CD Spectroscopy

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  • Anintroductiontocirculardichroismspectroscopy

    Circulardichroism(CD)isthedifferenceintheabsorptionoflefthandedcircularlypolarisedlight(LCPL)andrighthandedcircularlypolarisedlight(RCPL)andoccurswhenamoleculecontainsoneormorechiralchromophores(lightabsorbinggroups).

    Circulardichroism=A()=A()LCPLA()RCPL,whereisthewavelength

    Circulardichroism(CD)spectroscopyisaspectroscopictechniquewheretheCDofmoleculesismeasuredoverarangeofwavelengths.CDspectroscopyisusedextensivelytostudychiralmoleculesofalltypesandsizes,butitisinthestudyoflargebiologicalmoleculeswhereitfindsitsmostimportantapplications.Aprimaryuseisinanalysingthesecondarystructureorconformationofmacromolecules,particularlyproteinsassecondarystructureissensitivetoitsenvironment,temperatureorpH,circulardichroismcanbeusedtoobservehowsecondarystructurechangeswithenvironmentalconditionsoroninteractionwithothermolecules.Structural,kineticandthermodynamicinformationaboutmacromoleculescanbederivedfromcirculardichroismspectroscopy.

    Measurementscarriedoutinthevisibleandultravioletregionoftheelectromagneticspectrummonitorelectronictransitions,and,ifthemoleculeunderstudycontainschiralchromophoresthenoneCPLstatewillbeabsorbedtoagreaterextentthantheotherandtheCDsignaloverthecorrespondingwavelengthswillbenonzero.Acirculardichroismsignalcanbepositiveornegative,dependingonwhetherLCPLisabsorbedtoagreaterextentthanRCPL(CDsignalpositive)ortoalesserextent(CDsignalnegative).AnexamplecirculardichroismspectrumofasamplewithmultipleCDpeaksisshownbelow,demonstratinghowCDvariesasafunctionofwavelength,andthataCDspectrummayexhibitbothpositiveandnegativepeaks.

    Circulardichroismspectraaremeasuredusingacirculardichroismspectrometer,suchastheChirascan,whichisahighlyspecialisedderivativeofanordinaryabsorptionspectrometer.CD

  • spectrometersmeasurealternatelytheabsorptionofLandRCPL,usuallyatafrequencyof50kHz,andthencalculatethecirculardichroismsignal.

    1. Understanding Circular Dichroism

    Thebasicsofpolarisation

    Toreallyunderstandcirculardichroism,onemustfirstunderstandthebasicsofpolarisation.Linearlypolarisedlightislightwhoseoscillationsareconfinedtoasingleplane.Allpolarisedlightstatescanbedescribedasasumoftwolinearlypolarisedstatesatrightanglestoeachother,usuallyreferencedtotheviewerasverticallyandhorizontallypolarisedlight.

    Vertically Polarised Light

    Horizontally Polarised Light

    Ifforinstancewetakehorizontallyandverticallypolarisedlightwavesofequalamplitudethatareinphasewitheachother,theresultantlightwave(blue)islinearlypolarisedat45degrees.

  • 45 Degree Polarised Light

    Ifthetwopolarisationstatesareoutofphase,theresultantwaveceasestobelinearlypolarised.Forexample,ifoneofthepolarisedstatesisoutofphasewiththeotherbyaquarterwave,theresultantwillbeahelixandisknownascircularlypolarisedlight(CPL).Thehelicescanbeeitherrighthanded(RCPL)orlefthanded(LCPL)andarenonsuperimposablemirrorimages.

    Theopticalelementthatconvertsbetweenlinearlypolarisedlightandcircularlypolarisedlightistermedaquarterwaveplate.Aquarterwaveplateisbirefringent,i.e.therefractiveindicesseenbyhorizontallyandverticallypolarisedlightaredifferent.Asuitablyorientedplatewillconvertlinearlypolarisedlightintocircularlypolarisedlightbyslowingoneofthelinearcomponentsofthebeamwithrespecttotheothersothattheyareonequarterwaveoutofphase.ThiswillproduceabeamofeitherleftorrightCPL.

    Left Circularly Polarised (LCP) Light

  • Right Circularly Polarised (RCP) Light

    Thedifferenceinabsorbanceoflefthandandrighthandcircularlypolarisedlightisthebasisofcirculardichroism.AmoleculethatabsorbsLCPandRCPdifferentlyisopticallyactive,orchiral.

    2. Chiral Molecules

    Theoriginofopticalactivity

    Inthefirstsectionofthistutorial,westatedthatcirculardichroismisthedifferentialabsorbanceoflefthandcircularlypolarisedandrighthandcircularlypolarisedlight,andthatchiralmoleculesexhibitcirculardichroism.Butwhatarechiralmolecules?

    Chiralmoleculesexistaspairsofmirrorimageisomers.Thesemirrorimageisomersarenotsuperimposableandareknownasenantiomers.Thephysicalandchemicalpropertiesofapairofenantiomersareidenticalwithtwoexceptions:thewaythattheyinteractwithpolarisedlightandthewaythattheyinteractwithotherchiralmolecules.

    Circularbirefringenceandopticalrotation

    Chiralmoleculesexhibitcircularbirefringence,whichmeansthatasolutionofachiralsubstancepresentsananisotropicmediumthroughwhichleftcircularlypolarised(LCPL)andrightcircularlypolarised(RCPL)propagateatdifferentspeeds.Alinearlypolarisedwavecanbethoughtofastheresultantofthesuperpositionoftwocircularlypolarisedwaves,oneleftcircularlypolarised,theotherrightcircularlypolarised.Ontraversingthecircularlybirefringentmedium,thephaserelationshipbetweenthecircularlypolarisedwaveschangesandtheresultantlinearlypolarisedwaverotates.Thisistheoriginofthephenomenonknownasopticalrotation,whichismeasuredusingapolarimeter.Measuringopticalrotationasafunctionofwavelengthistermedopticalrotatorydispersion(ORD)spectroscopy.

  • Circular birefringence - the orange cuboid represents the sample

    Circulardichroism

    Unlikeopticalrotation,circulardichroismonlyoccursatwavelengthsoflightthatcanbeabsorbedbyachiralmolecule.AtthesewavelengthsLeftandrightcircularlypolarisedlightwillbeabsorbedtodifferentextents.Forinstance,achiralchromophoremayabsorb90%ofRCPLand88%ofLCPL.ThiseffectiscalledcirculardichroismandisthedifferenceinabsorptionofLCPLandRCPL.Circulardichroismmeasuredasafunctionofwavelengthistermedcirculardichroism(CD)spectroscopyandistheprimaryspectroscopicpropertymeasuredbyacirculardichroismspectrometersuchastheChirascan.

    Circular dichroism - the orange cuboid represents the sample

    Opticalrotationandcirculardichroismstemfromthesamequantummechanicalphenomenaandonecanbederivedmathematicallyfromtheotherifallspectralinformationisprovided.Therelationshipbetweenopticalrotatorydispersion,circulardichroism,absorptionspectra

  • andchiralityareshownbelow,withacomparisonofthetwoenantiomersofcamphorsulphonicacid.

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

    AlthoughORDspectraandCDspectracantheoreticallyprovideequivalentinformation,eachtechniquehasbeenusedforverydistinctapplications.Opticalrotationatasinglewavelengthisusedasageneralmeasurementtoolforchiralmolecules,todetermineconcentrationandasadeterminantofchiralpuritycomparedtoaknownstandard.Thesimplicityandlowcostoftheexperimentandinstrumentationmakesitidealforthisapplication.CirculardichroismspectraontheotherhandarebetterspectrallyresolvedthanORDspectra,andconsequentlymoresuitableforadvancedspectralanalysis.

    3. Chirality and Biology

    Circulardichroismandthestudyofbiologicalmolecules

    Circulardichroismisaconsequenceoftheinteractionofpolarisedlightwithchiralmolecules.Thevastmajorityofbiologicalmoleculesarechiral.Forinstance,19ofthe20commonaminoacidsthatformproteinsarethemselveschiral,asareahostofotherbiologicallyimportantmolecules,togetherwiththehigherstructuresofproteins,DNAandRNA.Thehighlychiralchemistryofbiologicalmoleculeslendsitselfwelltoanalysisbycirculardichroismandthestudyofbiologicalmoleculesisthemainapplicationofthetechnique.

    AlargesubsetoftheuseofcirculardichroisminbiochemistryisintheunderstandingofthehigherorderstructuresofchiralmacromoleculessuchasproteinsandDNA.ThereasonforthisisthattheCDspectrumofaproteinorDNAmoleculeisnotasumoftheCDspectraoftheindividualresiduesorbases,butisgreatlyinfluencedbythe3dimensionstructureofthemacromoleculeitself.Eachstructurehasaspecificcirculardichroismsignature,andthiscanbeusedtoidentifystructuralelementsandtofollowchangesinthestructureofchiralmacromolecules.

    Themostwidelystudiedcirculardichroismsignaturesarethevarioussecondarystructuralelementsofproteinssuchasthehelixandthesheet.Thisisunderstoodtothepointthat

  • CDspectrainthefarUV(below260nm)canbeusedtopredictthepercentagesofeachsecondarystructuralelementinthestructureofaprotein.SomeofthecommonproteinsecondarystructuralelementsandtheCDspectraassociatedwiththemareshownbelow:

    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.

    Therearemanyalgorithmsdesignedforfittingthecirculardichroismspectraofproteinstoprovideestimatesofsecondarystructure.TheproteinsecondarystructureCDanalysissoftwaredistributedwiththeChirascanisCDNN.

    Secondarystructurepredictionisonlypartofthepowerofcirculardichroismspectroscopy.Changesincirculardichroismspectraareverygoodproxiesforchangesinthestructureofamolecule.Couplethiswiththefactsthat(i)spectracanberecordedinminutesand(ii)singlewavelengthkineticscanberecordedfrommillisecondsonwards,CDisaparticularlypowerfultooltofollowdynamicchangesinproteinstructure.Forinstancechangesinducedbychangingtemperature,pH,ligands,ordenaturantsareallcommonlyused.

    Apowerfulapplicationofcirculardichroismistocomparetwomacromolecules,orthesamemoleculeunderdifferentconditions,anddetermineiftheyhaveasimilarstructure.Thiscanbeusedsimplytoascertainifanewlypurifiedproteiniscorrectlyfolded,determineifamutantproteinhasfoldedcorrectlyincomparisontothewildtype,orfortheanalysisofbiopharmaceuticalproductstoconfirmthattheyarestillinacorrectlyfoldedactiveconformation.

    4. CD Spectrometer Operating Principles

    Allthedetailsforafullunderstanding

    Circulardichroism(CD)isthedifferenceinlightabsorbancebetweenleft(LCPL)andrightcircularlypolarisedlight(RCPL)andcirculardichroismspectrometers(spectrophotometers)arehighlyspecialisedvariationsoftheabsorbancespectrophotometer.

    Acirculardichroismspectrophotometerisalsocommonlytermedacirculardichroismspectropolarimeteroracirculardichrograph.Mostmoderncirculardichroisminstrumentsoperateonthesameprinciples,whichisdemonst