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 S. cerevisiae pipeline - Eukaryo0c proteins http://www.nature.com/nmeth/journal/v3/n4/full/nmeth0406-303.html http://www.nature.com/nprot/journal/v3/n5/abs/nprot.2008.44.html 2. Op0miza0on of membrane protein crystals based on detergent stability (Talk 2)
GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR
GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR Wagner, S., Lerch- Bader, M., Drew, D., and De Gier J.W., Trends in Biotechnology (2006)
GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR periplasm cytoplasmic membrane cytoplasm membrane Fluorescent MP- GFP Overexpression inclusion bodies Not Fluorescent Drew, D, Nordlund, P, von Heijne, G, de Gier, JW. FEBS LeU (2001)
GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR Fluorescent Not Fluorescent periplasm cytoplasmic membrane cytoplasm Drew, D, Sjöstrand, D, Nilsson J, Urbig T, Chin CN, de Gier JW, von Heijne G. (2002). PNAS.
GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR ~ 80% of E. coli membrane proteins have Cin topology Daley D, Rapp, M, Granseth, E, Melen, K, Drew, D, von Heijne, G (2005) Science Hsieh JM, Besserer GM, Madej MG, Bui HQ, Kwon S, Abramson J. Protein Sci. 2010 Apr;19(4):868-80.
GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR In- gel fluorescence Drew, D, Lerch, M, Slotboom, D, Kunji, E, de Gier, JW. (2006). Nature Methods
GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR
GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR unfolded folded unfolded folded Drew, D, et al. Nature Methods. 2006 Geertsma ER, et al. PNAS. 2008
GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR Green: C41(DE3), Red: C43(DE3), Blue: BL21(DE3)pLysS, odd numbers: 0.1 mm IPTG and even numbers: 0.4 mm IPTG
Lemo21(DE3) Wagner et al. (2008) PNAS, 105 (38):14371-6.
Lemo21(DE3)
Autoinduction media Gordon et al, (2011). Protein Expr. Purif
Eukaryotic Membrane Protein Production Bill, R et al. Nat. Biotechnology (2011)
Eukaryotic membrane protein production in S. cerevisiae Newstead S., Hyun, K., von Heijne G., Iwata S., Drew D. (2007). PNAS. Drew, D., Newstead S., Sonoda, Y., Hyun, K., von Heijne G., Iwata S. (2008) Nature Protocols
Establishing screen condi4ons Eukaryotic membrane protein production in S. cerevisiae
Eukaryotic membrane protein production in S. cerevisiae
Eukaryotic membrane protein production in S. cerevisiae yeast cell suspension Add glass beads and break In 0ssue lyser for 10 minutes Spin in table- top centrifuge for 1 hour In- gel fluorescence Run standard SDS- PAGE Suspend crude membranes and measure fluorescence
S. cerevisiae overexpression screen strategy Eukaryotic membrane protein production in S. cerevisiae
Eukaryotic membrane protein production in S. cerevisiae
Screening monodispersity in crude samples by Fluorescence-detection Size-Exclusion Chromatography Kawate, T, Gouaux, E (2006). Structure
~ 60% of eukaryotic membrane proteins monodisperse in DDM From 15L 13 mg Yeast GDP-mannose tr. 4 mg Human Slc35b1 7 mg Mouse CMP-Sia tr.
Detergent solubilization efficiency is a poor criterion for selection 13 / 17 10 / 17 5 / 17
Detergent efficiency NOT good indicator Detergent solubilization efficiency is a poor criterion for selection
Detergent solubilization efficiency is a poor criterion for selection
Localization
~ 70% targeted to the correct organelle
Summary Membrane Proteins produced in S. cerevisiae 25 % of can be overproduced to > 1 mg/l 70 % of those are correctly localized Detergent-solubilization is not a reliable
MP- TEV- GFP- His 8 TEV10His 8His Drew, D, Slotboom, D, et al (2005). Prot. Sci.
Non- solubilized membranes Solubilized membranes Flow- through Ni- NTA bound LacY- GFP LacY- GFP eluate Natural light In- gel fluorescence Coomassie stained
Example case 1: Rat VGLUT2 Sonoda Y, Cameron A, Newstead S, Omote H, Moriyama Y, Kasahara M, Iwata S, Drew D. FEBS LeU.(2010)
Example case 2: human GLUT1
Crystalliza0on: Test- set of control membrane proteins Sonoda Y et al. Structure. (2011)
LDAO AmtB ammonium channel to 1.9-Å 7.0Å 3.5Å 2.3Å 1.8Å
9M Mhp1 hydantoin transporter outward facing conformation to 2.8-Å 11.2Å 5.6Å 3.6Å 2.7Å
12M NhaA physiological dimer to 3.6-Å 13.6Å 6.5Å 4.3Å 3.2Å
12M (BT-9) PepTso -40% identical to PepT at 3.6 / 4.0-Å 14 Å 6.9 Å 4.6 Å 3.4 Å Newstead, S, Drew D., Cameron, A.D., et al. EMBO J. (2011)
Benchmarking Stability for Op0mizing Crystalliza0on MP structures from cleavable GFP- His 8-10 tag
Transport Systems H + Secondary ac1ve transport Nutrients Symport Na + Nutrients Respira1on H + Na + H + An1port ADP Toxic molecules ATP synthase H + ATP ATP ATP H + Nutrient, K + Peter Henderson Primary ac1ve transport
S. Newstead, D. Drew et al. EMBO J. (2011) Secondary Transporters Large fraction of the membrane proteome Play a key role in drug pharmacokinetics Only a few secondary eukaryotic transporter structures known in PDB
Transporter structural summary? Crystalliza1on success rate similar between pro- and eukaryo1c transporters. Crystal op1miza1on is the biggest difference. Sonoda Y et al. Structure. (2011)
How detergent stable does the MP need to be? Sonoda Y et al. Structure. (2011)
Summary Membrane protein production is trial-and-error Fluorescence monitoring facilitates this process Obtaining membrane protein crystals is just the first step
Acknowledgements Stockholm University/ CBR Jan- Wllem de Gier (E. coli GFP pipeline) Joy Kim and Gunnar von Heijne (S. cerevisiae GFP pipeline) So Iwata s MPC/MPL group Yo Sonoda, Simon Newstead, Nein- Jen Hu, Alex Cameron, Norimichi Nomura, Hae- Joo Kang My Current group Chiara Lee, Hae- Joo Kang, Nein- Jen Hu