Crystallization and Stability of Membrane Proteins. David Drew
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1 Crystallization and Stability of Membrane Proteins David Drew
2 1. Produc)on, Purifica)on, Crystalliza)on (Talk 1) GFP- based E. coli pipeline - Bacterial proteins GFP- based S. cerevisiae pipeline - Eukaryo)c proteins Op)miza)on of membrane protein crystals based on detergent stability (Talk 2)
3 Whole-cell Drew et al., FEBS (2001) MPs tested rapidly for quantity and quality In-gel Drew et al., Nat Methods (2005), Newstead et al, PNAS (2007), Geertsma ER., PNAS (2008) FSEC Kawate T, Gouaux E. Structure (2006)
4 Plasmids and strains sent on request (no MTA)
5 Transporter structural summary? Crystallization success rate similar between proand eukaryotic transporters. Crystal optimization is the biggest difference. Sonoda Y et al. Structure. (2011)
6 The Emperical Triangle Y. Sonoda et al. Structure. (2011)
7 Micelle size is important Sonoda Y et al. FEBS (2010).
8 Micelle size correlates with dynamic radius Kunji ER. et al, Methods (2008)
9 Detergent (conformational) stability is important Serrano-Vega MJ, et al. PNAS (2008), Warne T et al. Nature (2008)
10 8Å 3Å DDM LDAO
11 How detergent stable does the MP need to be? Sonoda Y et al. Structure. (2011)
12 CPM assay to measure detergent MP stability DDM Different detergents Cys" CPM" 40 C Fluorescence Fluorescence CPM: the thiol-specific fluorochrome, N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide Alexandrov A., Mileni M., Chien E., Hanson M., Stevens RC., Structure (2008)
13
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15 Include test-set of control membrane proteins Sonoda Y et al. Structure. (2011)
16 Benchmarking Stability for Optimizing Crystallization CPM assay Rela)ve fluorescence (R.F.U) LDAO OG 9M 10M C 12 E 9 / 12M Time (min) Measure unfolding rate at 40 C for 130 min in proteins all purified In 12M and diluted 150- fold into buffer containing detergent at 3 x cmc
17 Calculate a relative unfolding rate (t 1/2 ) based on cysteine accessibility over time Fraction of folded protein = 1 - (fluorescence t =x) (max fluorescence) Sonoda Y et al. Structure. (2011)
18 Bacterial MPs more stable than Eukaryotic MPs Prokaryotic Eukaryotic N = 16 N = 8 Y. Sonoda et al. Structure. (2011)
19 Mean membrane protein detergent stability correlates with micelle-size Y. Sonoda et al. Structure. (2011)
20 Hydrophobic mismatch Olaf Anderson and Roger Koepe (2007). Annu. Rev. Biopys. Biomol. Struct
21 Comparing t 1/2 values to monodispersity
22 Decreasing stability
23 Benchmarking Stability for Optimizing Crystallization LDAO Eukaryo)c Prokaryo)c stable 17 min not- stable
24 Benchmarking Stability for Optimizing Crystallization 12M > > Eukaryo)c Prokaryo)c stable 17 min
25 MPs stable in harsh detergents are also more in general LDAO vs 9M
26 MPs stable in harsh detergents are also more in general 9M vs 10M 12M vs C 12 E 9
27 Stable MPs are more likely to crystallize in small detergent
28 All our structure success are only the most stable MPs LDAO min
29 4 bacterial and 1 plant transporter crystals diffract well
30 How do we tackle less stable transporters? LDAO min 5?
31 Benchmarking Stability Lipid for addition Optimizing Crystallization Rat sugar transporter
32 Benchmarking Stability for Optimizing Crystallization? Solved
33 wt turkey β1-receptor ronn prediction (xenopus)
34 Wt turkey β1 (N- and C-terminal truncations) Δ30aa ICL3 Turkey β1 structure sequence
35 Mammalian proteins can also screen for more stable homologue
36
37 Mammalian transporter-fab complex
38 H1 structure Shimamura et al. Nature (2011)
39
40 Other strategies 3. LCP in combination with T4-L (Ray Stevens, Brian Kolbilka, Martin Caffrey) 4. Stabilization ligands/ mutagenesis (Chris Tate) e.g. human β2-receptor e.g. turkey β1-receptor Cherezov V et al., Science (2007) Warne et al. Nature (2010)
41 The Emperical Triangle Y. Sonoda et al. Structure. (2011)
42 Acknowledgements So Iwata s MPC/MPL/Kyoto/RaCH group Yo Sonoda, Simon Newstead, Nein-Jen Hu, Emmanuel Nji, Alex Cameron, Norimichi Nomura, Hae-Joo Kang Bernadette Byrne Yilmaz Alguel, James Lee My Current group Chiara Lee, Hae-Joo Kang, Nein-Jen Hu
GFP-based pipelines for the overexpression and purification of membrane proteins. David Drew
GFP-based pipelines for the overexpression and purification of membrane proteins David Drew 1. Produc0on, Purifica0on, Crystalliza0on (Talk 1) GFP- based E. coli pipeline - Bacterial proteins GFP- based
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