Microfluidic Approaches for Membrane Protein Crystallization

Transcription

1 Microfluidic Approaches for Membrane Protein Crystallization Sarah L. Perry, Griffin W. Roberts, Sameer Talreja, Joshua D. Tice, Robert B. Gennis, Charles F. Zukoski, and Paul J.A. Kenis

2 Background and Motivation Membrane proteins: - signal transduction - material/energy transduction Murata, K. et al., Nature 2000, 407, Malfunction linked to many diseases drug targets Human genome: ~30,000 proteins Intrinsically unstructured proteins Unsolved soluble proteins 15% 20% 30% 35% Soluble proteins of known structure ~10,000 unresolved membrane proteins 2

3 Getting Crystals of Unknown Proteins Two Steps: (1) Identify promising conditions Precipitants, ph, T, (thermodynamics) Path to supersaturation (kinetics) tremendous screening effort How to determine the solubility boundary quickly? How to minimize material consumption per condition? (2) Grow actual crystals for X-ray diffraction How to avoid showers of small crystals? Microfluidics: kinetic control in-meso Membrane protein specific challenges: How to do 1 & 2 with even smaller amounts available? How to maintain membrane like environment? 3

4 Dealing with Amphiphilic Nature Hydrophilic intact membrane Hydrophobic In-surfo method In-meso method Detergent solubilized Cubic lipidic phase Ostermeier, C. and H. Michel, Current Opinion in Structural Biology, (5): p Landau, E.M. and J.P. Rosenbusch, PNAS, (25): p

5 Protein Crystallization Methods Our Approach: Directed Evaporation Guarantees phase change Kinetic Control More information per droplet (continuous monitoring) Hampton Research, Crystal Growth 101, Crystal Growth Techniques Talreja, Kim, Mirarefi, Zukoski, Kenis, J. Appl. Cryst

6 Evaporation-Based Crystallization 2 mm Droplet A ds J~ P Δ ~ L dt 10 wells, 10 µl each Channel of area A, length L Supersaturation 1 Initial Conditions Cp Cs Solubility Boundary Evaporation Platform: Talreja, Kim, Mirarefi, Zukoski, Kenis, J. Appl. Cryst., 2005, 38, 988 Critical Supersaturation: He, Bhamidi, Tan, Kenis, Zukoski, Crystal Growth & Design, 2006, 6, 1175 Polymorphs: He, Bhamidi, Wilson, Tan, Kenis, Zukoski, Crystal Growth & Design, 2006, 6, Time (hours) 6

7 Finding the Solubility Boundary Protein Concentration Initial state Metastable Zone (growth) 1 C S5 5 4 C S4 3 C S3 Labile Zone (nucleation) 2 C S2 1 5 C S5 2 Add precipitant to reservoir 3 Change reservoir Change reservoir 4 C S3 C S2 C S3 Precipitant Concentration C S5 C S4 500 µm µm µm µm µm 5 7

8 Phase Diagram of br 500 S = 1; Monomeric br S = 1; Trimeric br br Concentration (mg/ml) NaH 2 PO 4 Buffer Concentration (M) 8

9 Suppressing Showers of Crystallites Need a way to decouple nucleation and growth 1 Protein Concentration Initial state Metastable Zone (growth) 1 3 C S3 2 Labile Zone (nucleation) C S2 4 2 Add precipitant to reservoir 3 Remove precipitant from reservoir 4 C S3 C S2 Precipitant Concentration 9

10 Improving Crystal Quality RNase A C P /C S = 10 mg/(ml. M) C P0 = 20 mg/ml C S0 = 2 M X-ray 1.12Å Bacteriorhodopsin (a membrane protein) C P0 = 5 mg/ml Without Dilution 500 µm With Dilution 500 µm X-ray 7Å 10

11 High Throughput Screening Chip VLSI Microfluidics Chip: ~300x smaller 144 crystallization wells, ~30 nl each 10 wells, 10 µl each Screen 48 precipitants at 3 [protein] / [precipitant] ratios 4 Layers: Control layer on Metering + Mixing layer Layer with through-holes Crystallization wells and evaporation channels Control over ICs, ds/dt automatic loading & precipitant addition 11

12 Microfluidic Screening Chip 12

13 Mixing Array for 6 Wells Pneumatic Valves 13

14 Introducing Precipitant A 14

15 Introducing Precipitant B 15

16 Introducing Protein 16

17 Empty Mixtures into Wells work in progress 17

18 Dealing with Amphiphilic Nature Hydrophilic intact membrane Hydrophobic In-surfo method In-meso method Detergent solubilized Cubic lipidic phase Ostermeier, C. and H. Michel, Current Opinion in Structural Biology, (5): p Landau, E.M. and J.P. Rosenbusch, PNAS, (25): p

19 Hypothesis of in-meso Crystallization Addition of salt phase transition (dehydration of cubic phase) lamellar phase cubic phase crystal nucleation & growth Caffrey, M., Journal of Structural Biology, 2003, vol. 142(1), pp

20 Challenge of Mixing Lipid and Water η(lipid) = ~30x η(water) V = 20 μl Caffrey et al., Chemistry and Physics of Lipids, (1): p x volume reduction Lipid Protein (aq) Microfluidic Mixer Chamber Volume = 19 nl (9.4 nl protein nl pure lipid) Mixing at two time-scales -1- Injection of protein into lipid -2- Relaxation period 20

21 Direct Injection Mixer cycle rate is key (25 5 s/cycle) Scale bar: 500 µm = open = closed 21

22 In-meso br Crystallization 10 mg/ml Bacteriorhodopsin (br) in 25 mm NaH 2 PO 4 ph 5.5 mixed 1:1 (v/v) with monoolein (lipid) Salt to drive phase change: 2.5 M NaH 2 PO 4 ph O-H stretch C-H stretch amide I, II 1 Varian FTIR microscope (UMA 600) with Focal Plane Array (FPA) detector 32x32, connected to a FTIR spectrometer (FTS 7000). 22

23 Scaling Out: 4 Chambers per Chip Parallel mixing Separate precipitant lines Towards on-chip X-ray Show and Tell 23

24 Concluding Remarks How to minimize material consumption per condition? 144 wells, 30 nl each (48 precipitants, 3 conditions) How to determine the solubility boundary quickly? How to avoid showers of small crystals? br in-surfo + directed evaporation More information per droplet How to maintain membrane like environment? in-meso on a chip at 20 nl level br Ongoing: (i) Novel proteins (send us some!) (ii) Polymorph screening (iii) X-ray analysis on-chip and off-chip (iv) Array chips for in-meso and in-surfo 24