaction with the dianionic phosphoryl group (see Figure 2). Therefore, it may be suggested that the intramolecular H bond between the dianionic phosphoryl group and HAm is responsible for the increase in the relative population of β-strand upon double ionization of the phosphoryl group in pTD. It is likely that the methyl group on the β-carbon of pTD effects such an additional H-bonding interaction for conformational transition (see the detailed discussion in Section
III.D). This is the first experimental result demonstrating an important role of the methyl group on the β-carbon of pTD in determining the structural propensity of the peptide backbone upon double ionization of the phosphoryl group, which has not been found nor addressed before. C. IR and VCD Spectra. To search for other independent clues to the phosphorylation effects on the peptide backbone conformation, the IR and VCD spectra of SD, pSD, TD, and pTD were measured at 15 °C and near neutral pD (Figure 4). The amide I IR spectra of these four dipeptides are quite broad and featureless and do not provide any incisive information about the conformational changes upon phosphorylation (Figure 4, panels a and c). However, their amide I VCD spectra exhibit characteristic features of the PII structure and are quite similar to that of alanine dipeptide (Figure 4, panels b and d).27 The VCD spectrum of SD is nearly identical to that of pSD at pD 7.0, indicating no significant conformational change of SD upon phosphorylation (Figure 4b). This is fully consistent with the conclusion deduced from their CD spectra in Section III.B as well as the VCD spectra of GSSS and GSSpS in ref 10. On the other hand, the VCD spectrum of TD differs from that of pTD at pD 7.5, indicating the structural change of TD upon phosphorylation (Figure 4d). Comparing the VCD spectrum of TD with those of SD, alanine dipeptide,27 acetylproline amide,28,29 and other short peptides, we concluded that the solution structure of TD is mainly PII. However, the positive VCD peak of TD at 1652 cm-1 becomes very week in pTD. Note that β-sheet typically shows the weak amide I VCD band in comparison to PII or R-helix.30-32 This VCD spectral change is another strong evidence that TD undergoes conformational change from PII to β-strand upon phosphorylation. D. Comparisons with Previous Works: Side-chain Effects on the Peptide Backbone Conformation. Shi et al. used NMR spectroscopy to study the conformation of the XAO peptide,
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Figure 4. Amide I IR (a and c) and VCD (b and d) spectra of SD, pSD, TD, and pTD. The IR and VCD spectra were measured for 40 mg/mL SD (solid curve) or pSD (dashed curve) in D2O at 15 °C and pD 7.0; 40 mg/mL TD (solid curve) or pTD (dashed curve) in D2O at 15 °C and pD 7.5. The collection time for a VCD spectrum is 6 h for SD, pSD, and TD and 12 h for pTD.
an 11-mer with 7 alanine residues.23 Combined evidence from 3 JHNR coupling constants and NOE measurements demonstrated that its preferred backbone conformation is PII at 2 °C and the relative population of β-strand increases with temperature. Consistent with this result, there is a single isodichroic point at 205 nm in the CD spectra of XAO for varying temperature, which indicates that there exist two distinguished conformers, PII and β-strand, for this peptide. However, this view was challenged by others,33,34 who suggested that PII is likely to be just one of several conformations. Graf et al. carried out NMR and molecular dynamics simulation studies of the homologous series of alanine peptides.26 It was found that trialanine samples mainly PII (∼90%) and some β-strand (∼10%) conformations. Their fitting results for a series of short peptides with three to seven alanine residues showed that they all adopt dominantly PII (>80%) and some β-strand (
