acs poster final
TRANSCRIPT
SpectroscopicAnalysisandChemicalModificationoftheH134CMutantoftheThermusthermophius Rieske Protein
VictorRodríguezandLauraM.Hunsicker-Wang
TrinityUniversity,SanAntonio,TX
AbstractTheRieske proteinisfoundinthebc1complex(complexIII)andplaysanimportantroleintransportingelectronsandprotonsthroughtheelectrontransportchain.TheRieskeproteincontainsa[2Fe-2S]clusterligatedbya2-cystinesand2- histidines.ThereductionpotentialofthisclusterispH-dependentandvariesacrossspecies.AnH134CmutantoftheThermus thermophilus Rieske substitutesoneoftheligatinghistidines foracysteine,changingtheligationstructureoftheclusterfroma2Cys-2Histoa3Cys-1Hisenvironment.Tostudytheeffectsofthismutation,theproteinissubjectedtomodificationwithdiethylpyrocarbonate(DEPC)andtopHchanges.Thebehaviorofthisproteiniscomparedtothewildtypeproteinasobservedthroughcirculardichroism andUVvisiblespectroscopy.Becauseofthesimilarityintheiron-sulfurclusterligands,H134Cwillalsobecomparedtoanothermitochondrialprotein,mitoNEET,whichcontainsa3Cys-1Hisenvironment.
Rieske Proteins
• TheRieske proteinisfoundincomplexIII(cytochromebc1 complex)aspartoftheelectrontransportchain.
• Thiscomplexoxidizesubiquinol toubiquinoneandtransportstheelectronsthroughthecomplexintotheFe-SclusterinReiske.
http://www.scienceprofonline.org/metabolism/electron-transport-chain-cellular-respiration.htmlhttp://en.wikipedia.org/wiki/Coenzyme_Q_%E2%80%93_cytochrome_c_reductase
H134Cmutant&MitoNEET
• TheRieske proteinFe-Sclusterhasa2-His2-Cysligationenvironment.
• H134Cmutant- theligationstructureisalteredtohaveaonehistidine andthreecysteines.
• ThisligationenvironmentissimilartothatofMitoNEET,aknowndiabetesdrugactivator.
• ResultsfromtheseexperimentswillbeusedtocomparetothatofMitoneet tobetterunderstandthedifferenceswiththisligationenvironment.
Rieske
MitoNEET
PDBID3REE
pH-DependentUV-Visiblespectra
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
300 400 500 600 700 800
Absorbance(A)
Wavelength(nm)
4.39
6.63
7.54
8.65
9.61
10.41
10.97
11.99
• PreviousstudiesshowthatH134ChasalargerangeofpHstability
• H134ChasapKa of9.86.DatacollectedbyAbhishek Chhetri
H134C
H134CCrystals• CrystalsforH134CRieskemutantwereisolatedunderthefollowingconditions:30%PEG4000,0.2MMgCl2, 0.1MTris-HCl pH8.5or20%PEG2000MME,0.01MNiCl2·6H2O,0.1MTris pH8.5.
• Bestdatacollectedto1.66Åresolution!
• SpacegroupP2221Preliminaryelectrondensitymap(2Fo-Fc)showing3Cys,1His[2Fe2S]cluster
His154Cys 134
Cys 132 Cys 151
DEPCmodification
• Deprotonatedhistidines reactwithDEPC.• Intruncatedwildtype,notonlywasmodificationobserved,but
alsoreduction.
DEPC
DEPC=diethylpyrocarbonateKonkleetal.2010Biochemistry49,7272-7281
ReactionwithDEPC- UV-Vis
pH6
pH7
pH8
pH6
pH7
pH8
Konkleetal.2010Biochemistry49,7272-7281
truncTtRp H120Q/H162Q
• truncTtRpandH120Q/H162QaremodifiedbyDEPC• HigherpHcausesfastermodificationandmoremodificationintruncTtRp
EffectofpHonDEPCreaction- CD
pH=6 pH=7
pH=8 pH=9
H120Q/H162Q
Konkleetal.2010Biochemistry49,7272-7281• MorespectralchangeswithhigherpH• Proteinsbecomereducedaftermodification
H134CReactionwithDEPC- UV
-0.05
0.05
0.15
0.25
0.35
0.45
0.55
200 300 400 500 600 700 800
Difference
Wavelength(nm)
pH6.0
-0.05
0.05
0.15
0.25
0.35
0.45
0.55
200 300 400 500 600 700 800
Difference
Wavelength(nm)
pH7.6
• H134CwasreactedwithDEPC• differenceUV-Visspectraover40minutesatdifferentpHvalues.
-0.05
0.05
0.15
0.25
0.35
0.45
0.55
200 300 400 500 600 700 800
Difference
Wavelelngth(nm)
pH8.2
H134CismodifiedbyDEPC,buttheLMCTbandsareminimallyaffected
-0.1
0
0.1
0.2
0.3
0.4
0.5
200 300 400 500 600 700 800
Difference
Wavelength(nm)
pH9.0
pH-dependenceofmodification
• ReactionrateincreaseswithpH,buttheextentofmodificationdecreasesabovepH7.6
• TheprofilediffersfromtruncTtRp (seepreviousslide)
ΔAb
s.240nm
Time(min)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 5 10 15 20 25
pH6.0
pH7.6
pH8.2
pH9.0
• H134CreactedwithDEPCover90minutes(upperfigure).
• AchangeinsignalisobservedovertimeandfollowasimilarpattertowhatwasobservedforreductioninwildtypeRieske.
• Tofullyunderstandthechangesofthesignals,themutantproteinwasreducedandoxidize(lowerfigure)
CDspectrachangebuttheproteindoesnotappeartobecomereduced
H134CReactionwithDEPC- CD
24hourreactionwithDEPC
• TheCDsignalat450nmincreasesuntil3hours,andthenreturnstotheoriginallevel.
• OneinterpretationisthattheDEPCmodificationreversesovertime,indicatingalabileadduct.
-13
-11
-9
-7
-5
-3
-1
1
3
5
230 280 330 380 430 480 530 580
mde
g
Wavelength(nm)
H134CatpH8.2reacetdwithDEPCfor24hours
unreacted
0hours
3hours
10hours
23hours
27hours
0
2
4
0 3 6 9 12 15 18 21 24 27Absorbtion
Time(hours)
H134CreactedwithDEPCat450nm
Conclusions
• H134CpKa ofproteinis9.86.• Crystalstructureconfirmsthe3Cys1HisStructure
• ProteinismodifiedbyDEPCandnotreducedwithina90minutereaction.
• DEPCmodificationmayreversesoverlongertimes,indicatingalabileadductinthis3Cys,1Hisligationenvironment.