Discrepancies between Conformational ... - ACS Publications


Dec 7, 2010 - force field ff03* (Best, R.; Hummer, G. J. Phys. Chem. B 2009, 113, 9004-9015). The simulations yielded a statistical coil distribution ...
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J. Phys. Chem. B 2010, 114, 17201–17208

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Discrepancies between Conformational Distributions of a Polyalanine Peptide in Solution Obtained from Molecular Dynamics Force Fields and Amide I′ Band Profiles Daniel Verbaro,† Indrajit Ghosh,‡ Werner M. Nau,‡ and Reinhard Schweitzer-Stenner*,† Department of Chemistry, Drexel UniVersity, 3141 Chestnut Street, Philadelphia, PennsylVania 19104, United States, and School of Engineering and Science, Jacobs UniVersity Bremen, Campus Ring 1, D-28759 Bremen, Germany ReceiVed: September 30, 2010; ReVised Manuscript ReceiVed: NoVember 13, 2010

Structural preferences in the unfolded state of peptides determined by molecular dynamics still contradict experimental data. A remedy in this regard has been suggested by MD simulations with an optimized Amber force field ff03* (Best, R.; Hummer, G. J. Phys. Chem. B 2009, 113, 9004-9015). The simulations yielded a statistical coil distribution for alanine which is at variance with recent experimental results. To check the validity of this distribution, we investigated the peptide H-A5W-OH, which with the exception of the additional terminal tryptophan is analogous to the peptide used to optimize the force fields ff03*. Electronic circular dichroism, vibrational circular dichroism, and infrared spectroscopy as well as J-coupling constants obtained from NMR experiments were used to derive the peptide’s conformational ensemble. Additionally, Fo¨rster resonance energy transfer between the terminal chromophores of the fluorescently labeled peptide analogue H-Dbo-A5W-OH was used to determine its average length, from which the end-to-end distance of the unlabeled peptide was estimated. Qualitatively, the experimental 3J(HN,CR), VCD, and ECD indicated a preference of alanine for polyproline II-like conformations. The experimental 3J(HN,CR) for A5W closely resembles the constants obtained for A5. In order to quantitatively relate the conformational distribution of A5 obtained with the optimized AMBER ff03* force field to experimental data, the former was used to derive a distribution function which expressed the conformational ensemble as a mixture of polyproline II, β-strand, helical, and turn conformations. This model was found to satisfactorily reproduce all experimental J-coupling constants. We employed the model to calculate the amide I′ profiles of the IR and vibrational circular dichroism spectrum of A5W, as well as the distance between the two terminal peptide carbonyls. This led to an underestimated negative VCD couplet and an overestimated distance between terminal carbonyl groups. In order to more accurately account for the experimental data, we changed the distribution parameters based on results recently obtained for the alanine-based tripeptides. The final model, which satisfactorily reproduced amide I′ profiles, J-coupling constant, and the end-to-end distance of A5W, reinforces alanine’s high structural preference for polyproline II. Our results suggest that distributions obtained from MD simulations suggesting a statistical coil-like distribution for alanine are still based on insufficiently accurate force fields. Introduction The canonical view of the unfolded state of proteins and peptides suggests that they are unstructured in that all natural amino acid residues with the exception of proline sample the entire sterically allowed region of the Ramachandran plot with comparable probability. This view is generally termed the random or statistical coil model.1-3 However, recent experiments on short peptides and analyses of truncated coil libraries (helices and sheet structures were omitted) indicated that some amino acids have a much larger preference for polyproline II (PPII) like conformations than predicted by respective Ramachandran plots obtained from molecular mechanics and dynamics calculations.4-11 The canonical PPII conformation adopted in crystals of poly-L-proline exhibits dihedral angles of about φ ) -70 and ψ ) 150.12 Among the amino acids for which preferences for PPII-like conformations have been proposed, alanine has attracted particular attention. The focus on this amino acid residue reflects its abundance in proteins, its high helical propensity in folded * Corresponding author. Phone: 1-215-895-2268. Fax: 1-215-895-1265. E-mail: [email protected] † Drexel University. ‡ Jacobs University Bremen.

structures, and the simplicity of its side chain, which facilitates computational modeling. Two-dimensional IR, analyses of the amide I band profiles of IR, Raman, and vibrational circular dichroism (VCD) spectra, NMR, and electronic circular dichroism (ECD) spectroscopy on alanine-based peptides have provided ample evidence for the notion that alanine has a preference for PPII, but attempts to quantitatively determine the propensity for this conformation yielded rather different results (i.e., mole fractions between 0.3 and 0.9 per residue).13-19 Only recently, a very thorough NMR study by Graf et al., which utilized the φ and ψ dependence of seven different dipolar J-coupling constants in a study of various oligo-alanines, resolved the issue by providing strong evidence for a very high PPII propensity of alanine (i.e., 0.9).20 This result was recently confirmed by a reanalysis of amide I profiles for A3 and by a combined NMR/ vibrational spectroscopy study on GAG.21,22 The above results for alanine are still at odds with the results of many molecular dynamics simulations on short alaninecontaining peptides which generally predict rather statisticalcoil-like distributions with a substantial fraction of right-handed helical conformation.9,10,23 This is at variance with the rather small nucleation constants generally observed from investigations of helix T coil transitions.22 The respective distribution

10.1021/jp109404r  2010 American Chemical Society Published on Web 12/07/2010

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J. Phys. Chem. B, Vol. 114, No. 51, 2010

depends heavily on the choice of the force field.9,10 Only a modified AMBER force field, for which Garcia and co-workers eliminated the torsional energy terms, predicts a rather high PPII and a comparatively low helical fraction.24,25 Another attempt to reconcile experimental data with MD simulations has recently been undertaken by Best and Hummer.23 They used two modifications of the original AMBER force field termed ff99B and ff03. They were both obtained from reparametrizing the AMBER force field based on results from quantum chemical calculations. Best and Hummer optimized these force fields further by trying to account for the helix content of short peptides. They subsequently checked the validity and applicability of their new force fields (termed ff99SB* and ff03*) by calculating the J-coupling constants, which Graf et al. obtained for pentaalanine in water.20 This analysis yielded a much lower PPII content than that reported by Graf et al. (0.5 for ff03* and 0.4 for ff99SB*). Generally, the distributions emerging from the calculations with both force fields resemble a classical statistical coil. Besides using different modeling techniques, Graf et al. and Best and Hummer used different coefficients for the Karplus equations employed to simulate the different J-coupling constants. Whereas Graf et al. used empirically determined coefficients, Best and Hummer obtained their values from DFT calculations on short alanine peptides (AcA and AA).26 In view of the discrepancy between the results obtained from these two sets of Karplus equations, a further experimental check of the respective distributions seems to be a necessity. To this end, we measured the amide I′ band profile of the IR and VCD spectrum of H-A5W-OH which closely resembles the oligoalanine peptide investigated by Graf et al.20 1H NMR measurements were performed to determine the 3J(HN,HR) constants of its residues. The amide I′ band profile and the NMR coupling constants reported by Graf et al. were calculated in terms of a statistical ensemble built on the results of the MD simulations of Best and Hummer.23 In order to calculate the J-coupling constants, we followed these authors by utilizing the abovementioned DFT-based Karplus parameters reported by Case et al.26 The results of these calculations were compared with a direct conformational analysis of the above oligo-alanine based on a global fit of a conformational distribution model to amide I′ band profiles and J-coupling constants. The additional tryptophan residue at the end of the peptide serves two purposes. First, it allowed us to exactly determine the concentration of the peptide, which is important for obtaining the amide I profiles in absolute units. Second, it facilitated the comparison with another experiment designed to explore the peptide’s conformational ensemble. Recently, Nau and co-workers introduced Fo¨rster resonance energy transfer (FRET) as an ideal tool for determining the average length of short peptides by using tryptophan as donor and Dbo (2,3-diazabicyclo[2.2.2]oct-2-ene) as acceptor. This pair has a very short Fo¨rster radius (R0 ) 9 Å), which allows FRET measurements of short distances in the 10 Å domain.27,28 We used this technique to determine the average distance between the fluorophores of the labeled peptide Dbo-A5W (see Scheme 1), which can be regarded as a measure of the average end-to-end distance. We compared this value with estimations derived from the different conformational models used to account for the amide I′ profiles and NMR coupling constants. Eventually, this led to a reliable, experimentally based conformational model, which considers a high PPII propensity for alanine residues, thus supporting the results of Graf et al.20 Our results suggest that further modifications of

Verbaro et al. SCHEME 1: Molecular Structures of Investigated Acceptor Chromophore, Labeled Amino Acid, and Pentaalanine Peptide

force fields are necessary for an accurate description of the conformational manifold of even very simple and short peptides. Methods and Materials Unblocked L-alanyl-L-alanyl-L-alanyl-L-alanyl-L-alanyl-Ltryptophan (H-AAAAAW-OH, A5W) was custom synthesized by Celtek Peptides with >99.3% purity and further purified through lyophilization and dialysis using a MW500 dialysis bag to remove residual TFA (trifluoroacetic acid). With the exception of the C-terminal tryptophan, which we used for an exact determination of the peptide concentration and Fo¨rster resonance energy transfer (FRET) measurements, the peptide is analogous to the unblocked pentaalanine peptide investigated by Graf et al.20 The J-coupling constants obtained for this peptide were used by Best and Hummer to check the validity of the conformational ensembles obtained from their AMBER ff03* and ff99SB* force fields. For IR and VCD, the peptide was dissolved in D2O with a concentration of 20 mM. This is the highest achievable possible concentration in aqueous solution, which is sufficient for IR and VCD.29,30 A pD of 6.9 was obtained using the Glasoe and Long31 method with an Accumet micro size standard glass combination electrode with Ag/AgCl and an Accumet pH meter (Fisher Scientific). Vibrational spectra were taken on a BioTools Chiral IR with a 20 µm cell and an 8 cm-1 resolution. The combined spectra were collected over 720 min (VCD 680 min and IR 72 min). The temperature was kept at 25 °C using a Biotools water-cooling temperature controller. For ECD, a peptide concentration of 5 mM was prepared in D2O, which was used for direct comparison to vibrational data since conformational propensities have been shown to change if D2O is replaced by H2O.32 Spectra were taken every 5 °C between the wavelengths 180 and 280 nm on a J-810 spectropolarimeter (Jasco, Easton, MD). A 0.05 mm cell was used with 0.5 nm resolution and a scan speed of 500 nm/min. An average of 10 scans was taken at each temperature. Also, for the 1H and COSY experiments of A5W, the peptide was dissolved at 20 mM in 90% H2O and 10% D2O. The 3J(HN,HR)coupling constants were obtained with a Unitylnova 500 MHz NMR spectrometer at 25 °C. The FRET experiments were performed on the peptide H-Dbo-AAAAAW-OH (custom synthesized by Biosyntan in 96.1% purity), which contained an additional N-terminal Dbo (2,3-diazabicyclo[2.2.2]oct-2-ene)-labeled asparagine. Measurements were performed at ambient temperature (25 °C) in aerated H2O at pH 6.8 ((0.2), with reference to the donor-only labeled peptide, A5W. The fluorescence lifetimes of the peptides were

Conformational Distributions of Polyalanine Peptide

J. Phys. Chem. B, Vol. 114, No. 51, 2010 17203

Figure 1. Far-UV circular dichroism spectrum of 5 mM A5W in D2O. The arrows indicate the spectral changes with increasing temperature, which was changed in increments of 5 °C. The inset shows the difference spectrum ∆ε(80°C) - ∆ε(5°C).

measured by time-correlated single-photon-counting (FLS 920, Edinburgh Instruments Ltd.) using a PicoQuant pulsed LED (PLS-280, λexc ) 280 nm, λobs ) 350 nm, fwhm ca. 450 ps) for excitation of Trp. Steady-state emission spectra and intensities (λexc ) 280 nm) were recorded with a Cary Eclipse fluorometer (Varian). For the steady-state measurements, peptide concentrations (ca. 20 µM) were adjusted to an optical density of ca. 0.10 at the excitation wavelength. Results 3

N

R

The J(H ,C ) constant was measured for all residues in order to compare the alanines of A5W with A5. Correlation spectroscopy (COSY) only allowed identification of tryptophan 3 J(HN,CR), which was 7.53 ( 0.03 Hz. The alanine coupling constants were 5.49 ( 0.02, 5.71 ( 0.06, 5.39 ( 0.09, and 6.16 ( 0.06 Hz in no particular order. The coupling constants seem to be in close agreement with the values Graf et al. reported for A5.20 None of the alanines’ 3J(HN,CR) could be assigned to a distinct residue; however, all of the coupling constants are indicative of a φ value in the PPII region of the Ramachandran space. Electronic circular dichroism was utilized to qualitatively gauge the secondary structures adopted by A5W. The respective spectra measured as a function of temperature are displayed in Figure 1. At lower temperatures, the extrema at 220 and 190 nm are together diagnostic of a significant PPII fraction.22,29,35 The ∆ε values at these wavelengths are comparable with those reported for GAG and AAA,22,29,32 which strongly suggests a similar PPII content. Apparently, the influence of the terminal tryptophan on the far-UV CD signal is very barely detectable. As the temperature increases, the couplet amplitude decreases. The spectra clearly depict an isodichroic point, suggesting that the peptide predominantly samples only two minima of its Gibbs energy landscape. The difference spectrum ∆ε(80°C) - ∆ε(5°C) exhibited in the inset clearly suggests that β-strand-like conformations become more populated at high temperatures, in agreement with expectations.33 It is noteworthy that these ECD spectra, though yielding only qualitative information, are certainly at variance with the conformational distributions obtained from MD simulations of A5, in that the latter suggest at least a three-state model (PPII, β, right-handed helical). In what follows, this paper focuses on analyzing the conformational distribution, which A5W exhibits at room temperature. Figure 2 exhibits the amide I′ profiles of the FTIR and VCD spectra of A5W. The IR absorption band shows a peak at 1642

Figure 2. Amide I′ region of the infrared and vibrational circular dichroism spectra of A5W in D2O. The experimental conditions chosen for recording these spectra are described in Methods and Materials. The red lines result from a simulation using a conformational distribution reflecting the Ramachandran plot obtained from MD simulations with a ff03* force field. The black lines reflect the results of a fit with an adjustable conformational model describable as superposition of two-dimensional Gaussian distributions associated with PPII, β-strand, right-handed helical, and inverse γ-turn-like conformations. The conformational fractions are listed in Table 3 as amide I′based I. The blue line was computed with a refined model, which additionally considered a further modified distribution as mentioned in the results.

cm-1 and a shoulder at 1660 cm-1. The corresponding VCD spectrum depicts a strong negative couplet with a positive extremum around 1663 cm-1 and a negative one at 1638 cm-1, which is indicative of a strong preference for a PPII-like structure.34 In the following, these two profiles are used to check the validity of the conformational distributions, which Best and Hummer obtained from their MD simulations with ff03* and ff99SB* force fields.23 To this end, we proceeded as follows. We used the Ramachandran plots obtained from ff03* simulation (kindly provided by Dr. Best) to construct the following distribution function. The upper left-handed quadrant was divided into two subspaces centered at (variable) coordinates assignable to PPII and β-strand conformations. Points between (-90 < φ < -40 and 110 < ψ < 180) were considered PPII, (-180 < φ < -100 and 110 < ψ < 180) were considered β, and (-160 < φ < -20 and -120 < ψ