Table Of ContentNediljko Budisa
Engineering the Genetic Code
Expanding the Amino Acid Repertoire
for the Design of Novel Proteins
NediljkoBudisa
Max-Planck-InstituteofBiochemistry
DivisionofBioorganicChemistry
AmKlopferspitz18A
82152Martinsried
Germany
82006WILEY-VCHVerlagGmbH&Co.
PrintedintheFederalRepublicofGermany
ISBN-13: 978-3-527-31243-6
ISBN-10: 3-527-31243-9
Foreword
,,VielleichtistdiephysikalischundchemischorientierteBiologieimAugenblickdie
zukunftsreichsteallerRealwissenschaften.‘‘
CarlFriedrichFreiherrvonWeizsa¨cker
Fromthelecture:,,WiewirddieWissenschaftdieWeltvera¨ndern?‘‘,Munich1969.
The emerging field of chemical biology is based on blending chemistry with mo-
lecularbiology,aprocessthatislargelystimulatedbythemostrecentadvancesin
genetic code engineering. While common methods of protein engineering and
site-directed mutagenesis allow to identify contributions of particular amino acids
tothestructure and/orfunction ofproteins viapermutationof thetwentycanoni-
calaminoacids,expansionofthegeneticcodeofferstheperspectivesofenriching
this alphabet of building blocks with noncanonical a-amino acids of unique bio-
physical and chemical properties. In fact, chemistry should allow the access to a
nearlyinfinitearrayofdiversestructuralelementsforthedesignofnovelproteins
orforincorporationofnewchemicalfunctionalitiesthatcanbeexploitedatwillfor
bioorthogonal posttranslational protein tagging and transformations to study and
to enrich the chemistry of living cells. At the present stage of this forefront re-
search a comprehensive treatise on the state of the art was indeed required, and
the book Engineering of the Genetic Code, authored by our previous student and
now colleague Dr. Nediljko Budisa who decisively contributed to advances in the
field, elegantly fulfils this need. Since our own reseach addresses structure and
function of biomolecules, we are fascinated by the new perspectives offered and
we witness the fast development of this challenging field of research. We trust
thatthecriticaltreatmentandnovelconcepts describedinBudisa’sbook Engineer-
ing of the Genetic Code will provide stimulus and guidance for scientists, research-
ers, teachers and students to learn about and apply this fascinating research area
anditsinnovativetoolsfortheirquestionsattheinterfaceofchemistryandbiology.
RobertHuber
LuisMoroder
DieterOesterhelt
Contents
Foreword VII
Preface XV
1 Introduction 1
1.1 ClassicalApproachestoProteinModification 1
1.2 PeptideSynthesis,SemisynthesisandChemistryofTotalProtein
Synthesis 2
1.3 ChemoselectiveLigationsCombinedwithBiochemicalMethods 5
1.4 MethodsandApproachesofClassicalProteinEngineering 5
1.5 GeneticallyEncodedProteinModifications–ReprogrammingProtein
Translation 6
1.6 BasicDefinitionsandTaxonomy 8
References 10
2 ABriefHistoryofanExpandedAminoAcidRepertoire 13
2.1 The‘‘GoldenYears’’ofMolecularBiologyandTripletCode
Elucidation 13
2.2 EarlyExperimentsontheIncorporationofAminoAcidAnalogsin
Proteins 15
2.3 TestTube(Cell-free)SynthesisofProteinsandEarlyIncorporation
Experiments 18
2.4 NoncanonicalAminoAcidsasToolsforStudyingCellMetabolism,
Physiology,ProteinProcessingandTurnover 19
2.5 ProblemofProofsandFormalCriteriaforNoncanonicalAminoAcid
Incorporation 23
2.6 RecentRenaissance–GeneticCodeEngineering 26
References 28
3 BasicFeaturesoftheCellularTranslationApparatus 31
3.1 NaturalLaws,GeneticInformationandthe‘‘CentralDogma’’of
MolecularBiology 31
3.2 CellularInvestmentsinRibosome-mediatedProteinSynthesis 33
3.3 MolecularArchitectureofAARS 34
3.4 StructureandFunctionofthetRNAMolecule 40
3.5 AminoacylationReaction 44
3.6 AARS:tRNAInteractions–IdentitySets 45
3.7 TranslationalProofreading 48
3.8 RibosomalDecoding–ABriefOverview 50
3.9 CodonBiasandtheFidelityofProteinSynthesis 51
3.10 PreprogrammedContext-dependentRecoding:fMet,Sec,Pyl,etc. 53
3.11 BeyondBasicCoding–PosttranslationalModifications 56
References 59
4 AminoAcidsandCodons–CodeOrganizationandProteinStructure 66
4.1 BasicFeaturesandAdaptiveNatureoftheUniversalGeneticCode 66
4.2 MetabolismandIntracellularUptakeofCanonicalAminoAcids 68
4.3 PhysicochemicalPropertiesofCanonicalAminoAcids 69
4.4 ReasonsfortheOccurrenceofOnly20AminoAcidsintheGenetic
Code 73
4.5 WhatPropertiesofAminoAcidsareBestPreservedbytheGenetic
Code? 74
4.6 EvolutionaryLegacy:DualNatureofConservedCodeandFiniteNumber
ofProteinFolds 77
4.7 NaturalVariationsinAssignmentofCodonsoftheUniversalGenetic
Code 79
4.7.1 NucleosideModificationsandCodonReassignments 81
4.8 CodonReassignmentConceptsPossiblyRelevanttoCode
Engineering 81
4.8.1 GenomeSize,Composition,ComplexityandCodonReassignments 81
4.8.2 StopCodonTakeover,CodonCaptureandCodonAmbiguity 83
References 85
5 ReprogramingtheCellularTranslationMachinery 90
5.1 EnzymeSpecificityofAminoacyl-tRNASynthetases(AARS)andCode
Interpretation 90
5.1.1 LivingCellsasPlatformsforAminoAcidRepertoireExpansion 90
5.1.2 Uptake,ToxicityandMetabolicFateofNoncanonicalAminoAcids 91
5.1.2.1 GeneralConsiderations 91
5.1.2.2 AminoAcidTransport 93
5.1.2.3 MetabolicConversionsandToxicityofAnalogsandSurrogates 94
5.1.3 ConstrainsandLevelsinCodeEngineering 95
5.1.4 AuxotrophismandNaturalAARSwithManipulatedFunctions 95
5.1.4.1 Proteome-wideReplacements:‘‘UnnaturalMicroorganisms’’ 97
5.1.4.2 SubstitutionsattheLevelofSingleProteins–SelectivePressure
Incorporation(SPI) 100
5.1.4.3 KineticControl–EnhancedSystemforProteinTranslation 102
5.1.4.4 ExtensionoftheExistingSpecificitiesofAARS 104
5.1.4.5 AARSwithanAttenuatedEditingFunction 106
5.1.5 BeyondAuxotrophism:TowardsAARSwithDeNovoSubstrate
Specificity 108
5.1.5.1 KineticIssuesofOrthogonalAARS:CatalyticEfficiencyand
Selectivity 111
5.2 ReassigningCodingandNoncodingUnits 113
5.2.1 SenseCodonReassignment:MostCommonlyUsedSubstitutions 113
5.2.1.1 Tryptophan 114
5.2.1.2 Tyrosine 118
5.2.1.3 Phenylalanine 119
5.2.1.4 Histidine 122
5.2.1.5 Proline 124
5.2.1.6 Methionine 127
5.2.1.7 Leucine 130
5.2.1.8 ValineandIsoleucine 132
5.2.1.9 ArginineandCanavanine 133
5.2.1.10 LysineandLysyl-tRNASynthetases–EnzymeswithManyTalents 135
5.3 InVitroChemicalandEnzymatictRNAAminoacylation 137
5.3.1 ChemicaltRNAAcylationforSenseCodonReassignment 138
5.3.2 Ribozymes,RibosomesandMissenseSuppressions 140
5.4 NovelCodon–AnticodonBasePairs 142
5.4.1 InVitroandInVivoFrameshiftSuppressionof4-and5-base
Codons 142
5.4.2 TowardaThird,NoncanonicalBasePairinDNA 145
5.5 StopCodonTakeover 146
5.5.1 TheConceptofSuppressioninProteinTranslation 147
5.5.2 ChemicalAminoacylationofAmberSuppressortRNA 148
5.5.3 InVitroversusInVivoTranslationforExtensionoftheAminoAcid
Repertoire 150
5.5.4 GeneralLimitsofSuppression-basedApproaches 151
5.6 InVivoNonsenseSuppression-basedMethods 153
5.6.1 InSearchforOrthogonaltRNA 153
5.6.2 Species-specificAminoacylationFeaturesandOrthogonalAARS:tRNA
Pairs 154
5.6.3 OrthogonalAARS:tRNAPairsinE.coli 156
5.6.4 OrthogonalPairsinYeastandMammalianCells 158
5.7 OutlookandVisions 159
5.7.1 CouplingReprogrammedTranslationwithMetabolicEngineering
159
5.7.1.1 CatalyticPromiscuityandSyntheticCapacityExtensionofMetabolic
Pathways 159
5.7.1.2 ImportingNaturalProductMetabolicPathwaysandPossibleDeNovo
Design 160
5.7.2 ShuttleOrthogonalPairandHybridTranslationSystemswithCodon
Capture 162
References 165
6 ImplicationsandInsights:FromReprogrammedTranslationandCode
EvolutiontoArtificialLife 184
6.1 CodeEngineeringandSyntheticBiology 184
6.2 NovelFeaturesofProteinTranslationthathaveEmergedfromResearch
inCodeEngineering 185
6.2.1 CodeMalleability,CatalyticPromiscuityandtheAminoAcid‘‘Identity’’
Problem 185
6.2.2 ABarrierbetweenAllowableandNonpermissiveAminoAcids–An
IndexforEntryintheGeneticCode 187
6.2.3 ProteinStructuralTypesandAminoAcidSubstitutionCapacity 190
6.2.4 BuildingaDirectLinkbetweenMetabolismandReprogrammed
Translation 192
6.3 TheAminoAcidRepertoireanditsEvolution 193
6.3.1 ‘‘CopernicanTurn’’andtheLastSacrosanctinBiochemistry 193
6.3.2 SpontaneousTerrestrialandExtraterrestrialGenerationofAmino
Acids 194
6.3.3 MetabolicRoutesforAminoAcidSynthesesandCo-evolution
Theory 197
6.4 ArtificialGeneticSystemsandCodeEngineering 200
6.4.1 CellswithChemicallyAmbiguousProteomes–CodonReassignment
Issues 200
6.4.2 IsitPossibletoImprovetheAdaptiveFeaturesoftheGenetic
Code? 201
6.4.3 PossibilitiesforDeNovoDesignofOrganismswiththeir‘‘Own’’Genetic
Codes 203
6.4.4 CodeEngineeringandSociety–PhilosophicalandEthical
Implications 205
6.4.5 FutureChallenges,ChancesandRisks 207
References 208
7 SomePracticalPotentialsofReprogrammedCellularTranslation 213
7.1 PracticalChoiceofMethodsandSomeControversiesintheField 213
7.2 ThePlasticityoftheTranslationMachinery,AminoAcidGenericTypes
andProteinStructure 215
7.3 DNANucleotideAnalogs:FromSequencingtoExpandedCodeand
Therapy 217
7.4 NoncanonicalAminoAcidsinMaterialScience 217
7.5 IsomorphousReplacementandAtomicMutationsinStructuralBiology
andBiophysics 221
7.5.1 ProteinX-rayCrystallography 221
7.5.2 AtomicMutationsandPredictablePerturbationsintheProtein
Structure 223
7.5.3 ProteinsEnrichedwithChalcogen,HydroxylandAzaAnalogsand
SurrogatesofTrp 225
7.6 TranslationallyActiveAmino-TrpAnalogs:NovelSpectralWindowsand
ProteinSensors 228
7.6.1 ProvidingProteinswithpHSensitivity 228
7.6.2 Novel‘‘Golden’’ClassofAutofluorescentProteins 230
7.7 FluorinatedAminoAcidsinProteinEngineeringandDesign 233
7.7.1 MonofluorinatedAminoAcidsinProteinStudies,Engineeringand
Design 233
7.7.2 NonstickingEggsandBio-Teflon–TrifluorinatedAminoAcidsinProtein
EngineeringandDesign 241
7.8 ProteinProcessing,BioorthogonalityandProteinSurface
Diversifications 245
7.8.1 ChemoenzymaticalControlofProteinProcessingandPosttranslational
Modifications 245
7.8.2 Staudinger–BertozziLigationand‘‘Click’’ChemistryonProteins 246
7.8.3 Tagging,Caging,CrosslinkingandPhotoswitchingattheProtein
Surface 247
7.9 PharmacologicallyActiveAminoAcids 249
7.9.1 BioisostericCompounds,Antagonists,Agonistsand
Antimetabolites 249
7.9.2 NeuroactiveAminoAcidsandtheirDerivatives 251
References 253
Epilogue 261
Index 263
Preface
This book began as a review article in Angewandte Chemie (‘‘Prolegomena to fu-
tureexperimentaleffortsongeneticcodeengineeringbyexpandingitsaminoacid
repertoire’’) based on my Habilitation Thesis at Technical University in Munich.
Theideatowriteabookoncodeengineeringcrystallisedinmymindmuchearlier.
Finally, it was possible to transform my ideas into a book after intensive and con-
structive discussions with Dr. Peter Go¨litz and Dr. Frank Weinreich and their
strongencouragementandsupport.
TheEngineeringofthegeneticcodeisnotintendedtobetraditionaltextforunder-
graduates since code engineering unifies many research fields, each of them now
covered with at least several good introductory books and monographs. The main
aim of this book is to provide a balanced treatment of the major areas of organic
andbiologicalchemistryaswellasmolecularandtheoreticalbiology(Chapters1–
4) important to understand the basics, principles and potentials of code engineer-
ing (Chapters 5–7). Medical researchers and biologists, such as those engaged in
molecular biology, do need to know much more organic chemistry than is pre-
sentedespeciallyinfirstandlastchapters.Ontheotherhand,chemistsandphys-
icistsneedtoknowmuchmoremolecularbiologythanfoundinthisbook.Philos-
ophers, theologians and social scientists interested in possible general impacts of
codeengineeringonsocietyarerequiredtobefamiliaratleastwithbasicsofchem-
istry,biologyandphysics.Therefore, Ipresumetheyallwillconsult othersources
wherethesetopicsarecoveredmoreextensively.
Themajorityofthebookchapters(Chapters1–4)haveonlyalimitednumberof
references and many of them are reviews and book chapters. Although this
approachmightoffendsome,itshouldbekeptinmindthatitisalmostimpossible
togiveproperpriorityamongavastnumberofprimarysourcesinwellestablished
fieldssuchaschemicalmodifications(Chapter1),proteintranslationmechanisms
(Chapter 3) and theoretical biology dealing with genetic code (Chapter 4). The
same principle applies for the historical background of the field (Chapter 2). In
the last three chapters and especially in the central one, that is Chapter 5, I tried
mybesttoprovidealmostallreferencesthatappearedsincethebirthofthisyoung
researchfield(i.e.inthelastfifteenyears).Foralastchapterdealingwithapplica-
tive potentials of code engineering (Chapter 7), I may well imagine that a major
criticism from some of my colleagues working in the field might be that I have
left out some of their pet topics. I have tried very hard to describe everything that
prospective code engineers, biochemists, biophysicists and life scientists to my
opinionreallyneedtoknow.ButImightbewrong.Likemanyotherbookauthors,
Iam well aware that job of book-writing makes one prone to commit (mostly un-
intentionally) many sins, sins of commission and sins of omission. I have taken
particular caretoensurethat thetextisfreeof errors.Thisisdifficultinarapidly
changing field, such as code engineering. Therefore Iam eager to receive all sug-
gestions for improvement of any kind and promise to take them seriously. Of
course,allmistakesthroughoutthetextshouldbeassignedtome.
ThisbookisalsoaresultofmypersonalscientificdevelopmentandIconsiderit
as a work in progress. It was possible to write it only because I had luck to be at
right place at right time and surrounded with right people. By right time I mean
aluckyeventthatbeginningofmyPhDworkcoincidedwiththebirthofcodeen-
gineering.ByrightplaceImeanthoseplaces whereapreciousinstitutionoffree-
domiscultivated,i.e.whereheadsandleadersallowsometimesmembersoftheir
groupstostepbeyondthestrictboundariesoftheircharge.Suchheadsandleaders
areProf.RobertHuber,Prof.LuisMoroderandProf.DieterOesterhelttowhomI
would like to express my deep gratitude and thank. They generously provided me
not only with an excellent infrastructure and technical support, but also outstand-
inghelp,supportandadvices,whiletheirunyieldingfaithinmeandwillingnessto
give mefreedom inpursuing myworkallowedmeto followmy visions.Mytech-
nical assistants Traudl Wenger, Petra Birle and Tatjana Krywcun are also among
the most important people at Max-Planck Institute. With their positive attitudes
they provided friendly environment and created the circumstances that made
works on writing of this book much easier for me. Iapologise to my students for
the fact that I had less time as usual for them during book writing and thank
themfortheirpatience,understandingandsupport.Iamespeciallyindebttomy
student Prajna Paramita Pal who worked hard to ease my ‘‘Croatian English’’ and
colleague Dr. Markus Seifert for hisgenerous help inpreparation of somefigures
inthisbook.Andthelast,butnottheleastexpressionofheartfeltgratitudegoesto
my beloved wife Monika Franke and my children Lukas and Andreas for their
patience, tolerance and strong support. Finally, I would like to thank to Dr. Frank
Weinreich and his co-workers from Wiley-VCH for their assistance, patience and
support during the preparation of the manuscript and for the preparation of the
finishedbook.
Martinsried,September2005 NediljkoBudisa