I n 1959, Max Perutz and John Kendrew published an article on the three-dimensional structure of whale myoglobin, which is a small protein responsible for the transport of oxygen in whale cells. By investi- gating the protein’s structure, the two scientists wanted to understand the oxygen-carrying mechanism at the molecular level. They grew crystals from this protein and managed to determine its structure by analysing the X-ray diffraction pattern of the crystal. A number of myoglobins from other species had been tested before with little success, until Perutz and Kendrew obtained a useable diffrac- tion pattern with whale myoglobin crystals. This pioneering work was awarded the Nobel Prize in Chemistry in 1962 w1 . Fifty years later, however, it is still a challenge to obtain protein crystals for structural studies. What are proteins? Proteins are the largest group of non-aqueous components in living cells. Almost every biochemical reac- tion requires a specific protein, called an enzyme. Other types of proteins have 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 of small molecules (e.g. ion channels). The variety is immense: more than 20 000 different proteins are known to exist in humans alone. www.scienceinschool.org 30 Science in School Issue 11 : Spring 2009 Beat Blattmann and Patrick Sticher from the University of Zürich, Switzerland, explain the science behind protein crystallography and provide a protocol for growing your own crystals from protein – an essential method used by scientists to determine protein structures. Growing crystals from protein
Transcript
In 1959, Max Perutz and JohnKendrew published an article on
the three-dimensional structure ofwhale myoglobin, which is a smallprotein responsible for the transportof oxygen in whale cells. By investi-gating the protein’s 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 myoglobincrystals. 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 of
non-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 Zürich, Switzerland,explain the science behind protein crystallography and provide a protocolfor growing your own crystals from protein – 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 buildingblocks, 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 andRNA.
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 α-helices and β-sheets (see figurebelow), which are typically stabilisedby 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 theyare 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 togrow. Yet they are essen-tial for structural biologystudies by X-ray analysis
Image courtesy of G
aby Sennhauser, University of Z
ürich
a. Proteins are builtfrom aminoacids, which are 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 elementsare α-helicesand β-sheets
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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 structures‘visible’:
· 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 been determined by X-ray diffraction, wewill concentrate on this method. Tolearn more about the history of crys-tallography and the journey of a
www.scienceinschool.org32 Science in School Issue 11 : Spring 2009
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 the‘metastable zone’, inwhich the best condi-tions are found for thegrowth of large well-ordered crystals. Twocompeting processesdecrease the proteinconcentration in thesupersaturated state: (I) crystallisation, (II) precipitation
Prot
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conc
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Adjustable parameter (such as salt concentration)
Supersolubility curve
Solubility curve
Image courtesy of N
icola Graf
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 its structure is solved, see the article by Dominique Cornuéjols in this issue (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 onthe 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 (seeimage 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
www.scienceinschool.org 33Science in School Issue 11 : Spring 2009
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The vapour diffusionmethod is the mostfrequently used tech-nique to grow proteincrystals.
a. A small amount ofa crystallisation solu-tion is put into a smallreservoir.
b. A drop of proteinsolution and a drop of crystallisation solution 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 μlreservoirsolution
1.0 μlproteinsolution
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Lysozyme crystals in the classroomIn this practical activity, students learn more aboutmodern 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
· 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 solutionsThe 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.
www.scienceinschool.org34 Science in School Issue 11 : Spring 2009
1 2 3 4 5 6
A
B
C
D
pH increases from
3.5 to 6.5
Pipetting scheme for the crystal growth experiment
NaCl end concentration increases from 0.6 to 2.1 M
CL
AS
SR
OO
M A
CT
IVIT
Y 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 sodium citrate(end conc. 0.1 M),pH 6.5
2.0 ml of 3M NaClstock solution (endconc. 0.6 M)
7.0 ml DI-water
3.0 ml of 3M NaClstock solution (endconc. 0.9 M)
6.0 ml DI-water
4.0 ml of 3M NaClstock solution (endconc. 1.2 M)
5.0 ml DI-water
5.0 ml of 3M NaClstock solution (endconc. 1.5 M)
4.0 ml DI-water
6.0 ml of 3M NaClstock solution (endconc. 1.8 M)
3.0 ml DI-water
7.0 ml of 3M NaClstock solution (endconc. 2.1 M)
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 pH6.5.
Crystal growth experiment1. From the stock solutions, prepare the 24 reservoir
solutions 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 the corresponding reservoir solution into each of the 24reservoir 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 1μl of the reservoir solution into the crystallisa-tion cup on the sitting drop post in each well (‘b’ infigure on page 33).
4. Add 1μl of lysozyme stock solution to each 1μl 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 sealingtape 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-rayWhen your class has successfully grown protein crystals,please contact Dr Patrick Sticher at [email protected] 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-raymeasurements can be made either directly from schoolsamples, or, if shipment is a problem, by reproducing theoptimised crystallisation conditions found in your class andmeasuring those crystals. Together with the diffractionimage, the scientists offer to send additional information onwhat they would do next with this information to obtainthe actual structure, and a certificate if required.
Chat with scientistsStudents can chat online with the scientists via Skypew4,after performing their own experiments. To make anappointment, email Patrick Sticher ([email protected]) tochat 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 experiments are available onlinew5.
SuppliersThe following suppliersw6 provide
the 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.)
crystals: at the interface betweenphysics, chemistry and biology.Science in School 11: 70-76.www.scienceinschool.org/2009/issue11/crystallography
Web referencesw1 – Additional information about
the 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). The website offers a number of interest-ing teaching resources:
www.pdb.org
Another valuable resource for protein 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
ResourcesAbad-Zapatero C (2002) Crystals and
Life: A Personal Journey. La Jolla, CA,USA: International University Line.ISBN: 978-0972077408
Here are some recommendedprotocols for growing non-protein crystals with younger students:
Beat Blattmann is a chemist incharge of the high-throughput crystallisation facility at the NCCRStructural Biology. This system allows5000 crystallisation conditions to betested per day.
Patrick Sticher has a PhD in micro-biology. He is the scientific officer ofthe NCCR and is responsible for education, technology transfer andprogramme coordination.
www.scienceinschool.org36 Science in School Issue 11 : Spring 2009
This article provides agood introduction to thestudy of protein crystalsby X-ray diffraction. Assuch, it provides an inter-esting comprehensionexercise for biology,chemistry and physics –showing good linksbetween the three sci-ences. It can be used todiscuss how to look at thevery small, and why weneed to study things at thislevel. The article also pro-vides good backgroundreading for teachers whoare not aware of the use ofdiffraction as an analyticaltool.
The practical looks like itwill take a little time to setup and obtain results, butthe offer of having theresults analysed at a uni-versity gives it a differentdimension to other practi-cals.
