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Volume 15, 2013 No. 2, 13202 - 7 pages
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Spatial structure of tetrapeptide N-AC-Ser-Phe-Val-Gly-OMe in "protein-micelle of sodium dodecyl sulfate" complex and in solid state by NMR spectroscopy
D.S. Blokhin \ S. Berger 2, V.V. Klochkov 1 *
1 Kazan Federal University, Kremlevskaya, 18, Kazan 420008, Russia
2 University of Leipzig, Augustusplatz 10, 04109 Leipzig, Germany
*E-mail: [email protected]
(Received April 30, 2013; revised August 23, 2013; accepted August 28, 2013)
In the present paper the applicability of a structure determination for a four amino acid residues containing oligopeptide N-Ac-Ser-Phe-Val-Gly-OMe was investigated. The spatial structure of the "tetrapeptide (N-Ac-Ser-Phe-Val-Gly-OMe) - sodium dodecyl sulfate micelle" complex in aqueous solution was studied by 2D nuclear magnetic resonance (NMR) spectroscopy. The complexation was confirmed NOEs signs and values in the presence of sodium dodecyl sulfate. The spatial structure of the tetrapeptide in the complex was determined by 2D NOESY NMR spectroscopy. In present paper by the comparison 13C NMR chemical shifts was shown that tetra peptide's spatial structure in solid state and tetrapeptide structure in "peptide-micelle" complex are identical.
PACS: 82.56.Dj, 82.56.Ub, 87.64-t, 87.15.-v.
Keywords: oligopeptides, micelles, 2D NMR spectroscopy (TOCSY, NOESY), sodium dodecyl sulfate 1. Introduction
Proteins are extremely complex organic molecules - biopolymers (polypeptides) with amino acids as the structural units. It is well known that the biological activity of proteins is related to their spatial structure [1-4]. The study of oligopeptides conformations containing of two or more amino acid residues is also important because they can be considered as building blocks of proteins, and knowledge of their structure can be used to predict the polypeptide chain configuration. We also know that some of the short peptide sequences synthesized by the cell are parts of the immune system of a living organism [1-4].
Traditionally, the structural study of the relatively small organic compounds in solutions is based on 1D NMR (:H, 13C) spectroscopy, and some modern NMR spectroscopy approaches such as 2D COSY, TOCSY, HSQC, HMBC and NOESY NMR experiments [5, 6]. Note that ('H - !H) NOESY NMR spectroscopy allows to determine the interatomic distances up to 5 A, and thus, to establish the spatial structure of organic compounds in the solution [5]. However, a method based on 2D NOESY NMR spectroscopy is not always effective for relative small molecules [5, 6]. This is due to the rather small correlation time tc of these molecules in solution, which leads to a weak cross-peaks intensity in the NOESY NMR spectra and to some difficulty in obtaining quantitative information about interproton distances in the molecular systems.
In order to use the 2D NOESY NMR spectroscopy for the spatial structure determination of small oligopeptides, a method was introduced by authors [7]. This method is based on the mechanism of "oligopeptide - micelle" complex formation. Under this process the protein is transferred from the domain of small molecules (for which the condition of rapid tumbling is valid) to the category of large molecules (for which the condition of slow tumbling is effective) [5, 6]. The "efficiency" of the proposed method was confirmed by a number of structural studies for peptides with 7, 10 and 41 amino acid residues in length [7-10].
The aim of this study was to determine the applicability of 2D NOESY NMR spectroscopy to the spatial structure determination of oligopeptides in a complex with sodium dodecyl sulfate (SDS)
micelles and to establish the spatial structure of the tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe in a complex with SDS micelles in aqueous solution. The tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe was taken as a model. This oligopeptide was previously studied [11] and the inefficiencies of the NOESY spectroscopy to study the conformation of the tetrapeptide in aqueous solution has been shown.
2. Experimental
1H NMR (500 MHz) spectra of the tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe in complex with SDS in water solution were recorded on an ''AVANCE II-500'' spectrometer (Bruker). 1H NMR spectra were recorded using 90° pulses, with a spectral width of SW = 9.40 ppm, using 10 scans. 2D TOCSY [12] was used to assign signals in the 1H NMR spectra of the tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe.
The relaxation delay between subsequent runs of the NOESY sequence was 1.5 s. The spectra were recorded in a phase-sensitive mode with 1,024 points in the F2-direction and 256 points in the F1-direction. Exponential filtration was applied in both directions. Mixing time values, Tm, were 0.075, 0.10, 0.15, 0.20 and 0.25 s.
3. Results and discussion.
In this paper interproton distances which directly characterize the spatial geometry of the tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe (fig. 1) were defined by 2D NOESY NMR spectroscopy. On the basis of these experimental data the structure of a "protein - surface model of the cell membrane" (micellar systems of sodium dodecyl sulfate) complex was obtained. As the tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe is a short peptide with several aromatic and aliphatic components and polar groups, it may be of interest as a model system for peptide-solvent molecular interactions studies (for example water and trifluoroethanol).
The 1H NMR spectrum assignment of the tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe in solution H2O + D2O with SDS was done on the basis of previous data [13] and 2D TOCSY NMR experiments [12], and on the basis of the :H NMR chemical shifts of the tetrapeptide, dissolved in water [11, 14] (tab. 1).
Previously [11] the spatial structure of the tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe was determined by an approach based on the value of residual dipolar coupling constants [15] between the magnetic nuclei 13C and :H, separated by one chemical bond (:D). In this article we tried to use 2D NOESY NMR for the determination of the interproton distances that directly characterize the spatial geometry of the tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe in D2O solution, but there were not any intraresidue NOE's in the 2D NOESY NMR spectra (fig. 2a). So in this case the 2D NOESY NMR spectroscopy method is ineffective for the structural studies of relatively small molecules.
In 2D :H-:H NOESY NMR
spectrum of the tetrapeptide in
a mixture of H2O + D2O with
SDS in micellar state cross-
peaks of the same sign as the
diagonal signals were observed
(fig. 2b). This is typical of the
large molecules. This fact
confirm the tetrapeptide -
micelles of sodium dodecyl
sulfate complex formation. Figure 1 The structural formula of the tetraPePtide A-Ac-Ser-Phe-Val-
F Gly-OMe.
Figure 2. 2D 1H-1H NOESY NMR (500 MHz) spectra of the tetrapeptide N-Ac-Ser-Phe-Val-Gly-OMe dissolved: a) in a water, and b) in a mixture of H2O + D2O with sodium dodecyl sulfate in micellar state. Mixing time Tm = 0.250 s. T = 298 K.
Table 1. 1H NMR chemical shifts (S№ ppm relative DSS) for the tetrapeptide N-Ac-Ser-Phe-Val-Gly-OMe dissolved in H2O + D2O (90% + 10%) with sodium dodecyl sulfate (5.71 g/l) in the micellar state. T = 298 K.
« -, Chemical shifts (ppm)
Amino acid _11 '_
residue HN Ha Hb Others
Ser Phe Val Gly
7.45
7.63 7.73
4.30 4.60 4.05 4.43
3.70 3.05 1.98
2-6H 7.24 Hg 0.79
Processing and analyzing the intensity of cross-peaks in the NOESY NMR spectra were obtained as described elsewhere [16]. The dependence of the relative average integral intensities of cross-peaks for the Phe Ha - Val NH proton pairs on the mixing time are shown in figure 3. The slope value corresponds to the cross-relaxation rate constants (oiJ) in the proton pair. The latter one is directly
related to the interproton distance (riJ) [12, 16]. Interproton distances of the tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe in solution H2O + D2O with sodium dodecyl sulfate at a micellar state are presented in table 2.
Experimental values of the interproton distances were used as input data for the molecular dynamics method calculations by the XPLOR-NIH program [17]. Following structural calculations, the ensemble was subjected to restrained molecular dynamics using the XPLOR-NIH. Individual structures were minimized, heated to 1000°K for 6000 steps, cooled in 100°K increments to 50°K, each with 3000 steps, and finally minimized with 1000 steps of steepest descent followed by 1000 steps of conjugate gradient minimization. For a starting family of 200 structures, approximately 21-25 ones were kept for following molecular dynamics calculation and finally 8 lowest energy structures (fig. 4) were retained. The backbone RMSD for the extended core (Ser 2-Val 4) is 0.78 ± 0.25 A. The large value of RMSD is caused by the small size of the oligopeptide and its high mobility. The stereochemical validation of model was carried out with Ramachandran's plot (fig. 6). Psi and Phi dihedral angles is used for the Stereochemical evaluation of backbone of the protein revealing that 60.0, 30.0, 10.0 and 0.0% of residues were falling within the most favoured regions, additionally allowed regions, generously allowed regions and none in disallowed regions respectively of Ramachandran's plot. Coordinates of the atoms for the minimal energy conformation of tetrapeptide are presented in table 4. A model of the "tetrapeptide nAc-Ser-Phe-Val-Gly-OMe - SDS micelle" complex is presented in figure 5. This model was constructed on the basis of tetrapeptide chemical shifts differences in SDS and aqueous solution.
The 13C chemical shifts of the tetrapeptide in solid state [11] and 13C chemical shifts of tetrapeptide in complex peptide-micelle of SDS in water solution are shown in the table 3. Closeness of the 13C chemical shifts of tetrapeptide in isotropic solutions and solid state (AS < 1.5 - 2.5 ppm, with the exception of the 13C chemical shifts of 2,6 Phe, 3,5 Phe, 4 Phe) allows to conclude about the similarity of the conformations of the chain of the tetrapeptide in water solution with SDS and in the solid state.
Therefore the determined spatial structure of tetrapeptide in "peptide - micelle" complex (fig. 4) is corresponds with ones in solid state.
Table 2. Experimental interproton distances for the tetrapeptide N-Ac-Ser-Phe-Val-Gly-OMe in the (H2O + D2O) solution with SDS micelles. ("*" is the calibration distance).
Pair of protons Interproton
distances r, Â
Gly 5 Ha/Gly 5 HN 2.93 ± 0.5
Val 4 Hg1/Gly 5 HN 3.24 ± 0.5
Val 4 Hg2/Val 4 HN 3.14 ± 0.5
Val 4 Hb/Gly 5 HN 3.01 ± 0.5
Val 4 Hb/Val 4 HN 2.99 ± 0.5
Phe 3 Hb/Val 4 HN 3.30 ± 0.5
Phe 3 Ha/Val 4 HN 2.26 ± 0.5
Phe 3 Ha/Phe3 Hb 2.65*
Table 3. 13C NMR chemical shifts (SH, ppm relative DSS) for the tetrapeptide A-Ac-Ser-Phe-Val-Gly-OMe dissolved in H2O + D2O (90% + 10%) with sodium dodecyl sulfate (5.71 g/l) in the micellar state and tetrapeptide in solid state from Ref. [11]. T = 298 K.
Atom S Solid state, ppm S In water solution with SDS, ppm
Ser Ca 55.0 55.6
Ser Cb 60.0 60.8
Phe Ca 55.0 55.6
Phe Cb 36.9 37.0
Phe C 2,6 126.9; 126.3 128.6
Phe C 3,5 131.0 129.3
Phe C 4 124.4 126.9
Val Ca 59.4 59.2
Val Cb 29.8 30.2
Gly Ca 40.8 41.0
0.01
0 -1-1-1-1-1-1
0 50 100 150 200 250 300
tm, ms
Figure 3. The dependence of cross - peak average relative integrated intensity for pairs of Phe Ha - Val NH protons on the mixing time.
Figure 4. The full ensemble of 8 final structure of the tetrapeptide N-Ac-Ser-Phe-Val-Gly-OMe in SDS micelles calculated by the XPLOR-NIH program.
Ramachandran Plot
Figure 5. Final structure of tetrapeptide N-Ac-Ser- Figure 6. The full ensemble of 8 final structure of Phe-Val-Gly-OMe binding with an SDS the tetrapeptide N-Ac-Ser-Phe-Val-Gly-
micelle. OMe in SDS micelles calculated by the
XPLOR-NIH program.
Table 4. Coordinates of the atoms for the minimal energy conformation of tetrapeptide in complex "peptide - micelle of SDS" in pdb-format.
Atom Residue x y z Atom Residue x y z
1 CA 1ACE 6.348 4.166 4.611 34 CZ 3PHE 7.453 -0.672 4.909
2 HA1 1ACE 6.067 4.801 5.438 35 HZ 3PHE 8.317 -0.423 5.508
3 HA2 1ACE 7.263 4.532 4.170 36 C 3PHE 2.618 -0.964 0.570
4 HA3 1ACE 6.498 3.157 4.967 37 O 3PHE 2.223 -2.115 0.384
5 C 1ACE 5.239 4.175 3.564 38 N 4VAL 1.971 0.106 0.121
6 O 1ACE 4.392 5.069 3.550 39 HN 4VAL 2.335 0.998 0.300
7 N 2SER 5.251 3.176 2.688 40 CA 4VAL 0.729 -0.024 -0.633
8 HN 2SER 5.950 2.492 2.747 41 HA 4VAL -0.068 -0.285 0.047
9 CA 2SER 4.241 3.079 1.640 42 CB 4V AL 0.387 1.305 -1.308
10 HA 2SER 3.269 3.267 2.072 43 HB 4VAL -0.410 1.152 -2.021
11 CB 2SER 4.510 4.125 0.558 44 CG1 4VAL -0.064 2.314 -0.250
12 HB1 2SER 5.381 3.834 -0.014 45 HG11 4VAL -1.022 2.015 0.148
13 HB2 2SER 4.688 5.083 1.017 46 HG12 4VAL -0.150 3.293 -0.699
14 OG 2SER 3.377 4.219 -0.296 47 HG13 4VAL 0.662 2.347 0.549
15 HG 2SER 3.413 5.066 -0.745 48 CG2 4VAL 1.625 1.843 -2.029
16 C 2SER 4.246 1.687 1.017 49 HG21 4VAL 2.358 2.157 -1.301
17 O 2SER 4.527 1.529 -0.171 50 HG22 4VAL 1.345 2.686 -2.644
18 N 3PHE 3.933 0.681 1.827 51 HG23 4VAL 2.044 1.067 -2.651
19 HN 3PHE 3.718 0.866 2.765 52 C 4VAL 0.855 -1.115 -1.691
20 CA 3PHE 3.904 -0.695 1.343 53 O 4VAL -0.130 -1.757 -2.056
21 HA 3PHE 4.747 -0.854 0.688 54 N 5GLY 2.074 -1.320 -2.181
22 CB 3PHE 4.005 -1.665 2.522 55 HN 5GLY 2.822 -0.778 -1.853
23 HB1 3PHE 4.104 -2.674 2.150 56 CA 5GLY 2.316 -2.337 -3.197
24 HB2 3PHE 3.113 -1.590 3.126 57 HA1 5GLY 2.080 -3.308 -2.792
25 CG 3PHE 5.213 -1.317 3.358 58 HA2 5GLY 3.359 -2.313 -3.480
26 CD1 3PHE 5.060 -0.569 4.532 59 C 5GLY 1.455 -2.089 -4.431
27 HD1 3PHE 4.078 -0.240 4.838 60 O 5GLY 1.873 -1.363 -5.333
28 CD2 3PHE 6.486 -1.743 2.961 61 CA 6MEO -0.444 -2.378 -5.671
29 HD2 3PHE 6.604 -2.321 2.056 62 HA1 6MEO 0.149 -2.705 -6.514
30 CE1 3PHE 6.180 -0.246 5.307 63 HA2 6MEO -0.583 -1.307 -5.722
31 HE1 3PHE 6.062 0.331 6.212 64 HA3 6MEO -1.407 -2.866 -5.700
32 CE2 3PHE 7.606 -1.421 3.736 65 O 6MEO 0.221 -2.716 -4.458
33 HE2 3PHE 8.588 -1.749 3.430
4. Summary
In this paper the spatial structure of the tetrapeptide N-Ac-Ser-Phe-Val-Gly-OMe in a complex with SDS micelle in aqueous solution was determined by 2D NOESY NMR spectroscopy. We have shown that the 2D NOESY NMR spectrum of the tetrapeptide in complex with SDS micelles was informative and suitable for the oligopeptide molecular structure determination. The obtained results show that the proposed approach in [7] can be used for small oligopeptides with a few amino acid residues in length.
The identity of the tetrapeptide in solid state and in "peptide-micelle" complex structures was prooved by the invariability of 13C chemical shifts values.
Acknowledgments
This work was supported by the Ministry of Education and Science of the Russian Federation (state target of KFU, part 2, code 2.2792.2011) and by RFBRand the Tatarstan Academy of Sciences (project No 13-03-97041).
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