protein crystals starter kit

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Manual

JBS Protein Crystallization Starter Kit

Cat. No. Amount

CS-401EN 1 Kit

For in vitro use onlyQuality guaranteed for 12 monthsStore at 4°C

Introduction and Theory ofCrystallization

There are currently two main methods for determiningthe three-dimensional structure of a protein: NuclearMagnetic Resonance (NMR) Spectroscopy and X-raycrystallography. The NMR method is able to resolvethe atomic structure of proteins with an uppermolecular weight limit of approximately 25000 Da(25 kDa, or approximately 220 amino acids),whereas the X-ray method is more suitable for

determining the structure of larger proteins ormacromolecular complexes. The pioneering studies onthe X-ray crystal structures of myoglobin (1950) andhemoglobin (1955) were honored with the NobelPrize in Chemistry in 1962. This recognition not onlyunderscored the importance of X-ray crystallographybut also signalled the emergence of structural biologyas an essential field in the life sciences.

The first step in the determination of an X-ray crystalstructure, which is often also the most difficult step, isthe growth of sufficiently large protein crystals. Acrystal is a three-dimensional periodic arrangement ofbuilding blocks; in our case, these building blocks areprotein molecules. How does one manage to grow

Fig. 1: Schematic phase diagram of a proteinprecipitant mixture

The process of crystallization can be differentiatedinto two steps: (1) the nucleation process, and (2)crystal growth. With correctly selected conditions,crystal nucleation and growth can occur within thesupersaturated regions of the phase diagram. (seeFigure 1). As can be implied from the Figure 1,crystallization requires the formation of asupersaturated protein-precipitant solution, i.e. asolution which contains more protein molecules thanwould dissolve under normal conditions.

Unfortunately, one needs a higher degree ofsupersaturation for nucleation as for growth, andtherefore the different processes of crystal nucleationand growth are often difficult to individually control.The nucleation range is also called the labile zone,while the growth range is known as the metastable

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The transition from a stable solution to asupersaturated solution can be achieved by adjusting

the position of the protein-precipitant mixture in theexample phase diagram. This can be achieved byincreasing either the concentration of the protein orthe precipitant (vertical and horizontal axes in Figure1). The physical process of causing a change inconcentration can be carried out through dialysis ordiffusion. Both are characterized by the transport ofmaterial. The method of vapor diffusion is particularlysuitable for protein crystallization.

The experimental setup for a typical vapor diffusionexperiment is illustrated in Figure 2. This particularsetup is also known as the “hanging drop” method.The protein solution is located within a drop hangingon the underside of a microscope cover slip. The dropsize can vary from 0.5 µl up to approximately 20 µl.The reservoir solution (ca. 1 ml), which is located

beneath the hanging drop, contains a highconcentration of precipitant and thus has a lowervapor pressure than the protein solution. Over thecourse of time, water from the drop diffuses as watervapor into the reservoir solution. This raises theconcentration of the protein and precipitant in thedrop. With the correctly chosen conditions,crystallization of the protein will occur over the course

of days or weeks. In principle, one therefore uses asystem (drop plus reservoir) in which thermodynamicequilibrium is slowly adjusted over time.

Which Materials are Useful as Precipitants?

In principle any material which has an influence on

the solubility of the protein can be used as aprecipitant, provided that a high concentration of theprecipitant does not denature the protein.

One differentiates the different precipitants which areoften used in protein crystallization according to theeffect they have on the solution. Commonly usedprecipitants include salts, organic polymers, alcohols,and occasionally pure water.

Salts such as (NH4)2SO4, NaCl, LiCl, KH2PO4, etc.,change the ionic strength of the solution. The solubilityof proteins as a function of ionic strength is shown inFigure 3.

Fig. 3: Dependence of the solubility (S) on the ionicstrength (I)



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Organic precipitants such as ethanol, methanol,propanol, MPD (2-methyl-2,4-pentanediol) or

acetonitrile reduce protein solubility by lowering thedielectric constant of the solvent. Organic polymerswork the same way, such as PEGs(polyethyleneglycol), which are available in differentmolecular weights from 400 to 20000 Da.

Another important parameter which affects proteinsolubility is the pH-value of the solution. As a generalrule, solubility is lowest at the protein's isoelectric

point (IEP), since at the IEP the protein carries a netcharge of zero.

Due to the enormous number of possible parametersthat can affect crystallization, and because of therelatively small quantity of protein which is usuallyavailable, it is practically unfeasible to test allparameter combinations over their entire range. It istherefore important to develop a strategy to arrive at

your goal as quickly as possible and without using toomuch material.

Characteristics of Protein Crystals

Protein crystals are built up in principally the samemanner as crystals of small molecules or salt. Thesame rules of molecular packing and symmetry apply,however there are also some fundamental differences.These differences extend to mechanical and opticalcharacteristics of crystals as well as composition.Protein crystals are very soft and generally containbetween 30% and 70% water, most of which isrelatively unordered within the crystal.

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The JBS Protein Crystallization Starter Kit

The Jena Bioscience Protein Crystallization Starter Kit

contains all the materials necessary to explore thenucleation and crystal growth of an example protein(lysozyme from hen egg-white) using the hanging dropvapor diffusion method. On the one hand the pHvalue is changed in small steps, while on the otherhand the concentration of the precipitant (NaCl) isgradually varied. The experiment is set up in a gridorganization to allow direct visualization of the effects

of the two variables on the magnitude of nucleation,the crystal morphology (shape) and final crystal size.Moreover, the kit contains all the material tocrystallize lysozyme using the 'Batch Method'. In thismethod precipitant solution and protein solution aremixed together directly, and protein crystals grow inthe absence of vapor diffusion or a concentrationequilibrium.

Kit Components:

1. 2 24-well SuperClearTM plates for hanging-dropcrystallization

2. 1 syringe with 5 ml silicone grease to seal theplates

3. 100 cover slides for hanging-drop crystallization4. 1 microscope slide

5. 20 ml of 20% (m/v) NaCl solution6. 3 ml each of 250 mM Na-acetate buffer pH 4.0,

4.4, 4.8 and 5.27. 200 µl lysozyme solution (20 mg/ml) in water,8. 1 ml precipitant solution for batch-crystallization

(30% (m/v) PEG 5000-MME, 1 M NaCl, 50 mM



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Hanging-Drop Experimental Protocol

In the hanging-drop experiment (see Figure 2),

lysozyme is crystallized as the dilutedprotein/precipitant drop is equilibrated against theprecipitant solution in the reservoir within a sealedcrystallization plate. For this experiment a Na-acetate/NaCl system is used, whereby one canvisualize the dependence of protein crystallization onthe pH value of the buffer and the concentration ofNaCl. A pH range of 4.0-5.2 is tested in combination

with a NaCl concentration of 4-9%.Running the Experiment:

1. Grease the crystallization plates: using theenclosed syringe, apply silicone grease evenly tothe upper surface of the circular edges around theindividual reservoirs. Aim for a smooth and evenapplication, and try to avoid applying blobs of

grease to the edge or pulling threads of thesilicone across the reservoir opening.

2. Pipette the reservoir solution according to theaccompanying pipetting scheme (Figure 4): thetotal volume per reservoir amounts to 1 ml, andthe final buffer concentration after mixing withprecipitant and water is 50 mM. Note: beforepipetting, ensure that the crystallization plate is

correctly oriented by checking that the row (A-D)and column (1-6) identifiers are rightside up.

3. Pipetting the crystallization drop: pipette 1 µlprotein solution and 1 µl reservoir solution ontothe round glass cover slip. Pipette each row (A-D)separately First apply the drop of protein solution

lightly with your fingers (or other soft object) sothat the silicone grease forms a complete hermeticseal and isolates the interior reservoir chamber.

Store the prepared crystallization plates in a suitablearea with the temperature remaining as constant aspossible (20-22°C).

It is recommended to remove the plate cover whenobserving the drops under the microscope. Cautionshould be exercised when adjusting the microscopeobjective so as not to get it contaminated with grease.

Make sure to observe the entire depth of the proteindroplet under the microscope. Polarized light can alsobe applied in order to distinguish between amorphousprecipitate and crystalline material, since crystals areable to polarize the transmitted light.

Figure 5 displays an overview of the crystallizationdrops under the microscope (40 x magnifications)

after 20 hours. The coordinate numbering scheme(A-D, 1-6) corresponds to the coordinate system on thecrystallization plate.

It can be clearly seen that the number of crystals riseswith increasing NaCl concentration. An optimum ofcrystal quality is reached at a salt concentrationslightly lower than the maximum on the grid,according to the phase diagram shown in Figure 1

(see the relationship between nuclei formation andcrystal growth in the introduction of this manual). Alsonote that nuclei formation decreases with rising pHvalue, which expresses itself as a decrease in thenumber of crystals with increasing pH.

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Running the Experiment:

1. Pipetting the precipitant solution: pipette 4 µl of

the batch precipitant solution onto the microscopeslide.

2. Pipetting the protein solution: pipette 2 µl ofprotein solution onto the precipitant solution drop.

Now observe the drop under the microscope. Theprecipitant/protein volume ratio can also be varied,which will lead to a different speed of crystal growth

and overall quantity of crystals.

Acknowledgements

We thank Dr. Manfred Weiss for the original ideaand the assistance in organizing this kit. Largeportions of this manual are based on experimentalinstructions from Dr. Weiss.

CopyrightAll figures in this manual are copyright © Dr. ManfredS. Weiss. All rights to the figures remain with theowner.

Duplication of this manual, in whole or in part, isprohibited without express written permission of JenaBioscience GmbH.



Manual


Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100

of 7 www.jenabioscience.com Last update: December 04, 2012

20 % NaCl

4 % 5 % 6 % 7 % 8 % 9 %

Fig. 4: Pipetting scheme for the hanging-drop experiment

2 5 0 m M S

o d i u m A c e t a t e

200 µl600 µl200µl

200 µl600 µl200µl

200 µl600 µl200µl

200 µl600 µl200µl

250 µl550 µl200µl

250 µl550 µl200µl

250 µl550 µl200µl

250 µl550 µl200µl

300 µl500 µl200µl

300 µl500 µl200µl

300 µl500 µl200µl

300 µl500 µl200µl

350 µl450 µl200µl

350 µl450 µl200µl

350 µl450 µl200µl

350 µl450 µl200µl

400 µl400 µl200µl

450 µl350 µl200µl

400 µl400 µl200µl

400 µl400 µl200µl

400 µl400 µl200µl

450 µl350 µl200µl

450 µl350 µl200µl

450 µl350 µl200µl

50 mMpH 4.0

50 mM

pH 4.4

50 mMpH 4.8

50 mMpH 5.2

Finalconcentration

— 20 % NaCl — 250 mM Na-Acetate — distilled water



Manual


Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100

of 7 www.jenabioscience.com Last update: December 04, 2012

Fig. 5: Appearance of the protein droplets in the hanging-drop experiment after 20 hours (40x magnification)

A

2

C

B

3

D

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