Investigation of peptide lipid interaction by fluorescence correlation spectroscopy



INVESTIGATIONOFPEPTIDE‐LIPID
INTERACTIONBYFLUORESCENCE
CORRELATIONSPECTROSCOPY





GUOLIN
(B.Sc.)


ATHESISSUBMITTEDFORTHEDEGREEOF
DOCTOROFPHILOSOPHY

DEPARTMENTOFCHEMISTRY
NATIONALUNIVERSITYOFSINGAPORE

2010
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


I

This work was performed in the Biophysical Fluorescence Laboratory, Department of
Chemistry,NationalUniversityofSingaporeunderthesupervisionofAssociateProfessor
ThorstenWohland.

Theresultshavebeenpartlypublishedin:

Guo,L.,J.Y.Har,J.Sankaran,Y.Hong,B.KannanandT.Wohland(2008)."Molecular
diffusionmeasurementinlipidbilayersoverwideconcentrationranges:a
comparativestudy."Chemphyschem9(5):721‐8.
Kannan,B.,L.Guo,T.Sudhaharan,S.Ahmed,I.MaruyamaandT.Wohland(2007).
"Spatiallyresolvedtotalinternalreflectionfluorescencecorrelationmicroscopy
usinganelectronmultiplyingcharge‐coupleddevicecamera."AnalChem79(12):
4463‐70.
Yu,L.,L.Guo,J.L.Ding,B.Ho,S.S.Feng,J.Popplewell,M.SwannandT.Wohland
(2009)."Interactionofanartificialantimicrobialpeptidewithlipid
membranes."BiochimBiophysActa1788(2):333‐44.
Leptihn,S.,L.Guo,V.Frecer,B.HoandJ.Ding(2010)."Onestepatatime:Action
mechanismofSushi1antimicrobialpeptideandderivedmolecules."Virulence
1(1):42‐44.
Sankaran,J.,M.Manna,L.Guo,R.KrautandT.Wohland(2009)."Diffusion,transport,
andcellmembraneorganizationinvestigatedbyimagingfluorescencecross‐
correlationspectroscopy."BiophysJ97(9):2630‐9.



InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


II

Acknowledgement
Adoctoralthesislikethis,involvingvariousfields,wouldnotbepossiblewithout
the help of many people. I would like to take this opportunity to acknowledge the
personswhoprovidedgreathelpinmystudy.
First, I would like to acknowledge my supervisor Associate Professor Thorsten
Wohland from Department of Chemistry for providing such an interesting research

project.Iamalsogratefulforhisinvaluableguidance,supportandpatiencethroughout
theproject.
I would like to thank Professor Ding Jeak Ling from Department of Biological
Science and Associate Professor Ho Bow from Department of Microbiology for their
scientificsuggestionsanddiscussionsontheproject.
I am also grateful to all mycolleagues from Biophysical Fluorescence Laboratory
fortheirkindhelpandsupport.EspeciallyLanlanYuforhergreatadvicesontheproject
ofantimicrobialpeptides;LingChinHwangandXiaotaoPanfortheirhelpfuldiscussions
onFluorescenceCorrelationSpectroscopy;PingLiu,XiankeShi andSebastianLeptihnfor
theirkindsupportonbiologicalrelevanttopics;KannanBalakrishnan,JiaYiHar,Manna
ManojKumar and JagadishSankaran fortheir great helpon theImaging TotalInternal
ReflectionFluorescenceCorrelationSpectroscopyproject.
And last but not least I would like to thank my parents for their understanding,
supportandloveforalltheseyears.

InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


III

Acknowledgement II
TableofContents III
Summary VII
ListofFigures IX
ListofTables XI

Chapter1 Introduction 1
1.1 IntroductiontoAntimicrobialpeptides 3
1.1.1 Antimicrobialpeptides 5
1.1.1.1 Biologicalactivitiesofantimicrobialpeptides 5

1.1.1.2 Originsofantimicrobial peptides 5
1.1.1.3 Structuralfeaturesofantimicrobialpeptides 8
1.1.1.4 Therapeuticpotentialofantimicrobialpeptides 13
1.1.2 Designedantimicrobialpeptides 13
1.1.2.1 Designedantimicrobialpeptides 15
1.1.2.2 DenovodesignedVpeptidefamily 16
1.1.3 Mechanismofantimicrobialpeptides 18
1.1.3.1 Biologicalmembranes 19
1.1.3.2 Modelmembranes 24
1.1.3.3 Mechanismsofantimicrobialpeptides 26
1.1.3.4 Methodstostudymechanismofantimicrobialpeptides 30
1.2 ConventionalFluorescenceCorrelationSpectroscopy 34
1.2.1 BasicTheory–AutocorrelationFunction 36
1.2.2 BasicSetup‐ConfocalMicroscope 43
1.2.3 CombiningFluorescenceCorrelationSpectroscopywithaLaserScanning
Microscope 44
Chapter2 Investigationofthebindingaffinityofmodifiedantimicrobialpeptideto
membranemimics 46
2.1 Introduction 46
2.2 Materialsandmethods 47
2.2.1 Materials 47
2.2.2 Peptides 47
2.2.3 Smallunilamellarvesicles(SUVs)preparation 48
2.2.4 InteractionofmodifiedV4peptideswithLPS 48
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


IV

2.2.5 InteractionofmodifiedV4peptideswithSUVs 48

2.2.6 FCSInstrumentationandconfocalimaging 49
2.3 ResultsandDiscussion 50
2.3.1 CalibrationoftheFCSsetup 50
2.3.2 ModifiedAMPsaremoresolublecomparedwithV4 51
2.3.3 ModifiedantimicrobialpeptidecanbindtoLPSstrongly 55
2.3.4 ModifiedantimicrobialpeptidecanbindtoPOPGstrongly 59
2.3.5 ModifiedantimicrobialpeptidesshowlowbindingaffinitytoPOPC 61
2.3.6 ComparisonbetweendifferentVpeptides 62
Chapter3 Investigationofthemechanismsofantimicrobialpeptidesinteractingwith
membranemimics 66
3.1 Introduction 66
3.2 Materialsandmethods 68
3.2.1 Materials 68
3.2.2 Peptides 68
3.2.3 Fluorophoreentrappingv esiclepreparation 68
3.2.4 Fluorophorelabeledvesiclepreparation 69
3.2.5 InteractionofMV4swithrhodamine6GentrappedLUVs(REVs)andRho‐PE
labeledLUVs(RLVs) 69
3.2.6 FCSinstrumentationandconfocalimaging 69
3.3 Resultsanddiscussion 70
3.3.1 Modifiedantimicrobialpeptidesinduceleakageofrhodamine6Gentrappedin
POPGLUVs 70
3.3.2 ModifiedantimicrobialpeptidesinteractwithRho‐PElabeledPOPGLUVs 74
3.3.3 ModifiedantimicrobialpeptidesinteractwithRho‐PElabeledPOPCLUVs 78
3.3.4 VisualizationofModifiedpeptidesinteractingwithRho‐PElabeledLUVs 79
3.3.5 ComparisonbetweendifferentVpeptides 79
3.4 Confocalvisualizationofpeptide‐lipidinteraction 82
3.4.1 Materialsandmethods 83
3.4.1.1 Materials 83
3.4.1.2 GUVspreparation 83

3.4.1.3 ImmobilizationofGUVsoncoverslide 83
3.4.1.4 Confocalimaging 84
3.4.2 VisualizationofinteractionbetweenV4andGUVs 84
3.5 Invivomeasurements 87
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


V

3.5.1 Materialsandmethods 88
3.5.1.1 Peptides 88
3.5.1.2 Preparationofbacterialculture 88
3.5.1.3 Bacterialassay 89
3.5.2 MonitoringtheGFPleakagefromGram‐negativebacteria 89
Chapter4 ImagingTotalInternalReflectionFluorescenceCorrelationSpectroscopyasa
tooltomonitorthepeptide‐lipidinteraction 92
4.1 IntroductiontoITIR‐FCS 92
4.1.1 Totalinternalreflection(TIR)illumination 92
4.1.2 Imagingtotalinternalreflectionfluorescencecorrelationspectroscopy 94
4.1.3 Basicsetup 98
4.1.4 Basictheory‐AutocorrelationfunctionforITIR‐FCS 100
4.2 CharacterizationofITIR‐FCS 102
4.2.1 Introductiontodifferentfluorescencetechniques 103
4.2.1.1 Z‐scanFCS 104
4.2.1.2 Fluorescencerecoveryafterphotobleaching 105
4.2.1.3 Singleparticletracking 107
4.2.2 MaterialsandMethods 109
4.2.2.1 Lipidsanddyes 109
4.2.2.2 Peptides 109
4.2.2.3 PreparationofSLB 109

4.2.2.4 PreparationofGUVs 110
4.2.2.5 ImmobilizationofGUVs 110
4.2.2.6 FCSinstrumentationandmeasurement 110
4.2.2.7 FRAPinstrumentationandmeasurement 111
4.2.2.8 SPTandITIR‐FCSInstrumentation 111
4.2.2.9 SPTmeasurement 111
4.2.2.10 ITIR‐FCSmeasurement 112
4.2.3 ResultsandDiscussion 112
4.2.3.1 Results 112
4.2.3.2 Comparisonofdifferenttechniques 116
4.2.3.3 FeaturesofITIR‐FCS 121
4.3 UtilizingITIR‐FCStoinvestigatethebehaviorofanti microbialpeptidesonlipid
membrane 122
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


VI

4.3.1 Introduction 122
4.3.2 MaterialsandMethods 122
4.3.3 ResultsandDiscussion 123
Chapter5 ConclusionsandOutlook 127
5.1 Conclusion 127
5.2 Outlook 131
Reference 134



InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy



VII

Summary
In this work I investigate the action of antimicrobial  peptides(AMPs) with single
moleculesensitivefluorescencespectroscopymethods.
AMPsarenovelandpromisingcandidatesofantibiotics.AMPskillthepathogenby
permeabilizingthe bacterialmembrane. Soit is veryhard forbacteria to developdrug
resistance. De novo designed AMPs can greatly enlarge  the pool of available peptide
candidates,eliminatingsomeofthecytotoxicfeaturesofthenaturalones.Asadenovo
designedpeptide,V4originatedfromaLPS(lipopolysaccharide)‐bindingmotif,showed
itsgoodcombinationofstrongantimicrobialeffectandlowcytotoxic/hemolyticeffect.
However,itsapplicationislimitedduetoitslowsolubility.Toovercomethislimitation,a
seriesofmodifiedV4(MV4s)wasdesignedtohavebettersolubility.
Inthisstudy,theinteractionbetweenMV4sanddifferentlipidmodelmembranes
was investigated using single molecule sensitive fluorescence spectroscopy methods,
suchasfluorescencecorrelationspectroscopy(FCS)andimagingtotalinternalreflection
fluorescence correlation spectroscopy (ITIR‐FCS), togetherwith laser scanning  confocal
imaging. A similar mechanism of MV4s compared to V4 was observed: inducing lipid
aggregation before inducing the lipid membranes disruption. By comparing different
MV4s, we found that a) highly positively charged structure maintained preferential
bindingtonegativelychargedlipid,b)higherhydrophobicitygave risetoahigheractivity
againstbothnegativelychargedandzwitterioniclipid,andc)twobindingmotifsinMV4s
mayplay acrucialrole tomaintaintheiractivity.Agoodconsistencywasfoundbetween
predicted and actual property of peptides. Further study of AMPs on live E. coli
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


VIII


suggestedthatpeptideswithmediumhydrophobicityshowedthehighestantimicrobial 
activity.
ByinvestigatingdifferentmembersoftheV4peptidefamily,thisstudycontributes
toourunderstandingoftheirmechanismofantimicrobialactivityandselectivity.Itthus
providesfurtherguidelinesfortherationaldesignofantimicrobialpeptides.

InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


IX

ListofFigures
Fig1.1SchematicrepresentationoftheGram‐negativebacteriacellwall. 20
Fig1.2SchematicrepresentationofcellwallfromGram‐positivebacteria. 20
Fig1.3StructureoflipidA. 21
Fig1.4Schematicdrawingofdifferentmodelmembrane. 26
Fig1.5Thebarrel‐stavemodel. 27
Fig1.6Thecarpetmodel. 28
Fig1.7Thetoroidalmodel. 29
Fig1.8PrincipleofFCS. 37
Fig1.9PrincipleofACFcurves. 41
Fig1.10SchematicdrawingofatypicalFCSsetup 43
Fig2.1Principleofaffinitymeasurement. 47
Fig2.2ComparisonbetweenTV4‐TMRandTMR. 52
Fig2.3ComparisonbetweenV4norv‐TMR,V4abu‐TMR,V4ala‐TMRandTMR. 52
Fig2.4ACFandintensitytraceobtainedforV4norv‐TMR. 53
Fig2.5ConfocalimageofV4norv‐TMR. 55
Fig2.6ACFobtainedforV4ala‐TMR. 55
Fig2.7ACFcurvesobtainedfortitratingLPSintodifferentpeptides . 57
Fig2.8InteractionbetweenLPSanddifferentpeptides. 57

Fig2.9LPSdissolvedthepeptideaggregates. 58
Fig2.10V4norv‐TMR,V4abu‐TMR,V4ala‐TMRinteractingwithPOPGSUVs. 60
Fig2.11TV4showedalmostnoaffinitytoPOPGSUVs. 61
Fig2.12bindingaffinityofdifferentMV4‐TMRtoPOPGSUVs. 61
Fig2.13MV4sshowedalmostnoaffinitytoPOPCSUVs. 63
Fig3.1Principleofleakagemeasurement. 67
Fig3.2Principleofdisruptionmeasurement. 67
Fig3.3ComparisonbetweenPOPGREVandfreeR6G. 70
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


X

Fig3.4InteractionbetweendifferentpeptidesandPOPGREVs. 73
Fig3.5DifferentpeptidesshoweddifferentactivityagainstPOPGREVs. 74
Fig3.6InteractionbetweendifferentpeptidesandPOPGRLVs. 75
Fig3.7ACFandintensitytraceforPOPGRLVaggregatesandfragments. 76
Fig3.8DifferentpeptidesshoweddifferentactivityagainstPOPGRLVs. 77
Fig3.9InteractionofMV4swithPOPCRLVs. 78
Fig3.10ConfocalimagesofRLVsinteractingwithMV4. 82
Fig3.11ActivityagainstdifferentlipidsforMV4s. 83
Fig3.12V4evenlyandreversiblyboundonPOPCGUVs. 86
Fig3.13V4rapidlydisruptedPOPGGUVs. 87
Fig3.14PeptidesactivityagainstE.coli. 90
Fig4.1Totalinternalreflection. 93
Fig4.2Schematicdrawingofaprism‐basedTIR‐FCSsetup. 96
Fig4.3Schematicdrawingofanobjective‐basedTIR‐FCSsetup. 97
Fig4.4SchematicdrawingofITRF‐FCSsetupusedinthestudy. 99
Fig4.5Schematicrepresentationonz‐scanFCS. 104
Fig4.6SchematicillustrationofFRAPmeasurement. 106

Fig4.7SchematicillustrationonthecalculationofMSD. 108
Fig4.8Dataobtainedusingdifferentfluorescencetechniques. 114
Fig4.9HistogramofdiffusioncoefficientobtainedbySPT. 115
Fig4.10Dependenceofthelateraldiffusiontimeonthez‐positionofthefocus. 115
Fig4.11Comparisonofthediffusioncoefficientsobtainedwithdifferenttechniques.  119
Fig4.12AnexampleofACFcurvesobtainedusingITIR‐FCSsetup. 124
Fig4.13OnesetofITIR‐FCSresult. 125
Fig4.14AnothersetofITIR‐FCSdatashowingdifferentresult. 125
Fig4.15TIRFimageofunevendistributionofV4. 126

InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


XI

ListofTables
Table1.1Clinicaldevelopmentsofcationicantimicrobialpeptides. 14
Table1.2PredictedmolecularpropertiesofmodifiedV4. 18
Table2.1ComparisonbetweendifferentMV4‐TMRandTMR. 53
Table2.2DifferentMV4‐TMRuponsaturationbindingwithLPS. 59
Table2.3ComparisonofdifferentMV4‐TMRinteractingwithPOPGSUVs. 60
Table2.4MV4sshowedalmostnoaffinitytoPOPCSUVs. 62
Table4.1ITIR‐FCSresultsobtainedusingdifferentfittingmodelatvariousbinning. 116
Table4.2Comparisonoftheworkingconcentrationandareaof4differenttechniques. 117
Table4.3ComparisonofFCS,FRAP,SPTandITIR‐FCSresults. 120




InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy



1

Chapter1 Introduction
Due to drug resistance developed by bacteria, treatment of bacterial infections
using conventional  antibiotics is facing a serious challenge. Antimicrobial peptides
(AMPs) are considered to be promising candidates for solving the problem of drug
resistance. By directly targeting the membrane of the bacteria rather than proteins
crucial for bacteria survival, it is much harder for bacteria to develop drug resistance
sinceachangeinthemembranecompositionwouldalsorequirecorrespondingchanges
in many  membrane related proteins. However, due to their lack of selectivity, the
pharmaceuticalapplicationofnatural encodedAMPsislimited.Alternatively,designed
AMPs are able to overcome this problem. Nowadays, different mutations of natural
AMPscanbeeasilysynthesizedandtestedagainstdifferentbacterialstrainsinorderto
find AMPs with better selectivity. De novo designing of AMPs using computational
simulationfurtherenlargethepoolofavailablepeptidecandidates.
In a previous study (Freceret al. 2004) a series of de novo designed AMPs was
proposed.TheseAMPshaveacommonmotifofHBHPHBH(H:hydrophobic;B:basic;P:
polar)derived froma LA‐ (lipid A) or LPS‐ (lipopolysaccharide) binding pattern.Among
the7designedpeptides,V4hasthebestcombinationofhighantimicrobialactivity,low
cytotoxicandlowhemolyticactivity.Howeveritsapplicationislimitedduetoitsstrong
hydrophobicityasshowninpreviousstudy(Yuetal.2005).Toovercomethislimitation,
modificationsofV4peptideswereproposedinthecurrentstudy.Fluorescenceimaging
and spectroscopy were used in this study to investigate how peptides interact with
differentmembranesystem.Morespecifically,theaimsare:
 To investigate how different hydrophobicity would affect the solubility of the
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy



2

peptides.
 To study the binding affinity of modified V4 to LPS on the outer membrane of
Gram‐negative bacteria, and different phospholipids liposomes which mimic
different cytoplasmic membranes to elucidate the membrane activity and
specificityofdifferentpeptides.
 To compare the ability to induce  membrane permeabilization for different
peptides.
 To extend previous in vitro work to in vivo (measurement on Gram‐negative
bacteria) from which morebiological relevant results on activity of the peptides
willbeprovided.
 To elucidate the possible mechanisms of interaction between AMP and
membranes.
 Toapplynewtechniques(imagingtotalinternalreflectionfluorescencecorrelation
spectroscopy)tostudythepeptide‐lipidinteraction.
The study is aimed to enhance the understanding of the specificity of the V
peptide family and their mode of action on membranes. It may also provide more
evidence for the hypothetic mechanism of interaction between V peptides and lipid
membranes.Moreover,theresultscouldcontributetotherationaleofdesigningnovel
antimicrobialdrugsandprovideusefulinformationconcerningnewantimicrobialdrugs.
Information on the mechanism of the interaction between AMPs and lipid
membranes can be provided by fluorescence correlation spectroscopy (FCS), which is
the main technique used in this study. However FCS cannot distinguish between
insertion and adsorption when peptides interact with lipid membranes. Moreover
investigation could be performedon different typesand strains of Gram‐negative and
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


3


Gram‐positive bacteria, providing a much more comprehensive idea on the effect of
AMPsondifferentbacteria.
Inthisthesisfluorescenceimagingandspectroscopyareprovedtobeusefultools
to study the interactions between AMPs and membranes and related mechanism.
Differentmembranemodelsareappliedinthisstudy,includingmicelles,smallandlarge
unilamellar vesicles (liposomes). Micelles and small unilamellar vesicles are used for
investigating the binding affinity of peptides (chapter 2) and large unilamellar vesicles
areusedtostudymembranepermeation(chapter3).Detailsinthemethodologywillbe
presented in each related chapter. In chapter 4, a recently developed method called
imagingtotalinternalreflectionfluorescencecorrelationspectroscopy(ITIR‐FCS) willbe
characterized and applied to peptide‐lipid interaction study. A final conclusion of this
studywillbepresentedinchapter5includinganoutlookonfuturework.
In chapter 1.1, previous studies on AMPs will  first be reviewed, including the
general introduction of AMPs, de novo designed AMPs and proposed mechanisms of
peptide‐lipidinteraction.Afterthat,FCSasthemaintechniqueusedinthisstudywillbe
discussedindetail.

1.1 IntroductiontoAntimicrobialpeptides
Sincetheearly1900s,thewideusageofantibioticsalmostwipedoutalldiseases
caused by bacterial infection (Breithaupt 1999). The discovery of antibiotics has
drasticallyincreasedhumanlifeexpectancy.Butduetoindiscriminateuseofantibiotics,
bacteria develop multiple resistances to the currently available antibiotics (Breithaupt
1999; Lee 2008). The fact that bacteria can exchange plasmids, hence spread drug
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


4

resistance further exacerbates the situation (Hughes et al. 1983). On the other hand,

onlyafewclassesofantibioticshavebeenapprovedforclinicaluseinthelastfewyears,
including daptomycin, tigecycline and linezolid (Lee 2008). In some cases due to the
multi‐drugresistantpathogen,thetreatmentappearstogobacktothesocalled“pre‐
antibioticera”(Breithaupt1999;Lee2008).
Inthelastdecade,AMPshavebecomepromisingcandidatesfornovelantibiotics
(Breithaupt1999; McPheeetal.2005).AMPsaresmall,withupto50aminoacids,and
usually positively charged (due toarginine, lysine, or histidine in acidic condition) and
amphiphathic (contains >50% hydrophobic aminoacids) (Reddy etal. 2004; Gordon et
al. 2005). More information on AMPs can be found in the following section (1.1.1)
includingorigins,structuralfeaturesandtherapeuticpotentialofAMPs.
Normal antibiotics, which are usually bacteriostatic (prevent bacterial growth),
workbyinhibitingthesynthesisofbacterialcellwallorproteinswhichareessentialfor
cellgrowth.Ontheotherhand,AMPsbeingbactericidalkillbacteriadirectly.However
due to their lack of selectivity they are limited in pharmaceutical use, even though
designedAMPscanpossiblyovercomethislimitation(Freceretal.2004)(referto1.1.2).
AMPs kill bacteria (Andreu et al. 1998; Sitaram et al. 1999) by permeablizing the
membrane, so it is unlikely forbacteria todevelop drug resistancesince they have to
adapt themselves to the new drug by evolving newmembranes together with related
proteins. More recently, researchers also found that AMPs are also involved in the
immunomodulation acting as cytokines to modulate the adaptive immune response
(Brogden 2005). But in general, the mechanism of AMPs is still unclear, and further
investigationisneeded.

InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


5

1.1.1 Antimicrobialpeptides 
1.1.1.1 Biologicalactivitiesofantimicrobialpeptides

AMPsareancientdefensemoleculeswhichhave evolved overatleast2.6billion
years. Nowadays AMPs still remain effective as defensive weapons. The existence of
AMPs throughout the evolution disconfirm the idea that bacteria are able to develop
resistancetoanyfeasibledrugs(Zasloff2002).AMPshaveaverybroadspectrumagainst
different microbes, including Gram‐negative bacteria, Gram‐positive bacteria, viruses,
fungi and cancercells (Reddy et al. 2004), withvarious mode ofaction (Zasloff  2002).
AMPs can be found in different organism and species, including insects, amphibians,
mammals and plants, where they act as the first component to defend hosts from
pathogeninvasion.Inhighervertebrates,thisiscomplementedbytheresponseofthe
adaptive immune system which usually acts several days after bacterial infection
(Hancocketal.1998;Breithaupt1999).
1.1.1.2 Originsofantimicrobialpeptides
AMPs are present in a wide range of organisms.Till now, almost 1500 different
AMPs have been identified or predicted from nucleic acid sequences
( />).Thissectionwill brieflyreviewaselectiverangeof
peptidesoriginating frommammals, amphibians, insects,  crustaceans, plants,bacteria,
andviruses.
PeptidesfromMammals
ThemoststudiedmammalianAMPsaredefensins(Hancocketal.1999;Ganz2003;
Oppenheim et al. 2003). Defensins are divided into two main subfamilies, namely α‐
defensinsand β‐defensins. Inmammals, α‐defensinsare mainlypresent in neutrophils
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


6

and paneth cells,and the β‐defensinsare expressed in epithelial cells and leukocytes.
Both α‐ and β‐defensins have high arginine content.6 cysteine residues form
intramolecular disulfide bridges resulting in a triple ‐stranded β‐sheet structure
connected by a loop with a β‐hairpin hydrophobic finger. But the length of peptide

segmentsbetweencysteinesandpairingofthecysteinesaredifferentinthetwogroups.
Apart from α‐ and β‐defensins, another subgroup of defensins withdistinct structure
calledθ‐defensinhasbeenidentified(Tangetal.1999).Byanunknownprocess,cyclicθ‐
defensin is generated by splicing and cyclization of an α‐defensin‐like precursor.
Peptidesoriginatingfromhuman,suchascathelicidins,histatinsandprotegrins(Reddy
etal.2004;DeSmetetal.2005)werealsofound.Peptidesfromthefamilyofhistatins
aresmall,cationic,histidine‐richpeptidesisolatedfromhumansaliva.Cathelicidin(LL‐37)
isderivedproteolyticallyfromtheC‐terminalendofthehumanCAP18protein.Histatins 
andcathelicidinsformanα‐helicalstructureinahydrophobicenvironment.
Peptidesfromamphibians
FromthefirstdiscoveredAMPbombinin(Kissetal.1962;Csordasetal.1970),a
largenumberofAMPshavebeenidentifiedfromamphibians,includingmagainins.One
characteristicofamphibianpeptidesisthattheytendtolacksequencesimilarity(Kreil
1994).ThereisnohomologybetweenAMPsfromonespeciestoanother.However,all
amphibian peptides are predicted to form cationic amphipathic α‐helices (magainins,
dermaseptins,andbuforinII),orcysteine‐disulfideloops(ranalexinandbrevinins).
PeptidesfromInsects
SincethefirstcharacterizationofanAMPinmoth(Steineretal.1981),morethan
170peptideshavebeenidentifiedfromdifferentinsects(Buletetal.1999).InsectAMPs
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


7

are divided into two groups basedon their sources,namelypeptides expressed inside
thebody(cecropinfrommothhaemolymph)(Hultmarketal.1980)oroutsidethebody
(melittin from bee venoms) (Habermann 1972). Different insects generate a series of
AMPs when theysuffer an injury. Drosophila (drosophila melanogaster), for example,
contains 7 different AMPs in its hemolymph, namely drosomycin, cecropin, drosocin,
metchnikowin, defensin, diptericin and attacin (Hergannan et al. 1997). 15 peptides,

named ponericins, were isolated from the venom of a certain subfamily of ant called
Pachycondyla goeldii (Orivel et al. 2001), which show similarity with peptides such as
cecropins, melittins and dermaseptins. One interesting finding is that Drosophila can
differentially express several AMPs in response to various classes of microorganisms,
andalsoshowadaptedresponseto entomopathogenicfungibyproducingonlypepti des
withantifungalactivities(Lemaitreetal.1997).Morerecently(Leeetal.1989;Boman
1995),thebroaderdistributionofcecropinwasrevealedbythediscoveryofmammalian
cecropininporcinesmallintestine.
Peptidefromothersources
AMPs have also been isolated from other sources, such as crustaceans, plants,
bacteria, and viruses. Tachyplesin, polyphemusin, big defensin and tachycitin were
isolatedfromdifferentspeciesofhorseshoecrabs(Nakamuraetal.1988;Miyataetal.
1989;Saitoetal.1995;Kawabataetal.1996).Androctonin(Ehret‐Sabatieretal.1996)
andpenaeidin(Destoumieuxetal.2000)wereisolatedfromcrustaceansshrimp,oreven
fromscorpion.Thoininfromanumberofplantspecies(Floracketal.1994),bacteriocins
(Hancock et al. 1999) from Gram‐positive and Gram‐negative bacteria and LLPs from
virus(Tenczaetal.1997)areidentified.
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


8

Other promising sources of AMPs are synthetic peptides. A series of AMPs have
beensynthesizedbymodifyingthesequenceoftheirnaturalanalogues,oraccordingto
apredictionofamphipathicstructure.The ultimategoalsofinvestigationsonsynthetic
AMPsaretoproduceAMPswithhigherantimicrobialactivitiesandtogainmoreinsight
intothemechanismofAMPsinteractingwithlivingcells.

1.1.1.3 Structuralfeaturesofantimicrobialpeptides
WithinAMPsfromthesamethespeciesorhighertaxonomiclevels,theconserved

sequences are considered to regulate the translation, secretion and trafficking of the
peptides(Zasloff2002).However,diversityinthesequencesof AMPs ishighsuchthat
thesamepeptidesequenceisrarelyconservedintwodifferentspecies,evenwhenthey
are closely related.This diversity of AMP sequences indicatesadaptation of different
species to the environment. The biological activity of individual peptides can be
dramatically changed by a single mutation in its sequence. Thus the species could
surviveby emergence of beneficialmutations from differentindividuals (Zasloff 2002).
Due to the great diversity in AMPs, we can only categorize them based on their
structural features. Different reviews have provided various classifications based on
different criteria (Boman 1995; Blondelle et al. 1999; Epand et al. 1999; Reddy et al.
2004; Brogden 2005; McPhee et al. 2005). In this work, AMPs will be categorized
accordingtotheirsecondarystructure.
α
‐helicalantimicrobialpeptides
Cationic linear α‐helical AMPs may be the most widely spread and best
characterizedpeptides,includingmelittin,magainin,cecropin,cathelicidin(Blondelleet
al.1999;McPheeetal.2005)aswellasanumberofdenovodesignedAMPs(Datheet
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


9

al. 1996; Dathe et al. 1997; Wieprecht et al. 1997; Dathe et al. 2002). In aqueous
solution, many of these peptides exist in a disordered structure. However upon
interaction with hydrophobic solvents or surfaces such as trifluoroethanol, sodium
dodecyl sulphate (SDS) micelles or phospholipid vesicles, they fold into an α‐helical
conformation. α‐helical peptides are often found to be amphipathic, and thus can
adsorb onto bacterial membrane surfaces and insert into the lipid membranes as a
cluster of helical bundles. However, there are also α‐helical peptides that are
hydrophobic(gramicidinA)orevenslightlyanionic(alamethicin)(Epandetal.1999).In

general,anamphipathichelicalstructureisconsideredtobeimportantforantimicrobial
andcytotoxicactivity.Forexamplemagaininlosesitshelicalstructureandantimicrobial
activity when only few amino acids were replaced with their D‐isomers (Chen et al.
1988),eventhoughexceptionstothisdoexist.ByaddingsomeD‐aminoacidresidues,
α‐helical pardaxin (Oren et al. 1999) was converted to β‐structure, losing hemolytic
activity but keeping antimicrobial activity. Thus the structure‐activity relation is still
underdebate.
β
‐sheetantimicrobialpeptides
Thesepeptidesconstitutealargefamilyofcyclicpeptidesinthepresenceoftwo
ormoreβ‐strandsstabilizedbyoneormoreintramoleculardisulfidebonds.Inaqueous
solutiontheymainly existas β‐sheets,whicharefurtherstabilizeduponinteractionwith
lipid surfaces(Blondelle et al. 1999). Among all β‐sheet AMPs, defensins are the best‐
characterized subgroup. Both α‐defensins and β‐defensins contain 3 β‐strands and 6
cysteineswhichform3disulfidebonds,namelybetweenC1‐C6(disulfidebondbetween
1
st
cysteineand6
th
cysteine),C2‐C4,C3‐C5forα‐defensinsandC1‐C5,C2‐C4,C3‐C6for
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


10

β‐defensins(McPheeetal.2005).Anotherfamilycalledθ‐defensinsdiscoveredin1999
iscyclic(Tangetal.1999).
β
‐hairpinantimicrobialpeptides
β‐hairpin AMPs form a hairpin like structure composed of antiparallel β‐strands

interconnectedbyβ‐turns(Reddyetal.2004).Similartoβ‐sheetAMPs,thestructureof
β‐hairpinAMPsisalsostabilizedby1‐2disulfide bondsbetweenβ‐strands.Tachyplesins
and polyphemusins isolated from horseshoe crabs show a typical β‐hairpin structure
stabilizedbytwodisulfidebonds(Laederachetal.2002;Powersetal.2004).Theyshow
antimicrobial activity against both Gram‐negative and Gram‐positive bacteria, and
antifungal activity (Miyata et al. 1989; Tam et al. 2002). Protegrins from porcine
neutropilsalsoshowasimilarstructure(Harwigetal.1995).Thanatinsfrominsectsand
lactoferricinsderivedfromlactoferrin adopta β‐hairpinstructure stabilizedby a single
disulfidebond(Hwangetal.1998;Mandardetal.1998).
Cyclicantimicrobialpeptides
Asubgroupofthesepeptidesmayrelatetoβ‐hairpinAMPs,e.g.bactenecinsfrom
cattleneutrophilsarecyclicpeptidescontainingonedisulphidebondandaβ‐turn.There
are also peptides whose backbones are covalently cyclized, such as gramicidin S and
polymyxinB(Hancocketal.1999).
Extendedantimicrobialpeptides
ExtendedAMPsareaclassofpeptideslackingatypicalsecondarystructure.They
areusuallyrichinoneormorespecificaminoacids(McPheeetal.2005).Tritrpticinsand
indolicidins are examplesof this class. Theyare rich in tryptophan residues, namely 3
and5tryptophanresiduesintheirtotal13aminoacids.Theyformaboat‐likestructure 
whenbindingtodiphosphatidylcholine(DPC)(Schiblietal.1999;Rozeketal.2000).The
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


11

tryptophan‐rich region in the middle of the peptides interacts with one layer of the
membrane, and orients the two termini toward the aqueous environment. Other
peptides, such as histatin isolated from human saliva is rich in histidine residues (18‐
29%) and highly cationic (De Smet et al. 2005). Bac‐5 and Bac‐7 identified in bovine
neutropilsare rich in proline. PR‐39foundin porcineneutrophils is rich in proline and

arginine(Agerberthetal.1991).Peptidesrichinglycine canbefoundinamphibiansand
insects (Otvos 2000; Orivel et al. 2001). Glycine‐rich peptides may show structural
similaritytopeptidessuchasmelittin,cecropinorattacin.
Although AMPs show large variation in their structures, they do share some
commonfeatures(Brogden2005).
(1)TheusualsizeofAMPsissmall,andrangesfrom6to59aminoacids.
(2) Most natural AMPs arepositively charged. Theyare cationic peptides rich in
arginineandlysine.Thenetchargeusuallyrangesfrom+2to+9andvarieswithpH.The
positive charge facilitates the selective binding of peptides to negatively charged
membranesofbothGram‐positiveandGram‐negativebacteria.Anionicpeptidesrichin
asparticandglutamicacidsalsoexist.However,alocalcationicpartisneededtointeract
withnegatively chargedlipids. Subtilosin A is an anionic peptide, though itslysine‐rich
part facilitates its binding to the lipid membrane  (Thennarasu et al. 2005). Anionic
peptides complexed with zinc or highly cationic peptides are often more active than
neutral peptides or those witha lower charge. However there is no direct correlation
between the number and position of positive charged amino acids and antimicrobial
activity and specificity. Highly cationic peptides may have lower or even lose
antimicrobialactivity(Datheetal.1999).Theoptimalchargewasfoundtobebetween
+4and+6(Tossietal.2000).
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


12

(3)Studieshave shownthatamphipathicityofAMPsis crucialregardlessoftheir
secondarystructure (Blondelle et al. 1992;  Dathe etal. 1999; Kondejewski et al.1999;
Parketal.2005).InanAMP,hydrophilicaminoacidresiduesarelocatedononesideof
thepeptidemoleculeandhydrophobicaminoacidsarelocatedontheoppositeside.For
α‐helicalpeptides,amphipathicityisoftendefinedasthevectorsumofhydrophobicity
indices, which indicates the spatial separation between hydrophilic and hydrophobic

side chains. Usually, increasing amphipathicity can lead to an increasing hemolytic
activity and decreasing antimicrobial activity (Dathe et al. 1999; Kondejewski et al.
1999).
(4) The hydrophobic faces of the molecule enable soluble peptides in aqueous
solutiontopartitionintohydrophobiclipidbilayers.Thushydrophobicityisexpectedto
strongly regulate membrane activity of AMPs. Hydrophobicity is expressed as the
averageofthenumerichydrophobicityvaluesofallaminoacidresidues(Eisenbergetal.
1984).Increasinghydrophobicityisrelatedtoanenhancedhemolyticeffect(Datheetal.
1999). However, the relation between hydrophobicity and antimicrobial activityis still
debated.
(5) As mentioned above, AMPs can adopt a series of secondary structures.
Peptides with α‐helix and γ‐core motif (defensin‐like structure) often show higher
activity compared to those withless‐defined structures. For α‐helical peptides,higher
helicitycanfacilitateabetterspatialdistributionofhydrophobicandhydrophilicamino
acids, resulting in higher amphipathicity. However, in designing an AMP, helicity is
always closely related to both amphipathicity and hydrophobicity. The reduction of
disulfide bonds presented in β‐sheet structures may change the activity or even
mechanismoftheinteractionofpeptides(Andreuetal.1998).
InvestigationofPeptide‐LipidInteractionbyFluorescenceCorrelationSpectroscopy


13

1.1.1.4 Therapeuticpotentialofantimicrobialpeptides
Considering the possibilities of thousands of natural peptides and millions of
syntheticpeptides,relativelyfewpeptideshaveprogressedintoclinical trialsdespitethe
promising in vitro and animal tests. Although most researchers are optimistic, the
commercialvalueofthesepeptidesremainsambiguous.AlistofAMPsinclinicaltrials
wasreviewed(Hancock1997;Zasloff2002;Andresetal.2004)(Table1.1),eventhough
noneofthepeptideshasbeengrantedFDAapproval,forexampleduetolackofhigher

antimicrobialactivitycomparedtoconventionalantibiotics(Gordonetal.2005).
Withhighoccurrenceofbacterialresistancetowardsantibiotics,itisurgentneed
for discovering and developing novel classes of drugs to control bacterial infections.
AMPs which target the membrane make it difficult for bacteria to develop drug
resistance,andhencearepromisingcandidatestoachievethistarget.

1.1.2 Designedantimicrobialpeptides
TheprevioussectionbrieflyinducesresearchesonnaturalAMPsrecently.Alarge
numbers of natural AMPshave been identified whichshow a broad spectrum against
differentpathogens. For example,magaininsshow antimicrobialactivity againstGram‐
positive and Gram‐negative bacteria, fungi, protozoa and even viruses (Zasloff 1987;
Zasloff et al. 1988; Schuster et al. 1992). Gramicidin S, also show good performance
againstGram‐positiveandGram‐negativebacteria,andcertainfungi(Kondejewskietal.
1996; Prenner et al. 1999). However natural AMPs are often cytotoxic against
mammalian cells, and this limits their potential in pharmaceutical applications. Thus
large efforts have been undertaken to modify native AMPs or design new synthetic
peptidesinordertoobtainAMPsshowingbetterspecificityagainstmicrobeswithlower