© Johan Zeelen M.P.I. of Biophysics The crystallization of (membrane) proteins, using a flexible Sparse Matrix Screen. Soluble proteins and membrane proteins

© Johan Zeelen M.P.I. of Biophysics

The crystallization of (membrane) proteins,using a flexible Sparse Matrix Screen.

Soluble proteins and membrane proteins

Screening protocol for 3 D crystallization

- flexible sparse matrix

- adjusted screen

- optimisation

Membrane proteins and detergent

Excluding regions where nucleation is not evident



- Soluble proteins

Protein

- Membrane proteins

Protein

Detergent

(Lipid)



The purity of the starting material is a crucial factor.

- Purity of the protein:

SDS gel and TLC

- Sample heterogeneity:

Mass spectrometry

Dynamic light scattering

- Solubility 5-10 mg/ml

- Stability Storage conditions

SDS - page-

Thin-layer chromatography-> Proteolysis and post

translational modifications-> Oligomerisation, aggregation


Screening protocol for 3 D crystallization

1 2 3 4 5 6 7 8 9 10ABCD

FGHIJ

3.5Å

1.5Å.

8Å

E

Triosephosphate isomerase:

P212121 1.83ÅC2 (big) 2.1 ÅC2 (small) 2.1 ÅP1 1.5 Å

The screening for suitable crystallization conditions starts with the search in a multidimensional phase diagram for conditions that favor nucleation. Experiments with Triosephospate isomerase (soluble protein) and Light-harvesting complex II (membrane protein) indicate, that the proteins can crystallize in different space groups and resolution.

Two dimensional representation of the multi dimensional crystallization space


Initial screenThe systematic search of the multidimensional space requires large amount of protein and time.

1 2 3 4 5 6 7 8 9 10ABCDEFGHIJ

X XX XX XX XX XX X

To reduce the number of crystallization trails, the incomplete factorial is a powerful tool to identify the influence of different variables.

1 2 3 4 5 6 7 8 9 10ABCDEFGHIJ

1 2 3 4 5 6 7 8 9 10ABCDEFGHIJ

X

X

X

X

The conditions in the sparse matrix are not random but heavily biased towards published crystallization conditions. Most of the commercially available screens are sparse matrix screens.

1 2 3 4 5 6 7 8 9 10ABCDEFGHIJ

X

XX

X

1 2 3 4 5 6 7 8 9 10ABCDEFGHIJ

In the flexible sparse matrix also information from biochemistry (pH stability, salts) is used to exclude conditions where the protein denatures / is inactive.To simplify the interpretation of the results, the crystallization conditions are sorted by precipitant. The drop size is 1 µl protein and 1 µl well solution.

X

XX


Interpretation of the initial screen

The crystallization experiments are examined with a stereo microscope: 1) immediately after setup, 2) each day for the first week, and 3) once a week for several weeks.

Not precipitatedPrecipitated no birefringence no edgesPrecipitated with birefringence and edges


No nucleation with the initial screen

Try a different screen- random sampling

- based on published conditions

0

1

2

3

4

5

3 4 5 6 7 8 9 10 11

11 Commercially Available Screens

Am

mo

niu

m S

ulf

ate

pH0

1

2

3

4

5

3 4 5 6 7 8 9 10 11

Conditions from BMCD

Am

mo

niu

m S

ulf

ate

pH

As initial screen the sparse matrix is the most popular because it is commercially available. This does not mean that every protein will crystallize under these conditions. The strategy to try different sparse matrix screens will limit the search to the most common successful crystallization conditions.


No nucleation with the initial screen

Adjust the initial screen

Precipitant

soluble

supersaturation

slow precipitation /nucleation

Fast precipitation

[Pro

tein

]

No precipitate -> increase/double precipitant concentrationFast precipitation -> decrease/halve precipitant concentration

Repeat until the precipitation point for all wells is determined.

A different and possibly faster strategy is to use the solubility information from the first screen to exclude areas of the phase diagram where no crystallization will occur. This knowledge is used for the design of a new adjusted screen. In the adjusted screen only the precipitant concentration is changed.


Optimisation screen

After identification of conditions that favour crystal growth a new set of conditions is set-up by adjusting the:

A) precipitant 1 concentration with salt

B) precipitant concentration without salt

C) buffer and pH (4.5-9.5)

D) protein concentration

Initial screen

Adjusted screen

Optimisation

25 % PEG 6000, 200 mM LiSO4 at pH=6.5, 5 mg/ml protein -> small crystals after 3 days

A) 12.5% - 25 % PEG 6000, 200 mM LiSO4 at pH=6.5 B) 12.5% - 25 % PEG 6000 at pH=6.5 C) pH = 4.5 - 9.5, 25 % PEG 6000, 200 mM LiSO4 D) Protein 1 - 6 mg/ml, 25 % PEG 6000, 200 mM LiSO4 at pH=6.5

A - B -> is salt neededA or B -> precipitant concentrationC -> pH dependenceD -> protein concentrationIdentical conditions -> reproducibility


Detergent

The detergent plays a crucial role in the crystallisation of membrane

proteins.

Neurospora crassa plasma membrane H+-ATPaseprecipitant

dete

rgen

t

phase separation

CMC

Detergent phase diagram At high detergent and precipitant concentrations the micelles aggregate. The solution separates in a micelle rich and a micelle poor phase. Often 3D crystals are found, close to the condition where phase separation occurs.


2D and 3D crystallisation of LHC II

2D EM

3D X-ray


Flexible sparse matrix screenInitial screen

Initial screen

Adjusted screen

Detergent/biochemistry

Optimization Crystals

Optimization

Stock solutions

Initial screen Adjusted screen

Buffer Precipitant Additive

The flexible sparse matrix is an efficient method for searching the multidimensional phase diagram for conditions that favor nucleation, by excluding regions where nucleation is not evident. Although the preparation of wells from stock solutions is more labor intensive than ready-made solutions, it has the advantage that precipitant concentration, pH, and additive can be manipulated independently.


Acknowledgements

E.M.B.L Heidelberg

Group R.K. Wierenga

M.P.I. of Biophysics

Department of Structural Biology

W. Kühlbrandt

H+-ATPase: M. Auer and G.A. Scarborough

LHC II : M. Lamborghini

http://www.mpibp-frankfurt.mpg.de/~johan.zeelen/xtal.html

Garavito, R.M and Picot, D (1990) The Art of Crystallizing Membrane Proteins. Methods: A Companion to Methods in Enzymology Vol. 1 No. 1 pp. 57-69

Hjelmeland, L.M. and Chrambach, A. Solubilization of Functional Membrane Proteins. Methods in enzymology Vol 104 pp. 305-318

Zeelen, J.P. (1999) Strategy 1: A Flexible Sparse Matrix Screen. In Protein Crystallization, Techniques, Strategies, and Tips, chapter 9 (ed. T.M. Bergfors) IUL Biotechnology Series.

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© Johan Zeelen M.P.I. of Biophysics The crystallization of (membrane) proteins, using a flexible Sparse Matrix Screen. Soluble proteins and membrane proteins