Val-Pro-Gly-Val-Gly - American Chemical Society

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Macromolecules 2005, 38, 6038-6047

Structural Determination of an Elastin-Mimetic Model Peptide, (Val-Pro-Gly-Val-Gly)6, Studied by 13C CP/MAS NMR Chemical Shifts, Two-Dimensional off Magic Angle Spinning Spin-Diffusion NMR, Rotational Echo Double Resonance, and Statistical Distribution of Torsion Angles from Protein Data Bank Kosuke Ohgo,† Jun Ashida,‡ Kristin K. Kumashiro,§ and Tetsuo Asakura*,† Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan, Varian Technologies Japan Ltd., Minato, Tokyo 108-0023, Japan, and Department of Chemistry, University of Hawaii at Manoa, 2545 McCarthy Mall, Honolulu, Hawaii 96822 Received January 11, 2005; Revised Manuscript Received April 16, 2005

ABSTRACT: The structure of an elastin-mimetic model peptide, (VPGVG)6, was proposed by combining data obtained from quantitative use of the conformation-dependent 13C NMR chemical shifts, twodimensional off magic angle spinning spin-diffusion solid-state NMR with 13C double labeling of the peptides, rotational-echo double-resonance of 13C, 15N double labeling peptides, and statistical distribution of the backbone torsion angles of Val-Pro, Pro-Gly, and Gly-Val-Gly sequences from PDB. In essence, this approach drew upon several sets of data to formulate the resulting model, namely, that there is a distribution of conformations in this polypeptide. The Val-16 residue adopts torsion angles (φ, ψ) ) (-90 ( 15°, 120 ( 15°). In contrast, bimodal distributions for the central residues of the (VPGVG) subunit were detected. For Pro-12, (φ, ψ) ) (-60 ( 15°, 120 ( 15°), 80%, and (φ, ψ) ) (-60 ( 15°, -30 ( 15°), 20%; for Gly-13, (φ, ψ) ) (90 ( 30°, -15 ( 30°) and (φ, ψ) ) (-90 ( 15°, 0 ( 15°), 60%, and (φ, ψ) ) (-105 ( 75°, 150 ( 30°), (φ, ψ) ) (120 ( 60°, 150 ( 30°), (φ, ψ) ) (-120 ( 60°, -150 ( 30°) and (φ, ψ) ) (120 ( 60°, -150 ( 30°), 40%. For Val-14, (φ, ψ) ) (-110 ( 20°, 130 ( 20°), 70%, and (φ, ψ) ) (-75 ( 15°, -15 ( 15°) 30%. To reconcile the torsion angles corresponding to the main component in the conformational distributions, a type-II β-turn structure was assigned to about 40% of the Pro-Gly pair.

Introduction The reversible elongation and contraction of the blood vessels and connective tissue of vertebrates has been attributed to the amorphous protein elastin.1 Elastin is unusual in its composition, which is dominated by the presence of small hydrophobic amino acids, such as glycine (Gly, G), alanine (Ala, A), proline (Pro, P), and valine (Val, V). Generally, elastin is considered to have hydrophobic and cross-linking domains. The hydrophobic domains are characterized by long stretches of mostly small nonpolar residues. In contrast, the crosslinks are found in Ala-rich regions. The very nature of elastin, an extensive, cross-linked biopolymer with its predominantly hydrophobic composition, precludes the two most well-known methods for high-resolution structure elucidation, solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. Furthermore, insoluble or amorphous elastin is assembled from a soluble monomer tropoelastin, which has a molecular weight of 70-80 kDa. One could easily envision the complexities of doing a structural analysis of proteins such as elastin and tropoelastin. To circumvent the problems associated with the complexities of this protein, both in terms of its composition as well as its physical properties, many have focused their studies on the model peptides of elastin.2-6 The hydrophobic regions of elastin include a striking number of repeating polypeptide sequences, the most †

Tokyo University of Agriculture and Technology. Varian Technologies Japan Ltd. § University of Hawaii at Manoa. * To whom correspondence should be addressed. Telephone and fax: (+81)-423-83-7733 E-mail: [email protected]. ‡

well-known of which is poly(VPGVG). Some of the properties of this polypentapeptide mimic those of native elastin and tropoelastin.7 For example, the peptide is soluble at lower temperatures but aggregates, or coacervates, as the solution is warmed to near physiological temperatures. This property is well-known for tropoelastin and other soluble elastin peptides, such as R-elastin.8 Furthermore, the polypentapeptide can be cross-linked.9 Indeed, some have attributed the elasticity of elastin to arise from the poly(VPGVG) segments and others like it in the hydrophobic region.6,10 Although there are some concerns about its use as the most ideal model, there are, nevertheless, numerous advantages to using this repeating polypeptapeptide to answer questions in elastin structure-function. Other studies have included experimental approaches with CD, Raman, solution and solid-state NMR spectroscopy.4,11-14 In general, the experiments of the peptide in solution have supported the model first proposed by Urry,2,15 namely, that the repeating peptides form β-spirals, a regularized structure with repeating type II β-turns. Solid-state NMR spectroscopy is a powerful tool for determining molecular distances, torsion angles, and conformational distributions of solid proteins directly in their native environment. In our previous papers,16-18 13C solid-state NMR methods, including 2D off-magicangle-spinning (OMAS) spin-diffusion solid-state NMR coupled with double-13C-labeling of specific residues, have been successfully used to determine the torsion angles of the backbone amino acid residues for silk model peptides.16,18 The power of solid-state NMR spectroscopy is especially evident in our previous studies

10.1021/ma050052e CCC: $30.25 © 2005 American Chemical Society Published on Web 06/11/2005

Macromolecules, Vol. 38, No. 14, 2005 Table 1. Several

a

13C

and

15N-Labeled

An Elastin-Mimetic Model Peptide 6039 Peptides Synthesized for 2D Spin-Diffusion NMR and REDOR Experiments

peptide

method

1. (VPGVG)2VPGV[1-13C]G15[1-13C]V16PGVG(VPGVG)2 2. (VPGVG)2[1-13C]V11[1-13C]P12GVG(VPGVG)3 3. (VPGVG)2V[1-13C]P12[1-13C]G13VG(VPGVG)3 4. (VPGVG)2VP[1-13C]G13[1-13C]V14G(VPGVG)3 5. (VPGVG)2VPG[1-13C]V14[1-13C]G15(VPGVG)3 6. (VPGVG)2V[15N]P12[2-13C]G13VG(VPGVG)3 7. (VPGVG)2VP[15N]G13[2-13C]V14G(VPGVG)3

spin-diffusion spin-diffusion spin-diffusion spin-diffusion spin-diffusion REDOR REDOR

information Val16(φ,ψ) Pro12(φ,ψ) Gly13(φ,ψ) Val14(φ,ψ) Gly15(φ,ψ) Pro12(ψ) Gly13(ψ)

angle angle angle angle anglea distance distance

Reported previously (Asakura et al. Polym. J. 2003, 35, 293-296).

of (Ala-Gly)15, in which a high-resolution model for this silk fibroin peptide mimetic was obtained. Recently, we studied the local structure of residues Val-16, Val-14 and Gly-15 in the VPGVG pentapeptide in (VPGVG)6 by 2D spin-diffusion NMR.3 We showed that none of the spectra could be fit using the torsion angles corresponding to the β-spiral structure. A more detailed analysis on Gly-15 indicated that a conformational distribution was present. A shorter (but related) elastin peptide, (VPGVG)3 was investigated by Yao and Hong.4 They proposed a bimodal structure distribution for the PG residues in the central VPGVG subunit consisting of populations of a compact structure and an extended and distorted β-strand structure. In this paper, we synthesized the repeated pentapeptide, (VPGVG)6, with isotopic labels in key positions to obtain torsion angle information and the internuclear atomic distances of the backbone chain of the central pentapeptide subunit in the 30-mer using 2D OMAS spin-diffusion NMR and REDOR. The conformationdependent 13C chemical shift was also used to restrict the torsion angles, as reported previously.19,20 Our interpretation was tied strongly to the statistically allowed regions of the Ramachandran plots for the respective amino acids, based on information available in the Brookhaven Protein Databank (PDB). Experimental Methods Materials. We prepared the elastin-like model peptide (VPGVG)6 and its isotopically labeled peptides by solid-phase Fmoc chemistry on a fully automated Pioneer Peptide Synthesis System (Applied Biosystems Ltd.). The labeled sites of the peptides are summarized in Table 1. Fmoc amino acids and all reagents used in peptide synthesis were procured from PerSeptive Biosystems, Warrington, U.K. The resin and HATU were from Applied Biosystems and PE Biosystems, respectively. The solvents of high-purity grade and other chemicals were available locally from Wako Pure Chemical Industries Ltd. Typically the peptide was assembled on Fmoc-Gly-PEG-PS resin. The coupling of Fmoc amino acids was performed by HATU. After synthesis, the free peptides were released from the resin by treatment with a mixture of trifluoroacetic acid (TFA), phenol, triisopropylsilane, and water (88:5:2:5 vol %) for 2 h at room temperature. The crude peptide was precipitated with dry diethyl ether and washed repeatedly with cold ether. The precipitate, collected by centrifugation, was dried in vacuo. Then the crude peptides from the TFAcleavage were purified by HPLC using acetonitrile as an elution. After purification, acetonitrile was removed by evaporation at 42 °C and then the aqueous solution of the peptide was lyophilized for 24 h. The molecular weight of the polypentapeptide is 2475 Da. 2D OMAS Spin-Diffusion NMR. The 2D off magic angle spinning spin-diffusion NMR spectra were ob-

tained with a Varian MERCURYplus 400 NMR spectrometer operating at a magnetic field of 9.4 T, corresponding to a resonance frequency of 100.7 MHz for 13C. A 7 mm Jakobsen-type double-tuned MAS probe was used. Off-magic-angle-spinning (OMAS) conditions were (θm - 7°) and sample spinning of 6 kHz ((5 Hz). The angle θm between static magnetic field and sample spinning axes was determined by the measurements of scaled 13C chemical shielding anisotropy (CSA) spectra of β-quinol methanol under OMAS. The CSA of the benzene carbon of β-quinol methanol is 176 ppm and therefore the scaling factor and the angle θm were easily calculated from the scaled CSA powder pattern under OMAS. The principal values of the chemical shift tensors for the carbonyl carbon atoms were determined with the 1D OMAS spectra. The scaling factor of the 2D spin-diffusion spectra was 1/2 (3 cos2(θm - 7°) - 1) ) +0.2. The mixing time of two seconds was optimized for intramolecular spin-diffusion between enriched carbons in two adjacent residues, such that ∼100% of the 13C-13C spin pairs are selected. The contact time was set to 2 ms, and the variable-amplitude CP (VACP) technique was used. 21 All NMR experiments were conducted at room temperature. Analysis of 2D Spin-Diffusion NMR Spectrum. A calculation program developed in our laboratory was used to simulate the 2D spin-diffusion NMR spectra.18 An OCTANE (Silicon Graphics Inc.) workstation was used for the calculation of the theoretical spectra. For simulation of 2D spin-diffusion NMR spectra, the calculations were performed using a grid of 15° for φ and ψ values. To avoid the influence of a strong diagonal peak, the diagonal peaks were neglected in the best fit analysis. The RMSD value was used to quantify the difference between the experimental and calculated spectra. The definition of the RMSD was

RMSD(φ,ψ) )

x

N

[Ei - λ(φ,ψ)Si(φ,ψ)]2 ∑ i)1 N

where N is the number of intensities analyzed, Ei are the experimental intensities, Si(φ, ψ) are the calculated intensities, and λ(φ, ψ) is a scaling factor calculated to minimize RMSD at each φ, ψ pair.22 For the averaging of the calculated 2D spin-diffusion NMR spectra, Gaussian functions on the φ, ψ plane was assumed. Visualization of spectra and RMSD calculation were performed using MATLAB (The MathWorks, Inc.). REDOR Experiment. 13C-detected 13C-15N REDOR experiments were performed on a Chemagnetics Infinity 400 MHz spectrometer at 9.4T (100.0 MHz for 13C and 40.3 MHz for 15N), using the following conditions: a 4

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Figure 1.

Macromolecules, Vol. 38, No. 14, 2005

13

C CP/MAS NMR spectrum of (VPGVG)6.

Figure 3. 2D spin-diffusion NMR spectra calculated for Val16 residue as a function of (φ, ψ) using increments of 30°.

the scatter of the data points acquired for this system. Analysis of 13C Chemical Shift Map. The quantitative relationship between the 13C chemical shift and local structure in proteins19,20 was also used to impose constraints on the conformational parameters of this peptide. The results for CR and Cβ carbon chemical shifts were used to generate chemical shift error maps showing the root-mean-square difference between the observed CR and Cβ chemical shifts and the estimated shifts as a function of φ and ψ. The root-mean-square errors, ∆, in the chemical shift calculations are calculated as

∆) Figure 2. Combined CR and Cβ chemical shifts error map for Val-16, shown by black diagonal lines. The small RMSD regions corresponding to the 2D spin-diffusion NMR spectrum of peptide 1, (VPGVG)2VPGV[1-13C]G[1-13C]VPGVG(VPGVG)2, are superimposed as the area enclosed by black solid lines. Contour plot of the (φ, ψ) of the Val residues in the Val-Pro sequences obtained from the PDB data is also shown. The torsion angles of Val-16 summarized in Table 1 are confined to an area enclosed by the white square.

mm triple-channel magic-angle probe; spinning rate 6.666 kHz; π pulses for 13C and 15N channels, respectively, 6.5 and 8.2 µs; recycle delay 3.0 s. Phases of 15N π pulses were cycled according to the XY-8 scheme23 to minimize off-resonance and pulse error effects. REDOR evolution times ranged up to 18 ms. The REDOR results have been corrected for the natural-abundance background by calculation. Values of ∆S/S0 ) 1 - S/S0 were computed as the ratios of peak intensities in the REDOR spectra. The internuclear distance of 4.1 Å corresponds to the torsion angle, ψ ) 0° for Gly-13 or Pro-12 residues. The distance of 4.9 Å corresponds to ψ ) (180°. Simulations of REDOR curves (not shown) that are obtained for distributions of the population divided between the 4.1 and 4.9 Å distances do not account for

{

}

(CR - CRmap)2 + (Cβ - Cβmap)2 2

1/2

The random coil values reported in ref 24 were used here. PDB Analysis. X-ray crystallographic data from the Protein Data Bank25,26 at the Research Collaboratory for Structural Bioinformatics (RCSB) (http://www.rcsb.org/pdb/) were used to search the torsion angles of ValPro, Pro-Gly, and Gly-Val-Gly sequences in natural protein. The structures determined at 1.5Å resolution or better and R factor e15% were used in the case of Val-Pro and Pro-Gly and 2.0Å resolution or better and R factor e20% in the case of Gly-Val-Gly. A subset of 543 Val-Pro, 709 Pro-Gly, and 331 Gly-Val-Gly occurrences were obtained from the database after excluding multiple entries of proteins with a similarity greater than 50%. For an each pair, the torsion angles of both residues were calculated from atomic coordinate and were plotted on a Ramachandran plot. Results The 13C CP/MAS NMR spectrum of the elastinmimetic model peptide (VPGVG)6 is shown in Figure 1 together with the assignment of all resolved features.3


An Elastin-Mimetic Model Peptide 6041

Figure 4. Observed 2D spin-diffusion NMR spectrum of peptide 1, (VPGVG)2VPGV[1-13C]G[1-13C]VPGVG(VPGVG)2, (A) and the calculated 2D spin-diffusion NMR spectrum corresponding to (φ, ψ) ) (-90°, 120°) (B).

The chemical shifts of all major features in (VPGVG)6 are strikingly similar to those observed in another wellcharacterized elastin mimetic ([(VPGVG)4(VPGKG])39,27 indicating that their local structures are nearly identical. In addition, the positions of these peaks are similar to those reported for native elastin,28 indicating that these relatively simple peptides mimic the hydrophobic stretches found in nature. On the basis of these data, we assert that this shorter peptide is suitable for this study. 1. Determination of the Torsion Angles of Val16 Residue. The conformation-dependent chemical shifts and 2D spin-diffusion NMR were used for determination of the torsion angles of Val-16 residue. An additional restriction was imposed by the proximity of Pro from PDB data; i.e., the conformational distribution of Val-preceding-Pro is restricted due to the steric interactions between the Val side chain and the pyrrolidine ring of Pro residue.29 The chemical shift error map showing the root-mean-square difference between the observed CR and Cβ chemical shifts is shown in Figure 2. The observed Val-16 13CR and 13Cβ chemical shifts, 57.1 and 31.3 ppm,14 respectively, are indicated by the black diagonal lines. Experimental error is less than 0.5 ppm. Because of the existence of a β-carbon, the torsion angle φ of Val can be restricted to φ e 0°. The 2D spindiffusion NMR spectrum is used as an additional constraint in the Ramachandran map. As summarized in Figure 3, the 2D spin-diffusion NMR spectra calculated with the chemical shift tensor values of the carbonyl carbon of Val residue change largely depending on the torsion angles of φ and ψ of Val. Figure 4A shows the 2D spin-diffusion NMR spectrum of peptide 1: (VPGVG)2VPGV[1-13C]G[1-13C]VPGVG(VPGVG)2. The RMSD between the observed and simulated spectra was calculated according to the equation described in the Experimental Section, and the regions with small RMSD values are superimposed as the area enclosed by black solid lines in Figure 2. There are five lower RMSD regions located around (φ, ψ) angles of (-75°, -15°), (-90°, -90°), (-15°, -75°), (-120°, 75°), and (-90°, 120°). The region around (-15°, -75°) is easily removed from consideration, because these angles are not allowed in the Ramachandran plot of the Val residues.30 The torsion angles of (φ, ψ) ) (-90°, 120°) and (φ, ψ) ) (-120°, 75°) are consistent with both 13C chemical shift and 2D spin-diffusion NMR. The conformation of Val-preceding-Pro is restricted due to the steric interactions between the Val side chain

Figure 5. Combined CR and Cβ chemical shifts error map for Pro-12, shown by gray diagonal lines. The small RMSD regions corresponding to the 2D spin-diffusion NMR spectrum of peptide 2, (VPGVG)2[1-13C]V[1-13C]PGVG(VPGVG)3, are indicated as the area enclosed by the black solid lines. Also included are contour plots of the two most highly populated areas of the φ, ψ map for the Pro residues in the Val-Pro sequences obtained from the PDB data.

and the pyrrolidine ring of Pro residue.29 The contour plot of the torsion angles of the Val residues in the ValPro sequences in the proteins from PDB data is also shown in Figure 2. The torsion angles cluster in the region located around (φ, ψ) ) (-90°, 120°). The calculated 2D spin-diffusion NMR spectrum corresponding to the torsion angles of (φ, ψ) ) (-90°, 120°) is shown in Figure 4B, indicating that the agreement between the observed and calculated spectra is good. Judging from the overlapped range from the three constraints, the error in the torsion angles was evaluated to be ( 15° and the region (φ, ψ) ) (-90 ( 15°, 120 ( 15°), as shown by the white square in Figure 2. In a recent work by Yao and Hong,4 the torsion angles of the Val residue were reported as (φ, ψ) ) (-96°, 145°) or (-144°, 145°). The possibility that the torsion angles

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Figure 6. Observed 2D spin-diffusion NMR spectrum of peptide 2, (VPGVG)2[1-13C]V[1-13C]PGVG(VPGVG)3, (A) and the calculated 2D spin-diffusion NMR spectrum calculated by assuming (φ, ψ) ) (-60°, 105°) (B) and the 2D spin-diffusion NMR spectrum calculated by taking into account the conformational distribution (φ, ψ) ) (-60°, 135°) with fraction 0.8 and (φ, ψ) ) (-60°, -30°) with fraction 0.2 (C).

of Val are (φ, ψ) ) (-144°, 145°) becomes very low, because the RMSD value for these torsion angles is quite high (Figure 2). There is good agreement between our study and that of Yao and Hong for the φ angle measurement. However, there seems to be some disagreement for the angle ψ. 2. Determination of the Torsion Angles of Pro12 Residue. In Figure 5, the combined CR and Cβ chemical shifts error map is shown for Pro-12 residue. The Pro 13CR and 13Cβ chemical shifts, 60.8 and 30.5 ppm,14 respectively, satisfy two shaded areas, with an error of 0.5 ppm. The 2D spin-diffusion NMR spectrum of peptide 2: (VPGVG)2[1-13C]V[1-13C]PGVG(VPGVGV)3 is shown in Figure 6A. The corresponding RMSD map is also shown in Figure 5, respectively. There are four low RMSD regions located around the torsion angles (φ, ψ) ) (-60°, 105°), (-90°, 0°), (-15°, 105°) and (-120°, 45°) in the RMSD map. The regions located around (-120°, 45°) and (-15°, 105°) are removed from further consideration, due to constraints imposed by the ring in proline.30 Furthermore, these regions are not consistent with the chemical shift constraint. Thus, two regions, (φ, ψ) ) (-60°, 105°) and (-90°, 0°) with low RMSD values are possible, based on Ramachandran angles and the chemical shift constraints. The φ, ψ distribution of the Pro residues in Val-Pro sequences obtained from PDB recorded structures is also shown in Figure 5. The torsion angles of Pro residue in Val-Pro sequences adopt two distinct conformations between two theoretically predicted minima.29 The two general types of conformations are tightly clustered

around their mean values of (φ, ψ) ) (-60°, 135°) (region I) and (φ, ψ) ) (-60°, -30°) (region II), and the ratio of region I to region II is 0.8 to 0.2. Only region I overlaps with the shaded area obtained from the chemical shift data. This region is also close to the low RMSD region located around (φ, ψ) ) (-60°, 105°). As an additional constraint, distance information from the REDOR experiment23 was obtained. From the REDOR measurement of peptide 6, (VPGVG)2V[15N]P[2-13C]GVG(VPGVGV)3 shown in Figure 7, the internuclear atomic distance between [15N]Pro-12 and [2-13C]Gly-13 can be evaluated. This atomic distance is only dependent on the torsion angle ψ of Pro-12. If ψ is assumed to be 105°, the value determined by the method described above, then the internuclear distance between [15N]Pro-12 and [2-13C]Gly-13 nuclei corresponds to 4.7 Å. Figure 7 shows the theoretical REDOR curve, which fits well with the observed data. Thus, it might be possible to adapt region I for the torsion angles of Pro12. However, there is no overlap between the low RMSD region and φ, ψ distribution of the Pro residues in ValPro sequences obtained from PDB as shown in Figure 5. In addition, 2D spin-diffusion NMR spectrum calculated by assuming (φ, ψ) ) (-60°, 105°), shown in Figure 6B differs from the observed spectrum (Figure 6A). To address this issue, a model with a conformational distribution for the Pro-12 residue is used. Averaging of the calculated spectra was carried out. For the averaging, a conformational distribution consisting of two Gaussian functions on the φ, ψ plane was assumed. In each Gaussian, the ψ angle of its center was varied


Figure 7. Experimental REDOR plots ∆S/S0 ()1 - S/S0) for peptide 6, (VPGVG)2V[15N]P[2-13C]GVG(VPGVGV)3. ∆S and S0 are the REDOR amplitudes and full echo amplitudes, respectively. The solid line is the REDOR curve calculated by taking into account the conformational distribution of (φ, ψ) ) (-60°, 120°) with fraction 0.8 and (φ, ψ) ) (-60°, -30°) with fraction 0.2. The dotted line indicates the calculated line by assuming ψ ) 105° (the atomic distance between [15N]Pro-12 and [2-13C]Gly-13 nuclei is 4.7 Å).

by 15° increments, while the φ angle was held at -60°. The height of Gaussian was varied in 10% increments. Then, the RMSD values between the averaged and the observed 2D spin-diffusion NMR spectra and between the theoretical and the observed REDOR data were calculated. The result of this calculation is shown in Figure 8 by changing the ratio of ψ1 and ψ2 from 0.5: 0.5 (A) to 0:1.0 (F). On the basis of the φ, ψ distribution of the Pro residues in Val-Pro sequences obtained from PDB mentioned above, the ratio of region I ((φ, ψ) ) (-60°, 135°)):0.8 and region II ((φ, ψ) ) (-60°, -30°)): 0.2 was used. This ratio satisfies both the 2D spindiffusion NMR and the REDOR data (Figure 8D). The 2D spin-diffusion NMR spectrum calculated with (φ, ψ)


) (-60°, 135°) (0.8) and (φ, ψ) ) (-60°, -30°) (0.2) was also shown in Figure 6C and the calculated REDOR curve corresponding to the set of (ψ1,ψ2) ) (120°, -30°) with ratio 0.8:0.2 was shown as a solid line in Figures 7. Both the averaged spectrum and the calculated REDOR curve have better agreement with the observed data, in comparison to the ones calculated assuming only a single conformation of (φ, ψ) ) (-60°, 105°). Thus, (φ, ψ) ) (-60 ( 15°, 120 ( 15°), 80%, and (φ, ψ) ) (-60 ( 15°, -30 ( 15°), 20%, for the Pro-12 residue were obtained. 3. Determination of the Torsion Angle of Gly-13 Residue. To begin the determination of the torsion angles of Gly-13 residue, the observed Gly CR chemical shift was first considered. The correlation of structure to the Gly CR chemical shift is weak, but it is still utilized as an initial conformational constraint for this residue, as shown in Figure 9 as the gray diagonal lines. In the previous section, the torsion angles of the Pro residue were determined to be (φ, ψ) ) (-60 ( 15°, 120 ( 15°), 80%, and (φ, ψ) ) (-60 ( 15°, -30 ( 15°), 20%, similar to the Pro φ, ψ distributions obtained from the Val-Pro-Gly or Pro-Gly sequences in PDB recorded protein structures. The distribution of the Pro-Gly sequences obtained from PDB data of native proteins is shown in Figure 9 by contour plot. There are numerous regions that satisfy constraints imposed by both the chemical shift and PDB data. The 2D spin-diffusion NMR spectrum of (VPGVG)2V[1-13C]P[1-13C]GVG(VPGVG)3 was observed and is shown in Figure 10 A. The regions with small RMSD values are also shown in Figure 9 as the area enclosed by the black solid line. Because it is not possible to distinguish two calculated spectra corresponding to (φ, ψ) and (-φ, -ψ), the RMSD map of Gly is centrosymmetric with respect to the origin. Although there are regions with small RMSD values around φ ) 0°, these regions can be deleted because they are outside the energetically

Figure 8. RMSD plots between the calculated and the observed 2D spin-diffusion NMR spectra (areas colored by gray) and between the calculated and the observed REDOR data (vertical lines). For the calculation, a conformational distribution on the φ, ψ plane was used: the ψ angle of its center in Figure 5 was varied by 15° increments under the constant angle φ () -60°).

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Figure 9. CR chemical shift error map for Gly-13, shown by gray diagonal lines. The small RMSD regions corresponding to the 2D spin-diffusion NMR spectrum of peptide 3, (VPGVG)2VP[1-13C]G[1-13C]VG(VPGVG)3, are superimposed as the area enclosed by black solid lines. The contour plot of the φ, ψ distribution of the Gly residues in the Pro-Gly sequences was obtained from the PDB data. The torsion angles of Gly-13 summarized in Table 2 are confined to an area enclosed by the white square.

allowed regions of the Ramachandran map of Gly.30 Only the region around (φ, ψ) ) (-60°, 150°) seems to satisfy the requirements imposed by the constraints dictated by the Ramachandran plots, chemical shift, conformational distribution from PDB, and 2D spindiffusion NMR. We observed REDOR spectrum for obtaining further constraints for the determination of torsion angles of Gly-13 residue. The result of the REDOR measurement of peptide 7: (VPGVG)2VP[15N]G[2-13C]VG(VPGVGV)3 is shown in Figure 11. The dotted line indicates the calculated line by assuming ψ ) 150° (the atomic distance between [15N]Gly-13 and [2-13C]Val-14 nuclei is 4.8 Å). The agreement between the observed and theoretical REDOR curves is poor. Thus, although the region around (φ, ψ) ) (-60°, 150°) is removed from consideration. As a result of these experiments, it appears that a conformational distribution around the Gly-13 residue is likely, just as in the case of Pro-12 residue. The REDOR curve was calculated by taking into account the conformational distribution from PDB data.


There are roughly four regions on ψ values, around -15°, 0° and ( 150° in Figure 9, corresponding to the distances between [15N]Gly-13 and [2-13C]Val-14 nuclei of 4.13, 4.11, and 4.83 Å, respectively. The distances 4.13 and 4.11 Å were averaged to be 4.12 Å. By changing the ratio of the distances, 4.12 and 4.83 Å, the theoretical REDOR curves were calculated as shown in Figure 11. The fitted lines were obtained in the range from 0.7 to 0.4 for the distance 4.12 Å, although there are relatively large experimental errors. As an average we selected the case where the ratio of the distances, 4.12 and 4.83 Å, is approximately 0.6 and 0.4. Thus, a sum of the regions, (φ, ψ) ) (90 ( 30°, -15 ( 30°) and (φ, ψ) ) (-90 ( 15°, 0 ( 15°), is assumed to be 60%, and the sum of β-sheet region, (φ, ψ) ) (-105 ( 75°, 150 ( 30°), (φ, ψ) ) (120 ( 60°, 150 ( 30°), (φ, ψ) ) (-120 ( 60°, -150 ( 30°), and (φ, ψ) ) (120 ( 60°, -150 ( 30°) is assumed to be 40%. The distribution of the Pro-Gly sequences obtained from PDB data of native proteins in Figure 9 is about 70% for a sum of the regions, (φ, ψ) ) (90 ( 30°, -15 ( 30°) and (φ, ψ) ) (-90 ( 15°, 0 ( 15°), and about 30% for sum of β-sheet region; (φ, ψ) ) (-105 ( 75°, 150 ( 30°), (φ, ψ) ) (120 ( 60°, 150 ( 30°),(φ, ψ) ) (-120 ( 60°, -150 ( 30°) and (φ, ψ) ) (120 ( 60°, -150 ( 30°). Thus, the relative populations, whether obtained through this analysis of the REDOR data or via analysis of the statistical distribution data from the PDB, are reasonably consistent. To provide further support for this scenario, the calculated 2D spindiffusion NMR spectrum corresponding to the φ, ψ distribution is shown in Figure 10B. There is a good agreement between the calculated and the observed spectra. Hence, it appears that there is an even greater distribution about the central glycine residue in the VPGVG subunit, than observed for the Pro. 4. Determination of the Torsion Angle of Val-14 Residue. For a determination of the torsion angles of Val-14 residue, the conformation-dependent chemical shift was used. Figure 12A shows 13C CP/MAS NMR spectrum of (VPGVG)2VP[15N]G[2-13C]VG(VPGVG)3. The most downfield CR peak at 55-65 ppm is a superposition of the resonances of Val-14CR and Pro/ Val CR in natural-abundance. To obtain only the peak of Val-14 CR, a difference spectrum was obtained by subtracting the natural-abundance spectrum from the enriched spectrum. As a result, the Val-14 CR signal shows an asymmetric line shape (Figure 12B). Decon-

Figure 10. Observed 2D spin-diffusion NMR spectrum of peptide 3, (VPGVG)2VP[1-13C]G[1-13C]VG(VPGVG)3, (A) and the 2D spin-diffusion NMR spectrum calculated by taking into account the conformational distribution.



Figure 11. Experimental REDOR plots ∆S/S0 for peptide 7, (VPGVG)2VP[15N]G[2-13C]VG(VPGVGV)3. The solid lines are the REDOR curves calculated by assuming the bimodal distance distribution of 4.12 and 4.83 Å. The dotted line indicates the calculated line by assuming ψ ) 150° (the atomic distance between [15N]Gly-13 and [2-13C]Val-14 nuclei is 4.8 Å).

Figure 13. Combined CR and Cβ chemical shifts error map for Val-14, shown by the gray diagonal lines. The small RMSD regions corresponding to the 2D spin-diffusion NMR spectrum of peptide 4, (VPGVG)2VP[1-13C]G[1-13C]VG(VPGVG)3, are superimposed as the area enclosed by black solid lines. The contour plot of the φ, ψ distribution of the Val residues in the Gly-Val-Gly sequences obtained from the PDB data is also shown. The torsion angles of Val-14 summarized in Table 2 are confined to an area enclosed by the white square.

Figure 12. 13C CP/MAS NMR spectra of peptide 7, (VPGVG)2VP[15N]G[2-13C]VG(VPGVG)3 (continuous line), and (VPGVG)6 (dashed line) (A). The difference spectrum (dotted line) is prepared by subtracting the natural abundance spectrum from the spectrum of a 13C, 15N double labeled peptide (B). From the peak deconvolution of the difference spectrum, two peaks were found with isotropic chemical shifts of 62.2 ppm with fraction 0.3 and 59.0 ppm with fraction 0.7.

volution of this line shape indicates that there are contributions from two Gaussian peaks with isotropic chemical shifts of 62.2 ppm (with fraction 0.3) and 59.0 ppm (with fraction 0.7). It is well-known that the 14N quadrupolar interaction causes an asymmetric resonance of adjacent 13C nuclei.31 However, the splitting of this effect is only

Val-Pro-Gly-Val-Gly - American Chemical Society

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