Technologies for studying macromolecular complexes

1. Méthodes d’Etude des Complexes Technologies for studying macromolecular complexes Arnaud.Poterszman@igbmc.u-strasbg.fr

2. Une protéine n’agit jamais seule… …mais interagit avec des partenaires afin d’assurer sa fonction.

3. Classification of complexes (Nooren & Thornton, EMBO 2003) 1- Structural diversity • The interaction involves • proteins :homo- or hetero-oligomers • proteins and nucleic acids: protein/DNA protein/RNA • Isologous or heterologous association: • contact/interaction surfaces are identical (homo-oligomers) • contact/interaction surfaces differ (homo-/hetero-oligomers) identical chains different chains

4. Obligatory complexes : • Isolated proteins are not stable. • Isolated proteins are not functional. • Interaction is necessairy for stability and function. • Ex: large protein complexes (DNA polymérase, RNA polymerase, • transcription factors, ribosome…) • Non-obligatory complexes : • Isolated proteins are stable. • Isolated proteins are not functional. • Interaction produces an action. • Ex: antigen-antibody complexes, enzyme-inhibiteur, complexes aaRS-tRNA, intracellular signaliing complexes… 2- Fonctional diversity

5. 3- Dynamical diversity • Stable associations : • Only exists within a complex. • Obligatory PPI are generally permanent. • Transient associations : • In vivo association and dissociation. • Non-obligatory PPI can be transient or stable.

6. Quantitative data protomers oligomer - Contact area and polarity of various non obligate and obligate complexes. The ellipse denotes the aera-polarity space of weak transient interactions Relation between different types of PPIs, their binding energies and the localization of their protomers. The triggers that control transient oligomerization are in red.

7. Macromolecular assemblies in transcription and traduction

8. Complexes in transcription initiation and its regulation HDAC HAT CoR CoA Repression apo NR holo NR Derepression 19S Mediator RNA pol II TFIIH CAK Activation, Regulation by phosphorylation

9. Sample Bio-informatics NMR Biophysical approaches X-ray diffraction Mass Spectrometry Electron Microscopy

10. em-driven approches Cryo-negative staining negative staining

11. X-ray and NMR studies of fonctionals modules p44 Ring finger NMR P8/TTD-A NMR Cyclin H X-rays

12. structure-function studies

14. transient complexes PKA phosphoryle RARα (S369, LBD AF-2) Recrutement de TFIIH (via cycline H) TFIIH phosphoryle RARa (S77, AF-1) Augmentation de la transactivation C. Egly

15. Strategies for production of complexes Non-obligatory/Obligatory Transient/stable Yes No Produce (and purify) the proteins independently and reconstitute the complex in vitro Co-expression is mandatory Co-expression in E. coli or in insect cells Purification of Endogenous complexes • Single gene expression • E. coli • Insect cells baculovirus • Yeast • Mammalian

16. Recombinant multi-protein complexes Expression of subunits separately purifiy and mix mix and purify refold, mix and purify .... Co-transformation of bacteria with (compatible) expr vectors Use of multi-expression vectors Co-infection of insect cells by several viruses Generation of viruses that co-express several genes

17. Expression levels x < 100 mg/L 100 µg/L < x < 500 µg/L 500 µg/L < x < 2 mg/L 2 mg/L < x Hopeless (for stuctural studies but not for function assays) Micro-methods (Em, nanodrop crystallization, microfluidics) Standard approches endogenous, recombinant quantity mg mg g Poor expressors Difficult to handle proteins

18. Dynamic Light Scattering EMSA SAXS AUC ITC/DSC Electron microscopy Mass Spectrometry

19. Sample requirements In addition to chemical purity Native Gels, EMSA: mg MS:denaturing: 0.2 n mole MS: native: 5.0 n mole de-salted sample LC-MS: denaturing: 1 n mole DLS: # 1 mg/ml, 12ml/assay thermofluor: # a few mg ITC, DSC: # a few mg AUC: equilibrium OD 0.2-0.5, 120ml/assay AUC: velocity OD 0.2-0.5, 300ml/assay EM neg staining: # 50 ng/ml, 5ml/assay EM cryo: # 0.5 mg/ml, 5ml/assay SAXS: # 1 mg/ml, 100ml/assay

20. Characterization Strategies for obligatory complexes Purity ? SDS page/coomassie or silver staining MS in denaturing conditions Are the subunits associated ? Pull down Native gel eletroporesis Gel filtration Native MS Homogeneity/Monodispersity? DLS, gel filtration, EM, AUC, DSC….. Conformational homogeneity ? EM, SAXS, SANS, AUC, DSC…..

21. Characterization Strategies for non-obligatory complexes Isolated components are avalible and are pure/homogenous Pull down Native gel eletroporesis, EMSA Gel filtration AUC, SAXS, SANS Native MS, EM Association ? (biochemical analysis) Association ? (biophysical analysis ) Thermodynamic parameters ? EMSA, ITC, spectroscopy, AUC, SPR….. Conformational homogeneity ? EM, SAXS, SANS, AUC, DSC…..

22. - thermodynamics: the essentials Identification, production and purification - TAP tag/MS foot-printing - Single gene expression and in vitro assembly: - Co-expression techniques in E. coli and in insect cells - Purification strategies Analysis of assembly processes - spectroscopic methods - microcalorimetry - surface plasmon resonnance - equilibrium binding/allostery Size and shape - Dynamic Light scattering - SAXS and SANS - Analytical ultracentrifugation - non denaturing MS - Electron microscopy

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