Alone in the dark: Time-resolved fluorescence spectroscopy on single biomolecules

160 Pages·2012·5.91 MB·English

by  Victor I. Claessen

AALLOONNEE IINN TTHHEE DDAARRKK
Time-resolved fluorescence spectroscopy
on single biomolecules
Victor I. Claessen

Alone in the dark
Time-resolved(cid:282)uorescencespectroscopy
onsinglebiomolecules
VictorI.Claessen
Aloneinthedark:
Time-resolved(cid:282)uorescencespectroscopyonsinglebiomolecules
VictorI.Claessen
(cid:265)esisRadboudUniversityNijmegen-Illustrated
Withreferences-WithsummaryinDutch
ISBN:978-90-9026687-9
NUR-code:927
(cid:265)isthesiswasprintedongraphene-freepaper
©2012,VictorI.Claessen
CarrildelZorroPublishing
URL:www.aloneinthedark.nl
Cover:Amodelofthecowpeachloroticmo(cid:296)leviruscontainingahorseradishperoxidase
enzymemolecule,whichhasgenerated(cid:282)uorescentrhodamine6Gproductmoleculesthat
haveaccumulatedontheinsideoftheviruscapsid(Chapter5).
Alone in the dark
Time-resolved(cid:282)uorescencespectroscopyonsinglebiomolecules
Proefschri(cid:283)
terverkrijgingvandegraadvandoctor
aandeRadboudUniversiteitNijmegen
opgezagvanderectormagni(cid:279)cusprof.mr.S.C.J.J.Kortmann,
volgensbesluitvanhetcollegevandecanen
inhetopenbaarteverdedigen
opdonderdag21juni2012
om10.30uurprecies
door
VictorIljaClaessen
geborenop29september1981
teAmsterdam
Promotoren: Prof.dr.R.J.M.Nolte
Prof.dr.A.E.Rowan
Copromotor: Dr.H.Engelkamp
Manuscriptcommissie: Prof.dr.S.S.Wijmenga
Prof.dr.J.Ho(cid:281)ens(KULeuven)
Dr.K.Blank
Paranimfen: Drs.A.M.vanBuul
Ir.O.Lugten
Alone in the dark
Time-resolved(cid:282)uorescencespectroscopyonsinglebiomolecules
Doctoral(cid:265)esis
toobtainthedegreeofdoctor
fromRadboudUniversiteitNijmegen
ontheauthorityoftherectormagni(cid:279)cusprof.dr.S.C.J.J.Kortmann,
accordingtothedecisionofthecouncilofdeans
tobedefendedinpublic
on(cid:265)ursday,June21th2012
at10.30hours
by
VictorIljaClaessen
bornonSeptember29th,1981
inAmsterdam
Supervisors: Prof.dr.R.J.M.Nolte
Prof.dr.A.E.Rowan
Co-supervisor: Dr.H.Engelkamp
Doctoral(cid:265)esisCommi(cid:296)ee: Prof.dr.S.S.Wijmenga
Prof.dr.J.Ho(cid:281)ens(KULeuven)
Dr.K.Blank
Paranymphs: Drs.A.M.vanBuul
Ir.O.Lugten
Levéritablevoyagededécouverteneconsistepasàchercherdenouveauxpaysages,
maisàavoirdenouveauxyeux.
-MarcelProust
Contents
Contents viii
1 Anintroductiontosingle-moleculeenzymology 1
1.1 Enzymesastinybiologicaltools . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Whatdowealreadyknowaboutthewayenzymeswork? . . . . . . . . . 1
1.3 Whynowindividually? . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.4 Whathasbeendonebefore? . . . . . . . . . . . . . . . . . . . . . . . . 4
1.5 Aimandoutlineofthisthesis . . . . . . . . . . . . . . . . . . . . . . . . 6
1.6 Referencesandnotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2 Techniquesforopticalsinglemoleculeexperiments 7
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 Microscopyforsingle-molecule(cid:282)uorescencedetection . . . . . . . . . . 9
2.3 Dataanalysistechniquesforsinglemoleculeexperiments . . . . . . . . . 12
2.4 (cid:265)e(cid:282)uctuatingenzymemodel . . . . . . . . . . . . . . . . . . . . . . . 20
2.5 Strategiesforsingle-enzymeexperiments . . . . . . . . . . . . . . . . . 22
2.6 Surfaceimmobilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.6.1 Non-speci(cid:279)cadsorptionoftheenzyme . . . . . . . . . . . . . . 23
2.6.2 Site-directedenzymeadsorptionviaa“proteinfoot” . . . . . . . 24
2.6.3 Site-speci(cid:279)candcovalentenzymeimmobilisation . . . . . . . . 25
2.6.4 Enzymeimmobilisationviareceptor-ligandinteractions . . . . . 27
2.6.5 Immobilisationofthesubstrate . . . . . . . . . . . . . . . . . . 28
2.7 Spatialcon(cid:279)nement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.7.1 Encapsulationindroplets . . . . . . . . . . . . . . . . . . . . . 29
2.7.2 Femtoliterarray . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.8 Flowassayinacapillary . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.9 Referencesandnotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3 Insolution:studiesonfreelydiffusingenzymemolecules 39
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.1.1 (cid:265)econtainer . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
viii