Anintroductiontocirculardichroismspectroscopy

Circulardichroism(CD)isthedifferenceintheabsorptionoflefthandedcircularlypolarised light(LCPL)andrighthandedcircularlypolarisedlight(RCPL)andoccurswhenamolecule containsoneormorechiralchromophores(lightabsorbinggroups). Circulardichroism=A()=A()LCPLA()RCPL,whereisthewavelength Circulardichroism(CD)spectroscopyisaspectroscopictechniquewheretheCDofmoleculesis [Link] moleculesofalltypesandsizes,butitisinthestudyoflargebiologicalmoleculeswhereit [Link] conformationofmacromolecules,particularlyproteinsassecondarystructureissensitivetoits environment,temperatureorpH,circulardichroismcanbeusedtoobservehowsecondary structurechangeswithenvironmentalconditionsoroninteractionwithothermolecules. Structural,kineticandthermodynamicinformationaboutmacromoleculescanbederivedfrom circulardichroismspectroscopy. Measurementscarriedoutinthevisibleandultravioletregionoftheelectromagnetic spectrummonitorelectronictransitions,and,ifthemoleculeunderstudycontainschiral chromophoresthenoneCPLstatewillbeabsorbedtoagreaterextentthantheotherandthe [Link] bepositiveornegative,dependingonwhetherLCPLisabsorbedtoagreaterextentthanR CPL(CDsignalpositive)ortoalesserextent(CDsignalnegative).Anexamplecircular dichroismspectrumofasamplewithmultipleCDpeaksisshownbelow,demonstratinghow CDvariesasafunctionofwavelength,andthataCDspectrummayexhibitbothpositiveand negativepeaks.

Circulardichroismspectraaremeasuredusingacirculardichroismspectrometer,suchasthe Chirascan,[Link]

spectrometersmeasurealternatelytheabsorptionofLandRCPL,usuallyatafrequencyof 50kHz,andthencalculatethecirculardichroismsignal.

1. Understanding Circular Dichroism

Thebasicsofpolarisation Toreallyunderstandcirculardichroism,onemustfirstunderstandthebasicsofpolarisation. [Link] lightstatescanbedescribedasasumoftwolinearlypolarisedstatesatrightanglestoeach other,usuallyreferencedtotheviewerasverticallyandhorizontallypolarisedlight.

Vertically Polarised Light

Horizontally Polarised Light

Ifforinstancewetakehorizontallyandverticallypolarisedlightwavesofequalamplitudethat areinphasewitheachother,theresultantlightwave(blue)islinearlypolarisedat45degrees.

45 Degree Polarised Light

Ifthetwopolarisationstatesareoutofphase,theresultantwaveceasestobelinearly [Link],ifoneofthepolarisedstatesisoutofphasewiththeotherbya quarterwave,theresultantwillbeahelixandisknownascircularlypolarisedlight(CPL).The helicescanbeeitherrighthanded(RCPL)orlefthanded(LCPL)andarenonsuperimposable mirrorimages. Theopticalelementthatconvertsbetweenlinearlypolarisedlightandcircularlypolarisedlight [Link],[Link] [Link] convertlinearlypolarisedlightintocircularlypolarisedlightbyslowingoneofthelinear componentsofthebeamwithrespecttotheothersothattheyareonequarterwaveoutof [Link].

Left Circularly Polarised (LCP) Light

Right Circularly Polarised (RCP) Light

Thedifferenceinabsorbanceoflefthandandrighthandcircularlypolarisedlightisthebasis [Link],or chiral.

2. Chiral Molecules
Theoriginofopticalactivity Inthefirstsectionofthistutorial,westatedthatcirculardichroismisthedifferential absorbanceoflefthandcircularlypolarisedandrighthandcircularlypolarisedlight,andthat [Link]? [Link] [Link] ofenantiomersareidenticalwithtwoexceptions:thewaythattheyinteractwithpolarised lightandthewaythattheyinteractwithotherchiralmolecules. Circularbirefringenceandopticalrotation Chiralmoleculesexhibitcircularbirefringence,whichmeansthatasolutionofachiral substancepresentsananisotropicmediumthroughwhichleftcircularlypolarised(LCPL)and rightcircularlypolarised(RCPL)[Link] bethoughtofastheresultantofthesuperpositionoftwocircularlypolarisedwaves,oneleft circularlypolarised,[Link] medium,thephaserelationshipbetweenthecircularlypolarisedwaveschangesandthe [Link] opticalrotation,[Link] functionofwavelengthistermedopticalrotatorydispersion(ORD)spectroscopy.


Circular birefringence - the orange cuboid represents the sample

Circulardichroism Unlikeopticalrotation,circulardichroismonlyoccursatwavelengthsoflightthatcanbe [Link] [Link],achiralchromophoremayabsorb90%ofR CPLand88%[Link] [Link] dichroism(CD)spectroscopyandistheprimaryspectroscopicpropertymeasuredbyacircular dichroismspectrometersuchastheChirascan.

Circular dichroism - the orange cuboid represents the sample

Opticalrotationandcirculardichroismstemfromthesamequantummechanicalphenomena andonecanbederivedmathematicallyfromtheotherifallspectralinformationisprovided. Therelationshipbetweenopticalrotatorydispersion,circulardichroism,absorptionspectra

andchiralityareshownbelow,withacomparisonofthetwoenantiomersofcamphor sulphonicacid.

CD, ORD and Absorbance spectra of R and S forms of camphor sulphonic acid

AlthoughORDspectraandCDspectracantheoreticallyprovideequivalentinformation,each [Link] isusedasageneralmeasurementtoolforchiralmolecules,todetermineconcentrationandas [Link] [Link] spectraontheotherhandarebetterspectrallyresolvedthanORDspectra,andconsequently moresuitableforadvancedspectralanalysis.

3. Chirality and Biology

Circulardichroismandthestudyofbiologicalmolecules Circulardichroismisaconsequenceoftheinteractionofpolarisedlightwithchiralmolecules. [Link],19ofthe20commonamino acidsthatformproteinsarethemselveschiral,asareahostofotherbiologicallyimportant molecules,togetherwiththehigherstructuresofproteins,[Link] chemistryofbiologicalmoleculeslendsitselfwelltoanalysisbycirculardichroismandthe studyofbiologicalmoleculesisthemainapplicationofthetechnique. Alargesubsetoftheuseofcirculardichroisminbiochemistryisintheunderstandingofthe [Link] isthattheCDspectrumofaproteinorDNAmoleculeisnotasumoftheCDspectraofthe individualresiduesorbases,butisgreatlyinfluencedbythe3dimensionstructureofthe [Link],andthiscanbe usedtoidentifystructuralelementsandtofollowchangesinthestructureofchiral macromolecules. Themostwidelystudiedcirculardichroismsignaturesarethevarioussecondarystructural [Link]

CDspectrainthefarUV(below260nm)canbeusedtopredictthepercentagesofeach [Link] secondarystructuralelementsandtheCDspectraassociatedwiththemareshownbelow:

The secondary structure conformation and the CD spectra of protein structural elements. Right is an example of the backbone conformation of a peptide in an -helix and left is the conformation of a peptide in a -sheet. In the centre are the associated CD spectra for these different conformations.

Therearemanyalgorithmsdesignedforfittingthecirculardichroismspectraofproteinsto [Link] softwaredistributedwiththeChirascanisCDNN. Secondarystructurepredictionisonlypartofthepowerofcirculardichroismspectroscopy. Changesincirculardichroismspectraareverygoodproxiesforchangesinthestructureofa [Link](i)spectracanberecordedinminutesand(ii)single wavelengthkineticscanberecordedfrommillisecondsonwards,CDisaparticularlypowerful [Link] temperature,pH,ligands,ordenaturantsareallcommonlyused. Apowerfulapplicationofcirculardichroismistocomparetwomacromolecules,orthesame moleculeunderdifferentconditions,[Link] beusedsimplytoascertainifanewlypurifiedproteiniscorrectlyfolded,determineifa mutantproteinhasfoldedcorrectlyincomparisontothewildtype,orfortheanalysisof biopharmaceuticalproductstoconfirmthattheyarestillinacorrectlyfoldedactive conformation.

4. CD Spectrometer Operating Principles

Allthedetailsforafullunderstanding Circulardichroism(CD)isthedifferenceinlightabsorbancebetweenleft(LCPL)andright circularlypolarisedlight(RCPL)andcirculardichroismspectrometers(spectrophotometers) arehighlyspecialisedvariationsoftheabsorbancespectrophotometer. Acirculardichroismspectrophotometerisalsocommonlytermedacirculardichroism [Link] operateonthesameprinciples,whichisdemonstratedintheslideshowatthebottomofthe [Link]

eitherleftorrightcircularlypolarisedlightbypassingitthroughaquarterwaveplatewhose uniqueaxisisat45degreestothelinearpolarisationplaneasdescribedinthesectionabout polarisedlight. Insteadofastaticquarterwaveplate,acirculardichroismspectrophotometerhasa specialisedopticalelementcalledaphotoelasticmodulator(PEM).Thisisapiezoelectric [Link],whenthepiezoelectricelementisnot oscillating,thesilicablockisnotbirefringent;whendriven,thepiezoelectricelementoscillates atitsresonancefrequency(typicallyaround50kHz),andinducesstressinthesilicainsucha [Link] dynamicquarterwaveplate,retardingfirstverticalwithrespecttohorizontalcomponentsof theincidentlinearlypolarisedlightbyaquarterwaveandthenviceversa,producingleftand [Link] tunedsothattheretardationisappropriateforthewavelengthoflightpassingthroughthe silicablock. [Link] dichroicsampleinthelightpath,[Link] dichroicsampleinthelightpath,therecordedlightintensitywillbedifferentforrightand [Link],itispossibletomeasure thedifferenceinintensitybetweenthetwocircularpolarisations(vAC).Theaveragetotallight intensityacrossmanyPEMoscillations(vDC)canbeusedtoscalethesizeofthelockin [Link] andfromthemthecirculardichroismsignalcanbecalculatedeasilybydividingthevAC componentbythevDCsignal.

[Link] sectionaboutCDUnitsandtheirinterconversionexplainshowellipticityanddifferential absorbancearerelated.

5. CD Spectrometer Performance
Thedesignanditseffectonoperation Thelimitofdetectionofacirculardichroism(CD)spectrophotometer(orany spectrophotometer)isdeterminedbyitssignaltonoise(S/N)characteristics:thebetterthe S/N,thebetteritslimitofdetection. Thesignaltonoiseratioislimitedbyphotonshotnoise,whichisthestatisticalvariationabout [Link] quantisednatureofphotonsandtheirrandomarrivalatthedetectormeansthatalthoughthe averagenumberofphotonsdetectedpersecondmaybesay5,thenumberinanyparticular onesecondintervalmaybe0,2,[Link],ameasurementmustbe madeoverasufficientlylongperiodoftimetodeterminethetrueaverageandthetimetaken todeterminethetrueaveragewillbeinverselyproportionaltoS/[Link] designacirculardichroismspectrophotometertomaximiseitsS/Ncharacteristics.

Ageneralrelationshipbetweenthecontributingfactorstothesignaltonoiseinanoptical spectrometercanbewrittenas:

whereQ=detectorquantumefficiency,I=lightintensity,t=timescaleofthemeasurement. Fromthisitisapparenttherearethreewaystoimprovethesignaltonoiseofacircular dichroismspectrophotometer:increasetheintensityoftheincidentlinearlypolarised monochromaticlight,increasethequantumefficiencyofthedetector,orspendmoretime collectingandaveragingdatapoints. Thefirsttwofactors,lightintensityanddetectorperformance,arethosethatcanbe influencedbythedesignofacirculardichroismspectrophotometerandworktogetherto lowerthelastfactor,[Link] throughputandbetterthedetectorefficiency,thelesstimeittakestocollectqualitydataor, equally,thehigherthequalityofdatathatcanbecollectedintimelimitedexperimentssuch asstoppedflowmeasurements. Increasingtheintensityoftheincidentlightisthemainavenueforincreasingtheperformance ofcirculardichroismspectrophotometersandthisfindsitsultimateexpressionintheuseof [Link] intensityacrossaverywidespectralrangeofwavelengthsbutaccesstothemisexpensiveand limitedandtheiruseisrestrictedtothemorecuttingedgeapplicationsofcirculardichroism. ForthevastmajorityofCDexperiments,ahighintensitybenchtopsourceistheonlypractical option:AppliedPhotophysicsChirascanhasbeendesignedfromthegrounduptomaximise thelightthroughputfromitsXearclampsourcetothesample. TheChirascanmonochromatorusestwosynthetic,singlecrystalquartzprismsinsteadofthe diffractiongratingsthatmostpeoplearefamiliarwithfromnormalabsorbance [Link] rangeofwavelengths,[Link] disperselightintothecomponentwavelengthsbutalso,becauseoftheirbirefringence, dispersethelinearlypolarisedcomponents,oneofwhichisselectedforconversionto [Link] multiplesofthedesiredwavelength,whichisamajorsourceofstraylightingratingbased monochromators. Unlikethedispersionofagrating,whichislinearandhighlycustomisable,thedispersionofa [Link] andmechanicsofaprismmonochromatorhavetobemorecomplexthanagrating monochromator,withtheneedtoconstantlyvarytheslitwidthasafunctionofwavelengthto maintainaconstantbandpass,andacomplexrelationshipbetweenprismmovementand [Link],thelargewavelengthdispersionintheUVmeansthatwiderslitscanbe usedevenatasmallspectralbandpass,whichmeansgreaterlightcollectionefficiency [Link] intheUVanditisatthesewavelengthsthatthecharacteristicsofaprismareamajor advantage.

Inadditiontothehighintensitytoimprovesignaltonoise,thelightfromthemonochromator musthaveaverylowstraylightcontentandaveryhighpurityoflinearpolarisationtoprovide [Link] Chirascancirculardichroismspectrophotometerandhavebeenachievedbykeydesign featuresoftheChirascanmonochromator. ThesecondinfluenceontheS/[Link] [Link] spectrophotometers,thedetectorofchoiceinthelastfewdecadeshasbeenthe [Link],whicharetraditionallyused inspectroscopyforUVandvisiblelightdetection,hasremainedfairlystaticoverthatperiod. Recently,advancesinphotodiodetechnologyhaveresultedinnew,highgain,largeareasolid statedetectors,whichprovidesignificantimprovementsinquantumefficiencyintheultra violet,visibleandnearinfraredregionswhencomparedwithphotomultipliertubes(see Figure1).Oneofthesenewhighperformancesolidstatedetectorsisfeaturedinthenew ChirascanplusanditgivesfurtherS/Nimprovementsoverthephotomultiplierbased [Link] significantsignaltonoiseimprovementsoverandabovethealreadyhighperformanceofthe standardChirascan.

Figure 1. Quantum efficiency of Chirascan Plus detector vs a standard photomultiplier tube

6. CD Signatures of Structural Elements

Circulardichroismsignaturesofsecondarystructuralelements

7. CD Units & Conversions

Allrelationshipsexplained Circulardichroism(CD)isusuallyunderstoodandactuallymeasuredasthedifferential absorbanceofleft(ALCP)andrightcircularlypolarised(ARCP)light,andsocanbeexpressed as: A=ALCPARCP Takingintoaccountcellpathlengthandcompoundconcentration,wecanarriveatamolar circulardichroism(). =LCPRCP=A/(Cxl) WhereLCPandRCParethemolarextinctioncoefficientsforLCPandRCPlightrespectively, C=molarconcentration,andl=pathlengthincentimeters. [Link] forproteins,andreportsthemolarcirculardichroismforindividualproteinresiduesinsteadof [Link] [Link] informationisknownabouttheprotein. Theconcentrationofprotein(C)inmolarismultipliedbythenumberofaminoacids(N)inthe proteintoprovidethemeanresidueconcentration(CMR): CMR=CxN MR=A/(CMRxl) AnestimatecanbedeterminedforCMRifthesequenceoftheproteinisn'tknown,usingthe averageaminoacidresidueweightof113daltons,andtheconcentrationofprotein(P)ingL1 CMR=P/113 Aandarethemostintuitiveunitsformanybiochemists,astheyarederivedfromthe familiarconceptofUV/Visabsorbancespectroscopy,anditisalsohowmodernCDinstruments [Link](), whichisalegacyofpolarimetry,[Link] contextofmodernCDspectroscopy,[Link] relationshipbetweenAandareexplainedbelow. [Link] [Link] polarisedlightislightthatisnotfullycircularpolarised,[Link] becausethecircularpolarisedcomponentsoftheoriginallinearpolarisedlightarenownotof equalmagnitudesduetodifferentialabsorbance(circulardichroism).Theeffectis demonstratedbelow:

Circular dichroism as ellipticity - the orange cuboid represents the sample

Thedegreeofellipticity()isdefinedasthetangentoftheratiooftheminortomajorelliptical axis,andisillustratedbelow:

Linear polarised light has 0 degrees of ellipticity (), while fully LCP or RCP will have + or - 45 degrees respectively.

Theadvantageofcirculardichroismellipticityasameasurementunitisthatitismoreeasily [Link] aremeasurementsofchangesinpolarisationstateofalinearpolarisedanalyzerbeam,and [Link] comparisonofopticalrotationandcirculardichroismmeasurements,ausefulabilitywhen circulardichroismspectroscopyfirststartedtobewidelyused,backinthe1960's. FortunatelyitisveryeasytointerconvertbetweenandA: A=/32.982 Note:Duetothesmallsizeofmanymeasurements,isoftenquotedasmillidegrees(m)or 1/1000ofadegree. [Link] concentrationandcellpathlengthaccordingtoBeerLambertslaw,wecanderivea measurementofmolarellipticity[].Followingpolarimetricconventions,molarellipticityis reportedindegreescm2dmol1,ordegreesM1m1whichareequivalentunitsasshown below.

Molarellipticitycanbecalculatedusingthefollowingequation: []=100x/(Cxl) Cistheconcentrationinmolar,andlthecellpathlengthincm.Thefactorof100convertsto pathlengthinmeters. MolarCirculardichroismandmolarellipticitycanbeconverteddirectlyby: =[]/3298.2 Thisfactorisahundredfoldlargerthanbetweenrawabsorbanceandellipticityduetothe conversionbetweenmolarextinctiondefiningpathlengthsincentimetersandellipticityhaving pathlengthdefinedinmeters. Anotherimportantunitismeanresidueellipticity[][Link],and reportsthemolarellipticityforindividualproteinresiduesinsteadofwholeproteinmolecules. [Link] aretwowaystocalculatethisdependingonhowmuchinformationisknownaboutthe protein. Theconcentrationorprotein(C)inmolarismultipliedbythenumberofaminoacids(N)inthe proteintoprovidethemeanresidueconcentration(CMR): CMR=CxN []MR=100x/(CMRxl)

AnestimatecanbedeterminedforCMRifthesequenceoftheproteinisn'tknown,usingthe averageaminoacidresidueweightof113daltons,andtheconcentrationofprotein(P)ingL1 CMR=P/113 FortunatelytheProDatasoftwareforChirascancanconverteasilybetweenalltheseunits, withaminimumofuserintervention. WeatAppliedPhotophysicshopethatyouhavefoundthetutorialincirculardichroisma [Link]'rangeofcircular dichroismspectrometers,thenpleasecontactsales@[Link].