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Graduate
Category:PhysicalandLifeSciences
DegreeLevel:Ph.D
AbstractID#1182
TakingElectrostaticLessonsfromNaturalEnzymesforProteinDesign
TimothyA.Coulther,PennyJ.Beuning,andMaryJoOndrechen.NortheasternUniversity,Boston,MA
Enzymes
• 20differentaminoacids
- hydrophobic,polar,acid/base
Naturalversus designedenzymes
Acidremoval:
Effectonµ4
0
175150
225
sequence
50
sequence
0
-10
1100300500
-50
-20
-30
K83
-100
-150
-40
E358
H389
-200
100
NoEffects
0
-50
-100
-150
-200
Charge
pH
A
Answer
IfCisclosertoAthanB
thenhigherpKa preferred
IfCisfartherfromAthanB
thenlowerpKa preferred
B
• Complexbehaviorandinteractionsinnaturalenzymes
- Arrangedtoincreasemetricsofmultipleresidues
- Residuesbothhelpandhurtothers
• Behaviornotapparentindesignedenzyme
- Lowermoments,fewerinteractions
Morefocusonarrangementofresiduesneeded
forenzymedesign
• Prospectiveprinciplestodesignactivesites
• Retrospectiveanalysistounderstandactivity
1.M.J.Ondrechen,J.G.CliftonandD.Ringe,Proc Natl Acad Sci USA2001,98,pg
12473.
2.J.VanDurme etal.,Bioinformatics2011,12,pg 1712.
3.E.A.Althoff,etal. ProteinScience 2012,21(5),pg 717.
4.LGiger ,etal. NatureChemicalBiology 2013,9(8),pg 494
5.C.J.Jeffery,R.Hardre,andL.Salmon.Biochemistry 2001,40(6),pg 1560.
6.S.Somarowthu etal.,Biochemistry2011,50,pg.9283.
REFERENCES
0
C
Computationalmethodscanidentifyfunctional
residuesandinteractions
• Complexinteractionnetworkspresentin
naturalenzymes
• Arrangementallowsincreasedmu4of
multipleresidues,ratherthanjustone
• CatalyticresiduesdeviatefromstandardHendersonHasselbalch behavior
• Providesawidebufferrangeadvantageousfor
catalyticactivity
• Allowsgreateraccesstoboththeprotonated
anddeprotonatedstates
• Clustersofperturbedresiduestypicallyformanactivesite
THEMATICSandResidueScanning
• Analysisofallvariantsofenzyme
• Single-sitevariantsmadethroughFoldX2
• Determineeffectofresiduechangeonelectrostatic
propertiesofcatalyticresidues
• Breakdownbymutationtypestostudyinteractionsand
effects
C
50
THEoreticalMicroscopicAnomalous TheoreticalMicroscopicAnomalousTitrationCurveShapes
(THEMATICS)1
TItrationCurveShapes
PerturbedH-H
Curves
ExampleScenario
TwobasesatsamepKa (AandB)
Objective
Place3rd base(C)toincreaseµ4ofA
Choices
Location:closertoAthanB
orfartheraway
pKa:higherorlowerthanA
CONCLUSIONS
BACKGROUND
increasingmetrics
FullResidueScanningMutagenesis:
EffectsonCatalyticResiduesofPGIandRA95.5-5
• Catalyzeessentiallifereactions
- upto1026 rateenhancement
TypicalH-H
Curve
ProspectiveUse
• Identifymetricsthatcorrelate
Phosphoglucose Isomerase
withbeneficialmutations
• Naturalenzyme
5
• E358andH389arecatalytic
•
Computedifferentscenarios
• Biochemicallyverified
• Identifyprinciplesfor
6
extendedactivesite
Retro-Aldolase 95.5-5
3
• Designedenzyme(RA95)
• 5roundsofdirected
evolution4
• K83iscatalytic
Baseremoval:
Effectonµ4
Enzymeshaveapplicationsinawidearrayofindustriesduetotheirabilityto
catalyzereactionsathighratesundermildconditions.Enzymaticcatalysiscan
havemanyadvantagesoverconventionalcatalyticprocesses,namelylessenergy
consumptionandfewerunwantedby-products.However,anaturalenzymethat
cancatalyzethedesiredreactiondoesnotexistformostindustrialchemical
processes.Whiletherehavebeensuccessesintheproteindesignfield,including
denovo enzymedesignforafewdifferentreactionsthathavenoknownnatural
counterpart,theenzymesproducedhavelowactivitycomparedtonatural
enzymes.Laboratorytechniquessuchasdirectedevolutionhavebeenusedto
furtheroptimizedesignedenzymestoimproveactivitylevels,butthesemethods
arecostlyandverytimeconsuming,limitingtheirwidespreaduse.
THEMATICSisacomputationalmethodthatidentifiesactivesiteresidues
throughtheirperturbedionizationbehavior.Predictionsoftenincludenotonly
theresiduesthatdirectlycontactthesubstratebutalsoresiduesfartheraway.
Thesedistalresiduesmaynotcontactthesubstratedirectly,yettheycanstill
contributegreatlytocatalysis,aswehaveverifiedthroughbiochemicalassayson
single-sitevariants.Mostdesignedenzymes,however,lacktheseelectrostatic
propertiesthatweobserveessentiallyuniversallyinthelocalactiveregionsof
naturalenzymes.Whilenaturalenzymesmayhaveextensiveactivesite
networks,designedenzymesaretypicallylessconnected,withfewerresidues
seeminglycontributingtocatalysis.Thesedifferencesmaybeonereasonfor
theirlowactivity,presentingapathtowardsenzymeoptimizationbyincluding
thesepropertiesindesignprocesses.SupportedbyNSF-MCB-1517290.
205-R2-CX-0011(TAC)
RESULTS
ABSTRACT
NSFMCB-1158176
NSF-MCB-1517290
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