Cours MethodesBiolStruct RMN
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Méthodes de la Biologie structurale
Analyse de la structure des protéines(et autres biomacromolecules) par
resonance magnétique nucléaire (RMN)
Burkhard Bechinger
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Livres recommandés
La RMN des protéines
* Jeremy N. S. Evans, Biomolecular NMR spectroscopy, Oxford Univ. Press 1995
John Cavanagh, W.J. Fainbrother, A.G. Palmer III, N.J. Skelton: Protein NMR Spectroscopy, Principles and Practice, Academic Press (1996)
Kurt Wüthrich, NMR of proteins and nucleic acids, John Wiley & Son
Fondations de la RMN
recomande par un étudiant:
La RMN - Concepts, méthodes et applications (dunod) 2ème édition.
* Jeremy K.M. Sanders and Brian K. Hunter
Oxford University Press 1987 (Oxford, New York, Tokyo)
* Harald Günther: NMR-Spektroskopie, Thieme Verlag, Stuttgart, New York (traduit en Francais, English translation)
Robin K. Harris: Nuclear Magnetic Resonance Spectroscopy – A Physicochemical View, Longman Scientific & Technical, Essex
Daniel Canet : RMN - Concepts et méthodes, Inter Editions, Paris
P.J. Hore, J.A. Jones, S. Wimperis: NMR – The Toolkit, Oxford Chemistry Primers 92
Des chapitres sur RMN on retrouve aussi dans les livres chimie physique ou chimie organique comme:
www.protein-nmr.org.uk
recomande par des étudiants :http://mutuslab.cs.uwindsor.ca/schurko/ssnmr/ssnmr_schurko.pdf
http://www.rmn.uhp-nancy.fr/Mutzenhardt/CPM-701-RMN.pdf
http://www-keeler.ch.cam.ac.uk/http://www.kb.u-psud.fr/niveau2/enseignements/niveau3/etudmed/cours-irm/texte13.htm (aimantation nucléaire)
http://pharmacie.univ-lille2.fr/recherche/labos/physique/
http://www.chimie-sup.fr/rmn.html
La structure des biomolécules
livres de biochimie
voir aussi fichier avec images sur ENT
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Le déplacement chimique etl‘analyse structurale de biomolécules (protéines)
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Le déplacement chimique (protéines)
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Le déplacement chimique (protéines)
comparaison du spectre1H d‘une ,petite protéine‘
et de l‘éthanol129 aa, 14.3 kDa
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Les déplacements chimiquesdes acides aminés
Tableaux de référence:Wüthrich (livre) ou
http://www.bmrb.wisc.edu/
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!
!
L‘indexe de
déplacement chimique-> structure
secondaire
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Le champ magnétique Bo
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SpectroscopiesInteractions ondes electromagnétiques - matière
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Sanders et al., JACS 1957
Un avantage des champsmagnétiques elevés
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La fréquence v est proportionel au rapport-gyromagnetique (=isotope) et à Bo
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Transformé de Fourier et Relaxation
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Induction libre et
transformé deFourrier
•
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Superposition de plusieurs ondes
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Effet taille moléculaire du complexe
MW 25 kDa
2,5 kDa
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Residual Dipolar Coupling
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Bicelles = mixtures of long and short chainphospholipids (or detergents, petides etc.)
Δχ Bo2 > kT
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J J+RDC
106-92=14 Hz99-92= 7 Hz87-92= -5 Hz
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RMN en 2 dimensions et plus
L ( éi d 129 é id 14 kD
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Lysozyme (protéine de 129 résidues,14 kDagain en résolution
1D -----> 2D -----> 3D ---->
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RMN en 2 D:COSY, TOCSY, NOESY
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Systèmes des spinsdes acides aminés -RMN des protéines
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COSY d‘une petite protéine(BUSI - bull seminal inhibitor)
absolute value COSY DQF-COSY
incl. attribution des residues
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Total CorrelationSpectroscopy
(TOCSY)
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N a b g
COSY
g
b
a
N
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N a b g
TOCSY
g
b
a
N
N a b g
COSY
g
b
a
N
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COSY d‘une petite protéine
foculariser sur des regions
region methyl/methylenenon-résolu
frequences du système de spinpar TOCSY
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NOESYd‘un polypeptide
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NOESY d‘un polypeptide
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NOEs et structures sécondaires
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Couplage Jet expériences de correlation basé J
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Rappel: Couplage J
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Triangle de Pascal et Multiplicité des pics
ff
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Différentes allures à cause des couplages J
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Limitations du modèle vectoriel de Dirac (2)
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Couplage J et protéines
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Structure de la liaison peptidique
R2
R1
R3
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Relation deKarplus etprotéines
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Couplages J importants entre C-C, C-H, C-N, ...
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CaH(i-1)
NH(i)
C C
HNCA
HN(CO)CA
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Figure 4.10 : Strips des spectres HN(CO)CA (bleu) et HNCA (noir) pour les résidus 5 à 11 mettant en évidence l’attribution des Ca
( encadré noir) et du Cai-1) (encadré bleu) pour chaque résidu i et permettant ainsi l’attribution séquentielle ( pointillés rouges).
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Calcul des structures 3D des restraints géometriques
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RMN du solide (y inclus membranes, gels etc.)
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Rotation mécanique autour de l‘angle ,magique‘
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