Development of NMR methods for the study of protein folding [Elektronische Ressource] / von Kai Schlepckow

Description

FPrudyofeiNMRStMethoofdsotfonroldingtheDevelopmentDissertationzurErlangungdesDoktorgradesderNaturwissenschaftenvorgelegtbeimFachbereichBiochemie,ChemieundPharmaziederJohannWolfgangGoethe UniversitätinFrankfurtamMainvonKAI SCHLEPCKOWausKarlsruheFrankfurtamMain2008(D30)DemFachbereichBiochemie,ChemieundPharmaziederJohannWolfgangGoethe UniversitätalsDissertationvorgelegt.Dekan: Prof.Dr.HaraldSchwalbeGutachter: Prof.Dr.HaraldSchwalbeundProf.Dr.ClemensGlaubitzDatumderDisputation:NMRunfoldedoListroftheAbbTheoreviationsplingsviidipAbstractretationixfo1.FIntroContentsductionof1cou-Kineticolarefoldingresidualofofbinterpoviner-lactalbuminrk(BLA)ramew53retical92.roteins3.p1.1. ProteinFolding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2. NMRspectroscopyforthestudyofproteinfolding . . . . . . . . . . . . . . 51.2.1. Foldingintermediates . . . . . . . . . . . . . . . . . . . . . . . . . . 51.2.2. Unfoldedstatesofproteins . . . . . . . . . . . . . . . . . . . . . . . 61.3. Thesis: projectsandobjectives . . . . . . . . . . . . . . . . . . . . . . . . . 72.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.1.1. TheunfoldedstateanditsstudybyNMR . . . . . . . . . . . . . . . 92.1.2. Weakalignmentandresidualdipolarcouplings . . . . . . . . . . . . 112.1.3. Prediction and simulation of residual dipolar couplings under weaklyaligningconditions .

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 11 Mo

Extrait

Dissertation
zurErlangungdesDoktorgrades
derNaturwissenschaften
vorgelegtbeim
FachbereichBiochemie,ChemieundPharmazie
derJohannWolfgangGoethe Universität
inFrankfurtamMain
von
KAI SCHLEPCKOW
ausKarlsruhe
FrankfurtamMain
2008
(D30)
oldingDevelopmentotNMRnofoftheFreifoPrdsudyMethoStDemFachbereichBiochemie,ChemieundPharmazieder
JohannWolfgangGoethe UniversitätalsDissertationvorgelegt.
Dekan: Prof.Dr.HaraldSchwalbe
Gutachter: Prof.Dr.HaraldSchwalbeundProf.Dr.ClemensGlaubitz
DatumderDisputation:1.1. ProteinFolding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2. NMRspectroscopyforthestudyofproteinfolding . . . . . . . . . . . . . . 5
1.2.1. Foldingintermediates . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.2. Unfoldedstatesofproteins . . . . . . . . . . . . . . . . . . . . . . . 6
1.3. Thesis: projectsandobjectives . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1.1. TheunfoldedstateanditsstudybyNMR . . . . . . . . . . . . . . . 9
2.1.2. Weakalignmentandresidualdipolarcouplings . . . . . . . . . . . . 11
2.1.3. Prediction and simulation of residual dipolar couplings under weakly
aligningconditions . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.2. ResultsandDiscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.2.1. TheoreticalFramework . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.2.2. Predictionsandcomparisonwithexperiment . . . . . . . . . . . . . 37
2.3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
α
3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
iii
oofrKineticunfoldedrefoldingNMRthe3.Theo-lactalbuminplings(BLA)dip53retationductionfoIntroF1.pixofAbstractcou-viiolareviationsresidualAbbofofinterpListrContentsrk1ramew2.retical9roteinsbovineContents
3.1.1. Somegeneralaspects . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1.2. Measurementofproteinfoldingkineticsbystopped flowtechniques . 57
3.1.3.ofproteinfoldingkineticsbyNMR . . . . . . . . . . . 58
3.1.4. Bovineα lactalbuminasamodelsystem . . . . . . . . . . . . . . . 60
3.2. MaterialsandMethods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.2.1. CDspectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.2.2. Stopped flowspectroscopy . . . . . . . . . . . . . . . . . . . . . . . 63
153.2.3. Heterologous expression and purification of N labeled bovine α-
lactalbumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.2.4. NMR resonance assignment of methyl and methylene groups in the
nativestate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.2.5. Time resolvedNMRspectroscopy . . . . . . . . . . . . . . . . . . . 67
3.3. Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.3.1. TryptophanfluorescenceandCDspectroscopicsteady stateinvestiga
tions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.3.2. Foldingkineticsbystopped flowspectroscopy . . . . . . . . . . . . 71
153.3.3. Heterologous expression and purification of N labeled bovine α-
lactalbumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.3.4. Assignmentofbackboneamideandside chainaliphaticresonances . 79
3.3.5. Foldingkineticsbytime resolvedNMR . . . . . . . . . . . . . . . . 87
3.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
3.4.1. BLArefoldingis(more)complex . . . . . . . . . . . . . . . . . . . 94
3.4.2. BLAfoldsviaparallelpathways . . . . . . . . . . . . . . . . . . . . 97
3.4.3. BLAandHEWLshareacommonfoldingmechanism . . . . . . . . 98
3.4.4. AdvancesinNMRmethodology . . . . . . . . . . . . . . . . . . . . 100
3.4.5. Futureprospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
α
4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
4.1.1. Photochemicallyinduceddynamicnuclearpolarisation(Photo CIDNP) 103
iv
103inpa-lactalbuminagneticofbresencelanthanidespram-theonPhoto-CIDNPovine4.studiesContents
4.1.2. Paramagnetic lanthanides as shift and relaxation reagents and their
effectonCIDNP . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.1.3. Lanthanide inducedrefoldingofbovine α lactalbumin . . . . . . . . 108
4.2. MaterialsandMethods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4.2.1. Photo CIDNPNMRspectroscopy . . . . . . . . . . . . . . . . . . . 110
4.2.2. OrganicsynthesisofflavinI . . . . . . . . . . . . . . . . . . . . . . 111
4.3. Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
4.3.1. Photo CIDNPofN Acetylhistidine,N Acetyltryptophan,andN Acetyltyrosine
inthepresenceofytterbium(III) . . . . . . . . . . . . . . . . . . . . 112
4.3.2. OrganicsynthesisofflavinI . . . . . . . . . . . . . . . . . . . . . . 118
4.3.3. Photo CIDNPinvestigationsonbovine α lactalbuminusingflavinI . 120
4.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
15A.1. Heterologousexpressionandpurificationof N labelledbovine α lactalbumin 131
A.2. NMRresonanceassignments . . . . . . . . . . . . . . . . . . . . . . . . . . 134
v
Publications131Vitaeendix65AppListB.71A.Curriculum141167Zusammenfassungof1591Danksagung1Referencesvi1D,2D,3D one dimensional,two dimensional,three dimensional
ACBP acylcoenzymebindingprotein
AEBSF 4 (2 Aminoethyl)benzenesulfonylfluoridehydrochloride
apoMb apomyoglobin
αS α synuclein
BLA bovineα lactalbumin
BPTI bovinepancreatictrypsininhibitor
C8E5 n octyl penta(ethyleneglycol)
C12E5 n dodecyl penta(ethyleneglycol)
CmEn n alkyl poly(ethyleneglycol)
CARA computeraidedresonanceassignment
CD circulardichroism
CIDNP chemicallyinduceddynamicnuclearpolarization
CPBr cetylpyridiniumbromide
CTD C terminaldomain
DHPC dihexanoylphospahtidylcholine
DMN dimethoxy nitrophen
DMPC dimyristo
DNA desoxyribonucleicacid
DSS 2,2 dimethyl 2 silapentane 5 sulfonicacid
E.coli Escherichiacoli
fd bacteriophagefd
FMN riboflavin5’ mononucleotide
GAF Gaussianaxialfluctuation
vii
ListreviationsAbbofListofAbbreviations
GB B1domainofproteinG
GdnCl guanidinehydrochloride
HEWL heneggwhitelysozyme
HSQC heteronuclearsinglequantumcoherence
IMAC immobilizedmetalionaffinitychromatography
IPTG isopropylβ D 1 thiogalactopyranoside
LB Luriabroth
MG moltenglobule
mRNA messengerribonucleicacid
MS massspectrometry
NDBF nitrodibenzofuran
Ni NTA Nickelnitrilotriacetic
NMR nuclearmagneticresonance
NOE nuclearOverhausereffect
NOESY nuclearOveffectspectroscopy
OD opticaldensityat600nm600
PAG polyacrylamidegel
PALES PredictionofALignmEntfromStructure
Pf1 bacteriophagePf1
PP polyprolineIIII
ppm partspermillion
PRE paramagneticrelaxationenhancement
RDC residualdipolarcoupling
RNaseA ribonucleaseA
rpm roundsperminute
SDS sodiumdodecylsulfate
SDS PAGE sodiumdodecylsulfate-polyacrylamidegelelectrophoresis
SNase staphylococcalnuclease
States TPPI Statestime proportionalphaseincrement
UV ultra violet
WT wildtype
viiiTo decipher the principles that govern a given amino acid sequence to attain a well defined
three dimensional fold is one of the major goals in modern structural biology. The correct
folding of proteins assures their functionality and, thereby, cell homeostasis. It is also well
established that up to 30% of the proteins encoded in eukaryotic genomes exist in partially
or fully unfolded states which are functional. Moreover, partially unfolded states of proteins
are implicated in a number of diseases involving formation of fibrillar structures. The inves
tigation of protein folding therefore requires techniques which not only allow to follow con
formational transitions but to characterise the conformational states involved as well. In this
respect,NMRspectroscopictechniqueshaveprovenessentialinfurtheringtheunderstanding
ofproteinfolding.
The focus of this thesis has been to further advance and develop existing NMR techniques
for the study of protein folding. In order to do so, experimental as well as theoretical ap
proaches have been pursued. From the theoretical side, a successful attempt to the develop
mentofageneraltheoryforthetreatmentofresidualdipolarcouplingsinthecaseofunfolded
proteinshasbeenundertaken. Informationcontainedinresidualdipolarcouplingsisespecially
valuable due to its long range nature. The dynamic character of unfolded states of proteins,
which may be composed of distinct subsets of conformations, renders reliable interpretation
ofdataanon trivialtask. Statistical coil basedapproacheshavebeenshowntobepowerfulin
datainterpretation. Aconsistenttheorybasedonfundamentalpolymerphysics,however,had
notbeenpresentedsofar.
ThehereinpresentedmodeladdressesthisproblembuildingontheoriginalworkbyAnnila
andco workers. Inthiswork,severalshortcomingshavebeenidentified. Theseshortcomings
have been corrected here leading to a general approach for the treatment of residual dipolar
couplingsofunfoldedproteins. Morespecifically,itisshownthat,inthecaseoffullyunfolded
ix
AbstractAbstract
proteins aligned by a steric mechanism, basic dependencies of