Experimental mapping of protein precipitation diagrams

Morten O.A. Sommer (morten@ccs.ki.ku.dk) Centre for Crystallographic Studies University Of Copenhagen

Look at protein crystallography and liquid handling

Low volume liquid handling technology more experiments performed using less SAMPLE

Lab automation more experiments performed using less TIME

Low TIME and SAMPLE consumption enables new approaches to protein crystallization

Liquid handling for protein crystallization

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Microfluidic formulator technology

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Experiments done by: Carl Hansen, Morten Sommer and Stephen Quake. PNAS (2004) 101:14431-14436

Experiments done by: Carl Hansen, Morten Sommer and Stephen Quake. PNAS (2004) 101:14431-14436

Metering accurate and robust

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Injection volume: 80 pL +/- 0.6 pL

Metering accuracy determined by absorption measurements

Water at 20 degrees C

Motor oil (SAE 20) at 20 degrees C

Raw Linseed oil 20 degrees C

Ideal approach to protein crystallization

• GOAL: Further rationalization of protein crystallization

• Using minute amounts of protein sample to quantify:– Protein stability, folding & activity– Protein physical chemistry (solubility and

precipitation limits) – Protein - protein interactions (Virial

coefficients etc.)

Phase diagram of: aspartyl-tRNA synthetase-1

From Thermus thermophilus

Zhu et. al. 2001 Acta Cryst. D 57:552-558

Why use precipitation diagrams?

Detecting precipitation

Detection of Precipitation

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Towards a rational approach: Tailor made screens based on

precipitation diagrams

• Characterize protein solution and identify potential conditions

• Map protein precipitation diagrams

• Design and set up a tailor made crystallization screen based on the precipitation diagrams of the particular protein

Experiments done by: Carl Hansen, Morten Sommer and Stephen Quake. PNAS (2004) 101:14431-14436

Initial validation: Xylanase

1. Make solubility fingerprint identifying precipitating chemical conditions

2. Map precipitation diagrams for potential conditions

3. Set up crystallization experiments near precipitation boundary

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Initial validation: XylanaseCrystallization probability pr. trial

OPT (Tailor made screen): 27 hits out of 48 experiments = 56 %

Sparse matrix screens: 3 hits out of 384 experiments = 0.8 %

Experiments done by: Carl Hansen, Morten Sommer and Stephen Quake. PNAS (2004) 101:14431-14436

Further validation Membrane protein: SERCA

• Study the crystallization of membrane proteins using the previously crystallized calcium pump (SERCA)

• Crystallization conditions are know

• Reliable preparation and purification

Sørensen et.al., (2004) Science 304, 1672-1675

Further validation Membrane protein: SERCA

• Solubility fingerprint can be used to identify specific protein – precipitant interactions

• Identification of specific interaction between sodium acetate and SERCA

• Sodium acetate is an established crystallization agent for SERCA

Experiments done by: Morten Sommer and Sine Larsen. Journ. of Synchrotron Rad. (2005) in press

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Further validation Membrane protein: SERCA

• Based on the characterization of specific protein – precipitant interactions several chemical conditions were selected for precipitation diagram mapping

• Set up tailor made crystallization screen• Identification of well known and new

crystallization agents• Potentially useful for crystallizing previously

uncrystallized membrane proteins

Experiments done by: Morten Sommer and Sine Larsen. Journ. of Synchrotron Rad. (2005) in press

Process diagram

Proteinsample

Formulatorchip

Solubility fingerprint

Analysis ofprotein-precipitant

interaction

Precipitationdiagrams

Design rational crystallizationexperiments

Setupcrystallizationexperiments

Monitorexperiments

Crystals

PerspectivesRational approach to protein crystallization using minute sample volumes

Rational approaches are possible for many targets that are available in low amounts (Membrane proteins, protein complexes, and proteins purified from native tissue).

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Task Volume consumption (μL)

Solubility characterization 10

Setup of 300 crystallization exp. 25

Testing previously uncrystallized membrane proteins

The ultimate test of the rational approach: 3 previously uncrystallized membrane proteins are tested.

1. Voltage gated channel 2. DsbB: disulfide bond forming

membrane protein. 3. AIDA: adhesin autotransporter

protein

Voltage-gated channel:Solubility mapping

Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Jose Santos and Mauricio Montal

Voltage-gated channel in 0.1 M Linear Buffer pH 6.5

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Based on the solubility fingerprint 40 precipitation diagrams are mapped out.

Volume consumption pr. precipitation diagram: 100 nL

Total consumption for solubility screen and precipitation diagrams: 8 μL (44.8 μg)

Voltage-gated channel:Crystallization experiments

Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Jose Santos and Mauricio Montal

Voltage-gated channel in 0.1 M Linear Buffer pH 6.5

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A tailor made screen of 288 conditions is designed. The screen is set up as sitting drop exp. using an ORYX 6 at Douglas Instruments using 17 μL sample (95 μg of protein)

An additional screen is set up testing different additives

Voltage-gated channel:Crystallization experiments

Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Jose Santos and Mauricio Montal

Crystals tested at ESRF beamline ID 29.

Not protein crystals

Scalebars = 100 microns

DsbB:Solubility mapping

DsbB in 0.1 M Linear Buffer pH 9 and 80 mM Calcium Acetate

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Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Brian Vad and Daniel Otzen

DsbB in 0.1 M Linear Buffer pH 9 and 80 mM Calcium Acetate

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40 chemical conditions are chosen for determination of their precipitation diagram.

Using a total of 4 μL (40 μg of protein).

A tailor made screen consisting of 288 conditions was designed and set up using 18 uL (180 μg)

DsbB:Crystallization experiments

Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Brian Vad and Daniel Otzen

Crystals tested at ESRF ID 29

Some were not protein.

Some did not diffract cryo optimization

Scalebars = 100 microns

AIDA:Solubility characterization

AIDA with 0.1 M Linear Buffer pH 4 and 8 % v/v Glycerol

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Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Brian Vad and Daniel Otzen

40 precipitation diagrams are selected for mapping based on solubility fingerprint

Based on the diagrams a 576 experiment screen is designed and set up

Volume consumption:

Solubility mapping: 8 μL

Crystallization exp.: 22 μL

AIDA with 0.1 M Linear Buffer pH 4 and 8 % v/v Glycerol

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AIDA:Crystallization experiments

Experiments done by: Morten Sommer, Jens-Christian Navarro Poulsen, Sine Larsen, Brian Vad and Daniel Otzen

Crystals tested at ESRF ID 29

Some did not diffract optimize cryo conditions

Scalebars = 100 microns

Protein consumption

VGC DsbB AIDA

Solubility char.

>5000 exp. 8 μL (45μg)

>5000 exp. 8 μL (80μg)

>5000 exp. 8 μL (80μg)

Cryst. Exp ~576 exp.

34 μL(190μg)

~ 288 exp.

18 μL(180μg)

~ 576 exp.

22 μL(220μg)

Crystal Hits No Yes Yes

Total protein consumption

235 μg 260 μg 300 μg

Summarizing remarks

• As liquid handling technologies have achieved ~1 nL experimental volumes. A rationalization of protein crystallization in terms of

precipitation diagrams is possible

• Rational approaches to protein crystallization are performed using < 300 μg of protein sample.

• Hope: This method and technology will allow for a better understanding of the crystallization process - and that complementary low volume technology will be developed to address other aspects of protein crystallization

Acknowledgements• Univ. Of Copenhagen

– Jens-Christian Poulsen – Prof. Sine Larsen– Flemming Hansen– Centre for Crystallographic

Studies• Univ. Of Aalborg

– Prof. Daniel Otzen– Brian Vad

• Univ. Of Aarhus– Ass. Prof. Poul Nissen– Prof. Jesper Vuust Møller

• Tech. Univ. Of Denmark– Ass. Prof. Jörg Kutter– Detlef Snakenborg

• Stanford– Prof. Stephen R. Quake

• Univ. Of British Columbia– Ass. Prof. Carl L. Hansen

• Univ. of California – San Diego– Prof. Mauricio Montal– Dr. Jose Santos

• Douglas Instruments– James Smith – Peter Baldock – Patrick Shaw Stewart

• ESRF – ID29– Gordon Leonard

Experimental mapping of protein precipitation diagrams

Morten O.A. Sommer (morten@ccs.ki.ku.dk)Centre for Crystallographic Studies Univ. Of Copenhagen