Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

ProteinCrystallisationScreeningByUsing
DynamicLightScatteringUsingEquipmentFrom
MalvernInstruments
SponsoredbyMalvernInstrumentsLtd May132005

TopicsCovered
Background
GrowthofProteinCrystals
TheSecondVirialCoefficient
MeasurementoftheSecondVirialCoefficient
TheSecondVirialCoefficientandOptimalCrystalGrowth
ProblemsAssociatedDeterminationofTheSecondVirialCoefficient
DynamicLightScattering
DynamicLightScatteringandParticleSizeDetermination
DynamicLightScatteringandAggregation
DynamicLightScatteringandCrystallisationScreening
EndoPGIScreening
DynamicLightScatteringandEndoPGIScreening
ZetasizerNanoSystem

Background
Intheearly1990s,dynamiclightscattering(DLS)wasintroducedasascreeningtoolfor
crystallography.OverthepastdecadeDLShasbecomewidelyacceptedinthisrole,and
dynamiclightscatteringinstrumentsarenowroutinelyintegratedintoXraycrystallography
labs.Acommonmisconceptionhowever,isthatDLScantellonewhichsamplesarelikely
toyieldcrystals.Inpractice,DLSismoreappropriatelyusedasatoolforscreeningout
samplesthatareunlikelytoyieldcrystals.Inthisarticle,weaddressthesubtledifference
inthesepoints,andexaminethevalueoflightscatteringtechniquesascrystalscreening
tools.

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 1
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

GrowthofProteinCrystals
Theratedeterminingstepintheresolutionofcrystalstructuresisidentifyingtheoptimum
conditionsforhighqualitycrystalgrowth.Thereareamultitudeofreagentsforgenerating
thesoupsfromwhichhighqualitycrystalsaregrown.However,alltherecipestendtobe
proteinspecific,andwhilemodifyingafavoritemixtosuitanewproteintargetisthe
standardapproach,moreoftenthannot,luckplaysasignificantroleinthegenerationof
highqualityproteincrystals.

TheSecondVirialCoefficient
Thesecondvirialcoefficient(A2)isathermodynamicpropertydescribingtheinteraction
strengthbetweentheproteinandthesolvent.ForproteinsolventsystemswithA2>0,the
proteinlikesthesolventmorethanitselfandtendstobesoluble.WhenA2=0,the
solventisdescribedasathetasolvent,indicatingthatparticlesolventinteractionstrength
isequivalenttoparticleparticleinteractionstrength.WhenA2<<0,theproteinlikesitself
muchmorethanthesolvent,andtendstoaggregateintoamorphousprecipitate.WhenA2
isjustslightlynegativehowever,theconditionsareidealforproteincrystalgrowthat
saturatingconcentrations(Figure1).

Figure1.Crystalgrowthsuccessrateasafunctionofthe2ndvirialcoefficient(A2).

MeasurementoftheSecondVirialCoefficient
The2ndvirialcoefficientistypicallymeasuredusingstaticlightscattering(SLS)
techniques.TheRayleighexpressionusedtodescribethescatteringoflightfromparticles

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 2
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

smallerthanthewavelengthoftheincidentlightisgiveninEquation1,whereKisan
opticalconstant,Cistheparticleconcentration,Mistheweightaveragemolecularweight,
A2isthe2ndvirialcoefficient,andRistheRayleighratioofscatteredtoincidentlight
intensity.

(1)

AsevidentinEquation1,KC/Rshouldbelinearwithconcentration,withanintercept
equalto1/MandaslopethatisproportionaltoA2.

TheSecondVirialCoefficientandOptimalCrystal
Growth
Theuseofthe2ndvirialcoefficientasascreeningparameterforoptimalcrystalgrowth
conditionswasfirstsuggestbyWilsonetal.,whodefinedthecrystallizationwindowas0.8
x104>A2>8.0x104(molmL/g2).Thiscrystallizationwindoworcrystalslotis
highlightedaszoneIIinFigure2,whichshowsthestaticlightscatteringresultsfor
lysozymeatdifferentsaltconcentrationsinaceticacidbuffer(pH4).

Figure2.DebyeplotsforlysozymeatdifferentNaClconcentrationsinaceticacidbufferat
pH4.ThegreyhighlightinginzoneII,definesthecrystallizationslot,where0.8x104>
A2>8.0x104.

ProblemsAssociatedDeterminationofTheSecond
VirialCoefficient
P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 3
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

Whilecrystallographershaveenjoyedsuccessusingthecrystallizationwindowtoestablish
optimalsolutionconditions,thenumberofmeasurements,andhencethetimerequired
(upwardsof2hours),toevaluateA2forasinglesolventcanbeproblematic,particularlyfor
thoseseekingahighthroughputapproachtoproteincrystalscreening.

DynamicLightScattering
DLSisatechniquethatiscomplementarytostaticlightscattering,andcanbeperformed
onatimescalemeasuredinminutesratherthanhours.InDLS,scatteringintensity
fluctuationsaremonitoredacrossstimescalesandthencorrelated.Theintensity
fluctuationsareaconsequenceofparticlemotion,andthemeasuredpropertyinthe
correlationanalysisisthedistributionofdiffusioncoefficients.Thesizeisthencalculated
usingtheStokesEinsteinequation(Eq.2),whereRHisthehydrodynamicradius,kisthe
Boltzmannconstant,Tisthetemperature,isthesolventviscosityandDisthediffusion
coefficient.

(2)

DynamicLightScatteringandParticleSize
Determination
Figure3showsarepresentativeparticlesizedistributiondeterminedusingDLS.Sincethe
scatteringintensityis~proportionaltotheparticlemolecularweight,DLSisverysensitive
toaggregationandlargeimpurities,evenatlowconcentrations.

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 4
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

Figure3.RepresentativeDLSintensityparticlesizedistribution.

Assuch,thetechniqueisideallysuitedfordistinguishingsamplesthatareunlikelytoyield
highqualitycrystals,duetothepresenceofimpuritiesornonspecificaggregates,i.e.
solutionsresidinginzoneIIIofaDebyeplot(Figure2)whereA2<<0.

DynamicLightScatteringandAggregation
Figure4showsacomparisonofthetypesofaggregationobservedatbothhighandlow
concentrationfortypicalproteinsamples,alongwithtypicalsizedistributionsmeasured
usingDLS.TheruleofthumbtargetforcrystalscreeningusingDLSis~20%polydispersity
(Pd),i.e.ifthePd<~20%thesampleisconsideredtobeanidealsinglepopulationwith
noaggregatespresent.IfthePd>20%ontheotherhand,thepresenceofnonspecific
aggregates,variousoligiomericstates,and/orimpuritiesdecreasesthelikelihoodofhigh
qualitycrystalgeneration.

Figure4.Schematicshowingthetypesofaggregationobservedatlowandhigh
concentrationfortypicalproteinsamples.TheA2zonesarethosedetailedintheDebye
plotsshowninFigure2,andthesizedistributionsarerepresentativeofthosemeasuredfor
eachzoneatthediluteconcentrationsusedforstaticanddynamiclightscattering
measurements.Notethat%Pd=(PolydispersityIndex)x100.

DynamicLightScatteringandCrystallisationScreening
AsshowninFigure4,DLScanbeusedtoscreen2outofthe3solutionconditionsthat
areunlikelytoleadtohighqualitycrystalgrowth,i.e.sampleswithlargeimpuritiesand
4 2

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 5
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

sampleswithA2<8.0x104molmL/g2.Ingeneralthough,DLScannotdistinguish
samplesinzoneI,wheretheproteinistoosolubletoproducecrystals,fromthosewithin
zoneII(crystallizationslot),wheretheconditionsareoptimalforhighqualitycrystal
generation.Ontheotherhand,crystalgrowthisinherentlyanaggregationprocess,andas
such,researchersaregenerallyprobingtheboundarybetweenzonesIIandIII,ratherthan
theboundarybetweenzonesIandII.SowhileDLSmaynotbethesilverbullet
researcherswerehopingitwouldbe,itstillfulfilsanimportantroleinitsabilitytoscreen
outsamplesthatareunlikelytoproducehighqualitycrystals.

EndoPGIScreening
Acommonmechanismforthedisruptionofplantcellsbypathogenicmicrobialsisthe
enzymaticdegradationofpectininthecellwallbyEndoPGI,aglycosylatedmicrobial
hydrolase.Inrecentstudies,Katoetal.wereabletoresolvethecrystalstructuresofthree
formsofEndoPGIfromStereumpurpureumEndoPGIa,withtwosugarchains,EndoPG
Ic,withfoursugarchains,andDeEndoPGIa,thedeglycosylatedformofEndoPGIa.The
researcherswereunabletocrystallizeafourthformoftheenzyme,EndoPGIb,withthree
sugarchainsperprotein,andreporteddifficultyinobtainingqualitycrystalsforthe
EndoPGIcformoftheenzyme.

DynamicLightScatteringandEndoPGIScreening
TheprecrystalstudiesfortheEndoPGIprojectincludedDLScharacterizationunderdilute
conditions.TheDLSresultsforthestudyareshowninFigure5,whichindicates
polydispersityvaluesof7.4%,14.9%,and21.2%forthethreeproteinformswherecrystals
wereobtainedand29.1%fortheformthatfailedtoproducecrystals.Theseresultsare
consistentwiththe~20%PdruleofthumbforDLScrystalscreening.

Figure5.DLSresults(Pd=polydispersity)fromtheprecrystalcharacterizationofthefour
formsofEndoPGIandcrystalphotos.obtainedand29.1%fortheformthatfailedto
producecrystals.Theseresultsareconsistentwiththe~20%PdruleofthumbforDLS
crystalscreening.

ZetasizerNanoSystem
TheZetasizerNanosystemfromMalvernInstrumentsisthefirstcommercialinstrumentto

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 6
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

includethehardwareandsoftwareforcombineddynamic,static,andelectrophoreticlight
scatteringmeasurements.Thewiderangeofsamplepropertiesavailableformeasurement
withtheZetasizerNanosysteminclude,particlesize,molecularweight,andzetapotential.

TheZetasizerNanosystemwasspecificallydesignedtomeetthelowconcentrationand
samplevolumerequirementstypicallyassociatedwithpharmaceuticalandbiomolecular
applications,alongwiththehighconcentrationrequirementsforcolloidalapplications.
Satisfyingthisuniquemixofrequirementswasaccomplishedviatheintegrationofa
backscatteropticalsystemandthedesignofanovelcellchamber.Asaconsequenceof
thesefeatures,theZetasizerNanospecificationsforsamplesizeandconcentrationfar
exceedthoseforanyothercommerciallyavailabledynamiclightscatteringinstrument.

Figure6.MalvernInstrumentsZetasizerNanosystem.

Note:Acompletesetofreferencescanbeobtainedbyreferringtothesourcedocument.

Source:DynamicLightScatteringTheSilverBulletForProteinCrystalScreening?,ApplicationNotebyMalvernInstruments.

FormoreinformationonthissourcepleasevisitMalvernInstrumentsLtd(UK)orMalvernInstruments(USA).

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 7
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

MalvernInstrumentsLtd

Address

EnigmaBusinessPark,GrovewoodRd
Malvern
Worcestshire,WR141XZ
UnitedKingdom

Phone:44(1684)892456

Fax:44(1684)892789

Email:portal@malvern.com

VisitWebsite

RelatedProfiles

Nanosight

Malvernprovidesthematerialsandbiophysicalcharacterizationtechnologyandexpertise
thatenablesscientistsandengineerstoinvestigate,understandandcontroltheproperties
ofdispersedsystems.Thesesystemsrangefromproteinsandpolymersinsolution,
particleandnanoparticlesuspensionsandemulsions,throughtospraysandaerosols,
industrialbulkpowdersandhighconcentrationslurries.Usedatallstagesofresearch,
developmentandmanufacturing,Malvernsinstrumentsprovidecriticalinformationthat

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 8
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

helpsaccelerateresearchandproductdevelopment,enhanceandmaintainproduct
qualityandoptimizeprocessefficiency.

OurproductsreflectMalvernsdrivetoexploitthelatesttechnologicalinnovations.They
areusedbybothindustryandacademia,insectorsrangingfrompharmaceuticalsand
biopharmaceuticalstobulkchemicals,cement,plasticsandpolymers,energyandthe
environment.

Malvernsystemsareusedtomeasureparticlesize,particleshape,zetapotential,protein
charge,molecularweight,mass,sizeandconformation,rheologicalpropertiesandfor
chemicalidentification,advancingtheunderstandingofdispersedsystemsacrossmany
differentindustriesandapplications.www.malvern.com

MalvernStore

MaintainyourlaboratoryinstrumentsandprocesssolutionsfromMalvernInstruments.

Email:store.eu@malvern.com

DirectSubsidiaries

Canada
MalvernCanada
4995LevyStreet
Montreal
QuebecH4R2N9
Canada
Tel:+18009320101
Fax:+15087686403
SalesEmail:sales.canada@malvern.com
SupportEmail:support.canada@malvern.com
Website:www.malvern.com

China
MalvernInstrumentsChina


9913101

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 9
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

Unit101,XinAnPlaza
Building13
No99TianzhouRoad
Shanghai200233
China
Tel:
:+864006306902,+868008206902
:+864008206902
:+862161133777
Fax:+862161133778
SalesEmail:info@malvern.com.cn
SupportEmail:Support.China@malvern.com.cn

Denmark
MalvernInstrumentsNordicAB
Korskildelund6
DK2670Greve
Denmark
Tel:+4536915100
Fax:+4536915101
SalesEmail:info@malvern.se
SupportEmail:support.nordic@malvern.com
Website:www.malvern.com

Finland
MalvernInstrumentsNordicOY
Kumitehtaankatu5
Klondyke
FIN04260Kerava
Tel:+358(0)968995231
Fax:+358(0)968995232
SalesEmail:info@malvern.se
SupportEmail:support.nordic@malvern.com

France
MalvernInstrumentsSA
ParcClubdel'Universit
30RueJeanRostand
91893OrsayCedex

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 10
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

France
Tel:+33169351800
Fax:+33160191326
SalesEmail:contact@malvern.com
SupportEmail:support.france@malvern.com

Germany
MalvernInstrumentsGmbH
Rigipsstr.19
71083Herrenberg
Germany
Tel:+49(0)703297770
Fax:+49(0)703277854
SalesEmail:sales@malvern.de
SupportEmail:support.germany@malvern.com

India
MalvernAimilInstrumentsPvtLtd
BSELTechPark
11thfloorSector30A
OppVashiRailwayStation
Vashi
NaviMumbai400705
India
Tel:00912239646600
Fax:00912239646666
SalesEmail:info@malvernaimil.com
SupportEmail:helpdesk@malvern.com

Japan
(MalvernInstrumentsJapan)
6500047
552511
NorthBldg511KobeInternationalBusinessCenter
552MinatojimaminamimachiChuoku,Kobeshi
HyogoPrefecture6500047
Japan
Tel:+81(0)7830638060120571714
Fax:+81(0)783063807

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 11
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

SalesEmail:sales.japan@malvern.com
SupportEmail:support.japan@malvern.com
Website:www.malvern.jp

Korea
MalvernKoreaDivisionofSpectrisKorea
()

166,SH7
MalvernInstrumentsKorea
7FSHEnergyB/D
166SunaeDong
BundangGu,SeongnamCity
KyeonggiDo
Korea
Tel:+82317860840
Fax:+82317860950
SalesEmail:info.korea@malvern.com
SupportEmail:support.korea@malvern.com
Website:www.malvern.co.kr

Mexico
MalvernInstruments
Av.DeLaIndustriaNo300
SanPedroGarzaGarciaC.P.66279
Mexico
Av.DeLaIndustriaNo300
SanPedroGarzaGarciaC.P.66279
Mexico
Tel:+525556846548
018007272283
SalesEmail:ventas@malvern.com
SupportEmail:soporte@malvern.com

Norway
MalvernInstrumentsNordicAB.
Lokkeasveien22A
N3138Skallestad
Norway

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 12
/13
Protein Crystallisation Screening By Using Dynamic Light Scattering Using Equipment From Malvern Instruments

Tel:+4733387328
Fax:+4733387329
SalesEmail:info@malvern.se
SupportEmail:support.nordic@malvern.com

Sweden
MalvernInstrumentsNordicAB
Vallongatan1
75228Uppsala
Sweden
Tel:+46(0)18552455
Fax:+46(0)18551114
SalesEmail:info@malvern.se
SupportEmail:support.nordic@malvern.com

UnitedStatesofAmerica
117FlandersRoad
Westborough
Massachusetts,015811042
UnitedStates
PH:1(508)7686400
Fax:1(508)7686403
Email:info@malvernusa.com

PrimaryActivity
ComponentSupplier

TerritoriesServiced
Global

Services
Malvernareabletosupplyacomprehensiverangeofscientificequipmentforthe
characterisationofparticulatematerials.

P
Saved from URL: http://www.azonano.com/article.aspx?ArticleID=1240 13
/13