Obtaining spatial structure of cyclosporine (CsA) in chloroform using 2D NMR Текст научной статьи по специальности «Физика»

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Volume 13, No. 2 pages 21-26

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Obtaining spatial structure of cyclosporine (CsA) in chloroform using 2D NMR

A.Y. Manin*, S.V. Efimov, V.V. Klochkov

Kazan Federal University, Kremlevskaya, 18, Kazan 420008, Russia

*E-mail: [email protected]

(Received August 15, 2011; revised November 12, 2011; accepted November 25, 2011)

In this work the cyclic peptide cyclosporine was investigated. Signal assignment was made according to 2D NMR spectra. Using NOESY spectra and computer simulation the spatial structure was also obtained.

PACS: 82.56.Dj, 82.56.Ub, 87.64.-t, 87.14.ef.

Keywords: high-resolution NMR, structure determination by NMR, spectroscopic techniques in biophysics and medical physics.

1. Introduction

We have chosen cyclosporine (CsA) as an object for our research (CsA is one of the possible modifications of cyclosporine, for simplicity we will name it further simply as cyclosporine). Cyclosporine is a cyclic peptide which is used as immunosuppressive medication in transplantation. In cells, cyclosporine binds with the protein cyclophilin and, as a result, stops the reaction chain that is responsible for the immune response [1-3]. This peptide is poorly dissolved in water (23 ^M at 25°C), but in organic solvents its solubility is high [4]. Chloroform was used as a starting point in our studies, which we suppose to develop further by employing residual dipolar couplings measured in chloroform-based media. On the other hand, it is an example of an apolar solvent. Comparison of the cyclosporine’s structure and behavior in such a medium with the situation in different polar media (organic solvents mixed with water, micellar solutions) is of interest. The concentration of our sample was 17 mM.

The aim of our work was to analyze 1D and 2D NMR spectra, and to obtain spatial structure of cyclosporine using the structure calculation. We intend to obtain spatial parameters with high precision.

2. Experimental results and discussion

Schematic structure of cyclosporine molecule is shown in Fig. 1.

Figure 1. Schematic structure of cyclosporine (Bmt-Aba-Sar-Mle-Val-Mle-Ala-Dal-Mle-Mle-Val).

Let’s consider main spectra that were used for the assignment of NMR signals.

At first, we used selective TOCSY spectra. Amide protons were excited, and as a result, we observed individual subspectra of the corresponding amino acids. Besides, we recorded 2D TOCSY which yielded information about all signals of a certain amino acid. Due to this fact, it was used as the main method for assignment.

We obtained signals of carbons with the help of HSQC spectra and measured chemical shifts of attached protons. That method helped us in making assignment.

Also we employed an important experiment called HMBC. A peak of an N-methyl group within the 7-th residue, lying in the region of carbonyl resonances (chemical shift range S(13C) = 170...174 ppm), points to the CO group of the residue in position (7 - 1). Each cross-section at the chemical shift of an Ha atom contains two signals of carbonyl groups: that of the residue with concerned Ha atom and of the preceding one. Moreover, if we connect (Ha,CO)-signals with each other and obtain the cycle, we can prove that the assignment is correct. This is illustrated in Fig. 2.

Figure 2. HMBC (1H 500.13 MHz, 13C 125.76 MHz) spectrum of cyclosporine in chloroform at T = 293 K. Region of correlations between C = O and NCH3 groups is shown.

Having made the analysis of 1D and 2D NMR spectra, we obtained the chemical shifts of all protons, which you can see in Tab. 1.

At the next step we had to obtain the distances between protons. For this purpose, we recorded a set of NOESY spectra with different mixing times. Cross-peaks were integrated, and obtained intensities were normalized by the diagonal peaks. It was plotted as a function of the mixing time. The slopes of

obtained curves gave the cross-relaxation rates. Then we used correlation a = a\

( V6 r

pq

rj

, where rj

У j

is the reference distance between two nonequivalent a-protons of Sar assumed to be 1.75 A. After this calculation, interproton distances were obtained. They are listed in Tab. 2. below.

Table 1. Proton chemical shifts.

Amino acid residue Chemical shifts, ppm

MeBmt NCH3 а ß Y s є Z

3.52 CH CH CH 3 H ,C ,2 H C CH CH, CH3

5.5 3.S1 1.б2 0.71, 2.02 5.34 4.02, 1.03

Aba NH а ß Y

S.0 CH CH2 H3 C

5.03 1.б1, 1.б4 0.S7

Sar NCH3 а

3.41 CH

4.73, 3.2

Mle4 NCH3 а ß Y s

3.12 CH CH2 CH 3 H C

5.35 1.97 1.б4 0.S7, 0.S5

Val NH а ß Y

7.49 CH CH H3 C

4.бб 2.43 1.0S, 0.S9

M^ NCH3 а ß Y s

3.2б CH CH2 CH 3 H C

4.97 2.07 1.4 1.04, 1.01

Ala NH а ß

7.б7 CH 3 H C

4.54 1.3б

Dal NH а ß

7.1S CH 3 H C

4.S4 1.2б

Mle9 NCH3 а ß Y s

3.12 CH CH2 CH 3 H C

5.71 2.14 1.24 0.9б, 0.SS

Mle10 NCH3 а ß Y s

2.71 CH CH2 CH 3 H C

5.0S 2.07, 1.2S 1.2S 0.95, 0.SS

Mva NCH3 а ß Y

2.7 CH CH H3 C

5.13 2.15 0.S1, 0.9S

Table 2. Distance restraints, derived from the NOESY spectra.

Res name Atom name Res name Atom name Dist, A

Aba Ha Aba NH 3.2 ± 0.2

Aba Hb Aba Ha 2.53 ± 0.15

Ala Ha Ala NH 2.52 ± 0.14

Bmt Ha Aba NH 2.1 ± 0.1

Bmt Hb Bmt Ha 2.1 ± 0.1

Bmt Hd Aba Hb2 2.31 ± 0.12

Dal Ha Dal NH 2.99 ± 0.24

Dal Hb Dal Ha 1.92 ± 0.09

Mle10 Ha Mle9 Ha 1.9 ± 0.3

Mle10 Hb Mle10 Ha 2.43 ± 0.16

Mle10 Hg Mle10 Ha 2.6 ± 0.2

Mle10 Hg Mle10 Hb 1.54 ± 0.07

Mle4 Ha Val Hb 2.47 ± 0.13

Mle4 Hb Mle4 Ha 2.36 ± 0.17

Mle4 Hg Mle4 Ha 1.9 ± 0.2

Mle4 Hg Val Hb 1.63 ± 0.07

Mle6 Ha Ala NH 1.8 ± 0.4

Mle6 Hb Mle6 Ha 2.47 ± 0.12

Mle6 Hg Mle6 Ha 2.38 ± 0.08

Mle6 Hg Ala Ha 1.97 ± 0.09

Mle6 Hg Mle6 Hb 1.87 ± 0.08

Mle9 Hb Mle9 Ha 2.85 ± 0.39

Mle9 Hg Mle9 Ha 2.0 ± 0.1

Mva Hb Mva Ha 2.49 ± 0.13

Mva Hg1 Mva Hb 2.0 ± 0.1

Mva Hg2 Mva Hb 2.33 ± 0.15

Sar Ha2 Sar Ha1 1.8 ± 0.1

Val Ha Val NH 2.08 ± 0.11

Val Hb Val Ha 2.59 ± 0.21

Val Hg1 Val Hb 2.21 ± 0.11

Val Hg2 Val Hb 2.5 ± 0.2

Knowledge of chemical shifts and distances between protons allowed us to use structure calculation and to get the structure of the cyclosporine molecule, which we are interested in. For this purpose, we used DYNAMO package [5], which is widely used for structural researches.

Obtained spatial structure of cyclosporine with RMSD = 1.4 ± 0.4 A for backbone can be seen in Fig. 3.

V

X

i

'^r

.v*

VH Y

» •- * •>

iA, ,•

* I •

- X»

5v

Figure 3. Averaged spatial structure of cyclosporine in chloroform after final energy minimization.

As can be seen, there are only few interresidual NOESY cross-peaks. The reason for this is that the cyclosporine molecule adopts an elongated form, where residues are situated like in a beta-strand or a random coil. These structures are known to produce little or no interresidual NOEs. Only in the loop regions we can seen close contacts between nonadjacent amino acids. We can thus find out the loops, but cannot obtain a single best structure by means of molecular dynamics. That's the explanation for relatively high RMSD of the ten selected backbones (Fig. 4).

Figure 4. Backbones of 10 lowest energy structures.

Э. Summary

According to obtained spectra and signal assignment we got the spatial structure of cyclosporine in chloroform and will use this information in our further research.

References

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