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Comparison of rapid protein structure determination approaches driven by experimental NMR data

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Protein expression and isotopic labeling

Assessment of isotopic labeling

Conclusions

Structure calculation driven by NMR backbone chemical is an efficient alternative for 3D

structure determination while complete set of expérimental data cannot be obtain for

experimental approaches

These approaches are rapidly, provided good results in term of fitness to experimental structure

Comparison of rapid protein structure determination approaches driven by

experimental NMR data

M. Wanko

1

, C. Damblon

1,

, G. Feller

2

, A. Tchernychev, V. Volkov

1

Structural Biological Chemistry Unit, Department of chemistry, University of Liege, Allée du 6 Août 13, 4000 Liège, Belgium

2

Laboratory of Biochemistry, Centre for Protein Engineering (CIP), University of Liège, Allée du 6 Août 13, 4000 Liège, Belgium

e-mail:

amwanko@doct.ulg.ac.be

Introduction

Tridimensional structures of proteins are precious sources of information. They allow to

understand fundamental biological mechanisms, protein Interaction with other macromolecules.

3D structures are currently determines using NMR and

X-Ray Crystallography that faced to some limitations. In order to overcome time consuming

limitation of both NMR and Crystallography, modeling approaches driven by minimalist NMR

data have been developed. Indeed, it have been shown that NMR backbone chemical shifts are

secondary structure dependent.

Therefore, different modeling

approches driven by only NMR

backbone chemical shifts such

as Rosetta, RASREC

CS-Rosetta, CS-HM-Rosetta CS23D

a n d C h e s h i r e h a v e b e e n

developed. To assess whether if

these automated methods can

indeed produce structures that

closely match those manually

refined by experts using the

same experimental data, these

approaches were used to

determine 3D structure of a

benchmark of proteins.

Application to Cold Shock Protein

Comparison based on C

-RMSD

Well-defined regions ranges RMSD(Å) AA/AAt % of proteins coverage Gap CSPM from PDB 4..55, 63..70 0.55 60/70 85 % 1 Rasrec-CS-Rosetta 5..70 1.58 66/70 94 % 0 CS-Rosetta 1..32, 41..66 1.21 58/70 83 % 1 Rasrec-CS-Rosetta sans HA 4..38 1.27 35/70 35 % 0

• AA: Number of amino acids of

Well-defined regions

• AA

t

: Protein’s number of amino acids

• % of proteins coverage = (AA÷AA

t

) * 100

Backbone chemical shifts is structure dependent

+ IPTG

Inoculation in

unlabeled media

Massive culture in

unlabeled medium

Culture in

15

N and

13

C labeled medium

Harvesting

Purification

Protein

MW expected for

12

C

14

N: 7403 uma

MW expected for

13

C

15

N: 7816 uma

MW measured for

13

C

15

N: 7646 uma

13

C

–H coupling

H-

13

C

H-

13

C

H-

12

C

NMR Backbone Chemical shifts assignment

N (ppm)

H (ppm)

117.18 ppm

121.17 ppm

122.31 ppm

Gln 38

Ile 37

Ala 36

117.83

40.93

47.54

35.06

49.31

51.73

1

H=8.023

1

H=9.120

1

H=9.035

13

C

(p

p

m

)

15

N (ppm)

Gln 38

Ile 37

Ala 36

C

β

C

HNCACB

15

N-HSQC

HN(CO)CACB

Perspectives

Some regions are ill-defined

Is it due to modeling programs?

Is it because these regions are

dynamic?

Dynamic study using NMR will

be used to answer these

questions

To improve labeling, starvation

time should be increase

20

experimental NMR structur

es

Blue:

Others: Calculated structures

experimental NMR structur

e

References

Acknowledgements

A. Rosato, A. Bagaria, D. Baker, B. Bardiaux, A. Cavalli, J. Doreleijers, A. Giachetti, P. Guerry, P. Güntert, T. Herrmann, Y. Huang, H. Jonker, B. Mao, T. Malliavin, G. Montelione, M. Nilges, S. Raman, G. van der Schot, W. Vranken, G. Vuister and A. Bonvin, Nature Methods, 2009, 6, 625 - 626 A. Rosato, W. Vranken, H. Fogh, J. Ragan, R. Tejero, K. Pederson, H. Lee, H. Prestegard, A. Yee, B. Wu, A. Lemak, S. Houston, H. Arrowsmith, M.

Kennedy, B. Acton, R. Xiao, G. Liu, T. Montelione, W. Vuister, J Biomol NMR, 2015, 62, 413–424

Y. Shen, O. Lange, F. Delaglio, P. Rossi, M. Aramini, G. Liu, A. Eletsky, Y. Wu, K. Singarapu, A. Lemak, A. Ignatchenko, H. Arrowsmith, T. Szyperski, T. Montelione, D. Baker, and A. Bax, PNAS, 105, 2008, 4685–4690

M. Thompson, G. Sgourakisa, G. Liub, P. Rossib, Y. Tangb, L. Millsc, T. Szyperskic, T. Montelione, and D. Baker, PNAS, 2012, 109, 9875–9880

Blue:

experimental NMR structur

e

Red: RASREC CS-Rosetta structure

Green: CS-Rosetta structure

Cyan: Modeller structure

Magenta: Calculated structures

During protein expression, E. coli bacteria were transformed and grown in a unlabeled Terrific Broth media

After biomass production, cells were starved in M9 medium containing

15

NH

4

Cl and

13

C-6 D-Glucose as the

only source of carbon and nitrogen.

15

N and

13

C labeling are needed because

12

C which is the most

abundant source of carbon, has an even atomic number and even mass so therefore this nuclei is NMR

inactive while

13

C has an odd mass and is therefore NMR active.

14

N signals are usually significantly

broadened by quadrupolar interactions sometimes to the extent that they are unobservable on a high

resolution NMR spectrometer.

labeling % = 7646 - 7403

7816 - 7403

= 59%

S t a r t i n g f r o m

1 5

N - H S Q C ,

heteronuclear correlation

1

H-

15

N

is used to identifie C

and C

β

atoms of amino acid in position i

and C

and C

β

atoms of amino

acid in position i-1. Resulting

chemical shifts can be therefore

ordered to sequentially assigned

backbone atomes.

Calculated 3D structures driven by

NMR backbone chemical are close to

NMR experimental structure

It is also true for homology modeling

structures.

While homology modeling approaches

didn’t use experimental data, 3D

structures determined by calculation

approaches under the guidance of

NMR data can be used to validate

homology structure.

Despite the fact that both homology

and calculates approches provides

3D structures close to experimental

structure, somme regions were

ill-defined

It is because modeling approaches

aren’t powerful enough or it is due to

dynamic?

By integration,

Références

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