Growing Lysozyme Crystals

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In 1959, Max Perutz and JohnKendrew published an article onthe three-dimensional structure ofwhale myoglobin, which is a smallprotein responsible for the transportof oxygen in whale cells. By investi-

gating the proteins structure, the twoscientists wanted to understand theoxygen-carrying mechanism at themolecular level. They grew crystalsfrom this protein and managed todetermine its structure by analysingthe X-ray diffraction pattern of thecrystal. A number of myoglobins fromother species had been tested beforewith little success, until Perutz andKendrew obtained a useable diffrac-tion pattern with whale myoglobin

crystals. This pioneering work wasawarded the Nobel Prize in

Chemistry in 1962w1. Fifty years later,however, it is still a challenge toobtain protein crystals for structuralstudies.

What are proteins?

Proteins are the largest group ofnon-aqueous components in livingcells. Almost every biochemical reac-tion requires a specific protein, calledan enzyme. Other types of proteinshave mechanical and structural func-tions (e.g. collagen in connective tis-sue), or mediate cell signalling (e.g.hormone receptors), immune respons-es (e.g. antibodies) or the transport ofsmall molecules (e.g. ion channels).The variety is immense: more than

20 000 different proteins are known toexist in humans alone.

www.scienceinschool.org30 Science in School Issue 11 : Spring 2009

Beat Blattmann and Patrick Sticher fromthe University of Zrich, Switzerland,explain the science behind protein

crystallography and provide a protocolfor growing your own crystals fromprotein an essential method used byscientists to determine protein structures.

Growing crystalsfrom protein


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Despite this variety, all proteinsshare an identical structural principle.They consist of 20 different building

blocks, called amino acids, which arearranged in a linear chain connectedby covalent bonds between adjacentamino acids (see figure below). Thelength of the protein chain variesfrom a few dozen to thousands ofamino acids. In cells, each protein isassembled using the informationencoded in its corresponding gene.The assembly is performed by a ribo-some, which is a complex molecularmachinery consisting of proteins and

RNA.

Proteins are folded into distinctthree-dimensional structures

Under natural conditions, the linearchains of amino acids spontaneouslyfold into distinct three-dimensionalstructures. Stretches of amino acidsform typical secondary structural ele-ments. The most prominent elementsare -helices and -sheets (see figurebelow), which are typically stabilised

by hydrogen bonds between individ-ual amino-acid residues. The entireprotein forms a tertiary structure con-sisting of a variety of such structureelements.

Structure is function: what doesthe three-dimensional structureof a protein tell us?

The function of a particular proteindepends on its three-dimensional struc-ture. Only when the protein is folded,the specific amino acids of the proteinare close enough to enable the forma-tion of an active site. These sites cancatalyse biochemical reactions, as in thecase of enzymes, or form a specificbinding site, as in the case of antibod-ies. Investigating the structural detailsof a protein is of great importance tounderstand how fundamental process-

es of life function at a molecular level:this is the research area of structuralbiologists. One of the major challengesin structural biology today is the eluci-dation of the structure, function andinteraction of huge macromolecularcomplexes and membrane proteinsw2.Due to their complexity, these proteinsare experimentally extremely challeng-ing, and every time the structure of aprotein is determined, it is a majorachievement. Nevertheless, since they

are involved in fundamental biologicalprocesses, there is a great interest inbetter understanding their structureand function, and scientists keep tryingto crystallise them.

Teaching activities

www.scienceinschool.org 31Science in School Issue 11 : Spring 2009

Protein crystals are smalland fragile objects, lessthan a millimetre indiameter and difficult to

grow. Yet they are essen-tial for structural biologystudies by X-ray analysis

Imagecourtesyo

fGabySennhauser,UniversityofZrich

a. Proteins are builtfrom aminoacids, whichare covalentlylinked to form

a linear chain

b.Proteins arefolded to athree-dimension-al structure thatdetermines theirfunction. Smallstretches of theamino-acidchain form typi-cal folds. Twoprominent struc-tural elements

are -helicesand -sheets

Imagecourte

syofMarcLeibundgut,ETHZrich,andwww.p

db.org

a

b


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Proteins are too small for directobservation

Proteins are tiny structures, measur-ing only a few nanometres (1 nm = 1millionth of a mm). Particles that sizecannot be observed even with thestrongest light microscope, which hasa maximum resolution of 1 microme-

tre (1 m = 1 thousandth of a mm).Three major technologies are avail-

able to make protein structuresvisible:

X-ray diffraction of protein crystals

Nuclear magnetic resonance (NMR)

Electron crystallography

As more than 90% of all proteinstructures deposited in the publiclyaccessible protein database of bio-logical macromoleculesw3 have beendetermined by X-ray diffraction, wewill concentrate on this method. Tolearn more about the history of crys-tallography and the journey of a


Workflow for proteinstructure determinationby X-ray diffraction

Protein Crystal Structure

Image courtesy of Beat Blattmann and Patrick Sticher

Nucleation zone

Supersaturation

Undersaturated Zone

Metastable zone

Crystals grow from anaqueous protein solu-tion, which is broughtinto supersaturation.Crystallisation proceedsin two phases, nucle-ation and growth. Afternucleation, it is impor-tant to reach what isknown as themetastable zone, inwhich the best condi-tions are found for the

growth of large well-ordered crystals. Twocompeting processesdecrease the proteinconcentration in thesupersaturated state:(I) crystallisation,(II) precipitation

Protein

concentration

Adjustable parameter (such as salt concentration)

Sup

erso

lubility

curv

e

Solubility

curve

ImagecourtesyofNicolaGraf

Selection Protein Refinement

Production crystallization Validation

Purification Data acquisition Biological context

Analysis Structure solution

Precipitation zone

I

II


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protein from lab to lab, until itsstructure is solved, see the articleby Dominique Cornujols in thisissue (pages 70-76).

Crystallising proteins is a trickytask, because it is difficult to deter-mine the right conditions underwhich each new protein will crys-

tallise sometimes, it even seemsimpossible. So to ensure reproduciblecrystal quality (i.e. that equally goodcrystals can be grown again), scien-tists use controlled experimental set-ups to crystallise their proteins. Themost frequently used method in pro-tein crystallography is the vapour dif-fusion method (see image above): inthis method, a small amount of acrystallisation solution is added to thereservoir of the crystallisation cham-

ber. A drop of protein solution and adrop of the crystallisation solution are

pipetted onto the sitting drop postthat is located in the centre of thischamber.

Immediately after adding all solu-tions, the chamber is sealed to avoidevaporation. Since the concentrationof salt ions is higher in the crystallisa-tion solution than in the mixture on

the sitting drop post, solvent mole-cules will move from the protein dropto the reservoir by vapour diffusion inthe gas phase. During this process,the solubility of the protein in thedrop decreases. The protein solutionin the drop eventually becomessupersaturated, which is a thermody-namically unstable state. This causessome of the protein in the drop eitherto form crystal nuclei that finallygrow into larger protein crystals (see

image on page 32), or to precipitate asamorphous protein which is useless

for X-ray analysis. Crystallisation andprecipitation are competing processes,so it is extremely important to findthe optimal conditions favouringcrystallisation.

Teaching activities


ImagecourtesyofBeatBlattmannandPa

trickSticher

b ca

The vapour diffusionmethod is the mostfrequently used tech-nique to grow proteincrystals.

a. A small amount ofa crystallisation solu-tion is put into a small

reservoir.b. A drop of proteinsolution and a dropof crystallisationsolution are placedonto the sitting droppost in the chamber.

c. The chamber issealed to start thecrystallisation process

0.5 ml reservoirsolution

Sitting drop post

Clear sealing tape

1.0 lreservoirsolution

1.0 lproteinsolution


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Lysozyme crystals in the classroom

In this practical activity, students learn more about

modern X-ray crystallography by determining the opti-mal crystallisation conditions for a protein. They inves-

tigate the formation of lysozyme crystals as a functionof pH and salt concentration.

Lysozyme

Lysozyme is a protein belonging to a family of anti-

bacterial enzymes which damage bacterial cell walls.In humans, it is abundant in a number of secretions,

such as tears, saliva and mucus. Large amounts oflysozyme can also be found in chicken egg whites.

Equipment and materials

One or two Cryschem crystallisation plates(Hampton Research) per class

Crystal clear sealing tape (5 cm) (HamptonResearch)

1 ml and 1 l manual pipettes A microscope to observe the crystals

Storage space at 20 C

Chemicals

Lysozyme (SigmaAldrich Product #62971,BioChemika grade lysozyme from a differentsource will probably also do, but this one has beenthoroughly tested with the protocol, so it is recom-

mended, to be on the safe side)

Sodium chloride (NaCl) (table salt from the super-market will do)

Citric acid Sodium acetate

Sodium phosphate, monobasic

Sodium hydroxide solution Glacial acetic acid Deionised water (DI-water)

Stock solutions

The following aqueous stock solutions should be pre-

pared in advance by the teacher:

50 mg/ml lysozyme stock solution in water 3 M sodium chloride

Dissolve 17.53 g NaCl in 100 ml

DI-water.


1 2 3 4 5 6

A

B

C

D

pH

increasesfrom3.5to6.5

Pipetting scheme for the crystal growth experiment

NaCl end concentration increases from 0.6 to 2.1 M

C

LASSROOMA

CTIVITY 1.0 ml sodium

citrate(end conc. 0.1 M),pH 3.5

1.0 ml sodiumacetate(end conc. 0.1 M),pH 4.5

1.0 ml sodiumacetate(end conc. 0.1 M),pH 5.5

1.0 ml sodiumcitrate(end conc. 0.1 M),pH 6.5

2.0 ml of 3M NaClstock solution (endconc. 0.6 M)

7.0 ml DI-water


6.0 ml DI-water


5.0 ml DI-water


4.0 ml DI-water


3.0 ml DI-water


2.0 ml DI-water

A1 A2 A3 A4 A5 A6

B1 B2 B3 B4 B5 B6

C1 C2 C3 C4 C5 C6

D1 D2 D3 D4 D5 D6


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1 M sodium citrate, pH 3.5Dissolve 19.24 g citric acid in 100 ml DI-water. Adjustthe pH with sodium hydroxide solution to pH 3.5.

1 M sodium acetate, pH 4.5Dissolve 13.6 g sodium acetate in 100 ml DI-water.

Adjust the pH with glacial acetic acid to pH 4.5.

1 M sodium acetate, pH 5.5Dissolve 13.6 g sodium acetate in 100 ml DI-water.

Adjust the pH with glacial acetic acid to pH 5.5.

1 M sodium phosphate, pH 6.5Dissolve 15.6 g sodium phosphate in 100 ml DI-water.Adjust the pH with sodium hydroxide solution to pH

6.5.

Crystal growth experiment

1. From the stock solutions, prepare the 24 reservoirsolutions for the crystallisation experiments accordingto the table on the left. The students can be split intosmall groups, each preparing some of the 24 differentsolutions. All groups can use the same stock solutions.

2. Using the table for reference, pipette 0.5 ml of thecorresponding reservoir solution into each of the 24

reservoir wells of a Cryschem plate (a in figure onpage 33). The table on the left summarises the condi-tions in each well and shows the position of the wellson the plate.

3. Pipette 1l of the reservoir solution into the crystallisa-tion cup on the sitting drop post in each well (b infigure on page 33).

4. Add 1l of lysozyme stock solution to each 1l reser-voir solution drop (b in figure on page 33).

5. Immediately after adding the drops of protein solution,close the crystallisation vessel with crystal clear sealing

tape to prevent evaporation from the vessel (c in fig-ure on page 33).

6. Store the plate at 20 C. The crystals will start to growimmediately in some wells, and growth can beobserved directly under the microscope at 1-2 hourintervals. The plates may be stored until the next lessonfor final analysis. After about 1-2 weeks, crystals willhave grown to their final size. A sealed plate will keepup to a year, sometimes even longer.

7. Analyse the size, number and distribution of lysozymecrystals. The crystals may be too small to be observedwith the naked eye, so a good magnifying glass or

even better a microscope would be very useful.

8. By comparing the results from the 24 reservoirs, deter-mine the optimal conditions for crystallisation.

Have your crystals measured by X-ray

When your class has successfully grown protein crystals,please contact Dr Patrick Sticher at [email protected].

The Swiss NCCR (National Center of Competence inResearch) Structural Biologyw2 has offered to produce an X-

ray diffraction image for the first 10 school classes that suc-cessfully grow protein crystals using this protocol. X-ray

measurements can be made either directly from school

samples, or, if shipment is a problem, by reproducing theoptimised crystallisation conditions found in your class and

measuring those crystals. Together with the diffractionimage, the scientists offer to send additional information on

what they would do next with this information to obtainthe actual structure, and a certificate if required.

Chat with scientists

Students can chat online with the scientists via Skypew4,after performing their own experiments. To make an

appointment, email Patrick Sticher ([email protected]) to

chat with him using the Skype account proteincrystallog-raphy.

Teaching activities



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Download additional teachingmaterial

A set of Powerpoint slides,

images and further experimentsare available onlinew5.

Suppliers

The following suppliersw6 providethe required materials and chemicals:

Hampton Research:

Cryschem 24-1 SBS plate, Cat.No. HR1-002 (We recommendusing this type of plate. One platecosts about US$3.)

Crystal Clear Sealing Tape (5 cm),Cat. No. HR4-511

Gilson Inc:

1 ml and 1 l manual pipettesSigma Aldrich:

Lysozyme, Product #62971 Sodium chloride, Product #71380

Citric acid, Product #27488

Sodium acetate, Product #71190

Sodium phosphate, monobasic,Product #71502

References

Cornujols D (2009) Biologicalcrystals: at the interface betweenphysics, chemistry and biology.

Science in School 11: 70-76.www.scienceinschool.org/2009/issue11/crystallography

Web references

w1 Additional information aboutthe 1962 Nobel laureates in chem-istry and their pioneering work canbe found on the website of theNobel Prize Committee:http://nobelprize.org/nobel_prizes/chemistry/laureates/1962/

w2 The Swiss National Center ofExcellence in Research (NCCR)Structural Biology is a consortiumof scientists dedicated to the eluci-dation of structure-function rela-tionships of membrane proteins andsupra-molecular complexes:www.structuralbiology.uzh.ch

Selected research highlights can befound here:

www.structuralbiology.uzh.ch/research004.asp

w3 New structures of biologicalmacromolecules (proteins andnucleic acids) are deposited in theProtein DataBank (PDB). Thewebsite offers a number of interest-ing teaching resources:

www.pdb.org

Another valuable resource forprotein information is:

www.proteopedia.org

w4 To download and install Skype,

see: www.skype.com

w5 Additional teaching resourcesare available here:www.structuralbiology.uzh.ch/teacher

Login: crystallization,Password: xraybeam2008

This site will be updated regularly.

w6 The websites of suppliers are asfollows:

Hampton Research:www.Hamptonresearch.com

Gilson Inc.: www.gilson.com

Sigma-Aldrich:www.sigmaaldrich.com

Resources

Abad-Zapatero C (2002) Crystals andLife: A Personal Journey. La Jolla, CA,USA: International University Line.ISBN: 978-0972077408

Here are some recommendedprotocols for growing non-proteincrystals with younger students:

www.msm.cam.ac.uk/phase-trans/2002/crystal/a.html

www.waynesthisandthat.com/crystals.htm

http://chemistry.about.com/od/growingcrystals/Growing_Crystals.htm

Beat Blattmann is a chemist incharge of the high-throughputcrystallisation facility at the NCCRStructural Biology. This system allows5000 crystallisation conditions to be

tested per day.Patrick Sticher has a PhD in micro-

biology. He is the scientific officer ofthe NCCR and is responsible foreducation, technology transfer andprogramme coordination.


This article provides a

good introduction to the

study of protein crystals

by X-ray diffraction. As

such, it provides an inter-

esting comprehension

exercise for biology,

chemistry and physics

showing good links

between the three sci-

ences. It can be used to

discuss how to look at the

very small, and why we

need to study things at this

level. The article also pro-

vides good background

reading for teachers who

are not aware of the use ofdiffraction as an analytical

tool.

The practical looks like it

will take a little time to set

up and obtain results, but

the offer of having the

results analysed at a uni-

versity gives it a different

dimension to other practi-

cals.

Mark Robertson, UKREVIEW

Documents

Growing Lysozyme Crystals