Artificial Metalloenzymes through Chemical Modification of Engineered Host Proteins

Handle URI:
http://hdl.handle.net/10754/337069
Title:
Artificial Metalloenzymes through Chemical Modification of Engineered Host Proteins
Authors:
Zernickel, Anna ( 0000-0002-0567-2979 )
Abstract:
With a few exceptions, all organisms are restricted to the 20 canonical amino acids for ribosomal protein biosynthesis. Addition of new amino acids to the genetic code can introduce novel functionalities to proteins, broadening the diversity of biochemical as well as chemical reactions and providing new tools to study protein structure, reactivity, dynamics and protein-protein-interactions. The site directed in vivo incorporation developed by P. G. SCHULTZ and coworkers, using an archeal orthogonal tRNA/aaRS (aminoacyl-tRNA synthase) pair, allows site-specifically insertion of a synthetic unnatural amino acid (UAA) by reprogramming the amber TAG stop codon. A variety of over 80 different UAAs can be introduced by this technique. However by now a very limited number can form kinetically stable bonds to late transition metals. This thesis aims to develop new catalytically active unnatural amino acids or strategies for a posttranslational modification of site-specific amino acids in order to achieve highly enantioselective metallorganic enzyme hybrids (MOEH). As a requirement a stable protein host has to be established, surviving the conditions for incorporation, posttranslational modification and the final catalytic reactions. mTFP* a fluorescent protein was genetically modified by excluding any exposed Cys, His and Met forming a variant mTFP*, which fulfills the required specifications. Posttranslational chemical modification of mTFP* allow the introduction of single site metal chelating moieties. For modification on exposed cysteines different maleiimid containing ligand structures were synthesized. In order to perform copper catalyzed click reactions, suitable unnatural amino acids (para-azido-(L)-phenylalanine, para-ethynyl-(L)-phenylalanine) were synthesized and a non-cytotoxic protocol was established. The triazole ring formed during this reaction may contribute as a moderate σ-donor/π-acceptor ligand to the metal binding site. Since the cell limits the incorporation of boronic acids, an aqueous protocol for Miyaura borylation using a highly active palladacycle catalyst was established and can be transferred to a selective borylation of proteins. It allows subsequent Suzuki cross coupling and therefore broadens the possibilities for chemical modifications and the establishment of new metalloenzymes. Different metal chelating amino acids were investigated, such as Hydrochinolin-Alanine, Bipyridyl-Alanine, Dipyridine-Lysines and phosphorous containing amino acids.
Advisors:
Eppinger, Jörg ( 0000-0001-7886-7059 )
Committee Member:
khashab, niveen ( 0000-0003-2728-0666 ) ; Hamdan, Samir; Hadjichristidis, Nikolaos ( 0000-0003-1442-1714 )
KAUST Department:
Physical Sciences and Engineering (PSE) Division
Program:
Chemical Sciences
Issue Date:
Oct-2014
Type:
Dissertation
Appears in Collections:
Dissertations; Physical Sciences and Engineering (PSE) Division; Chemical Science Program

Full metadata record

DC FieldValue Language
dc.contributor.advisorEppinger, Jörgen
dc.contributor.authorZernickel, Annaen
dc.date.accessioned2014-12-11T12:03:42Z-
dc.date.available2014-12-11T12:03:42Z-
dc.date.issued2014-10en
dc.identifier.urihttp://hdl.handle.net/10754/337069en
dc.description.abstractWith a few exceptions, all organisms are restricted to the 20 canonical amino acids for ribosomal protein biosynthesis. Addition of new amino acids to the genetic code can introduce novel functionalities to proteins, broadening the diversity of biochemical as well as chemical reactions and providing new tools to study protein structure, reactivity, dynamics and protein-protein-interactions. The site directed in vivo incorporation developed by P. G. SCHULTZ and coworkers, using an archeal orthogonal tRNA/aaRS (aminoacyl-tRNA synthase) pair, allows site-specifically insertion of a synthetic unnatural amino acid (UAA) by reprogramming the amber TAG stop codon. A variety of over 80 different UAAs can be introduced by this technique. However by now a very limited number can form kinetically stable bonds to late transition metals. This thesis aims to develop new catalytically active unnatural amino acids or strategies for a posttranslational modification of site-specific amino acids in order to achieve highly enantioselective metallorganic enzyme hybrids (MOEH). As a requirement a stable protein host has to be established, surviving the conditions for incorporation, posttranslational modification and the final catalytic reactions. mTFP* a fluorescent protein was genetically modified by excluding any exposed Cys, His and Met forming a variant mTFP*, which fulfills the required specifications. Posttranslational chemical modification of mTFP* allow the introduction of single site metal chelating moieties. For modification on exposed cysteines different maleiimid containing ligand structures were synthesized. In order to perform copper catalyzed click reactions, suitable unnatural amino acids (para-azido-(L)-phenylalanine, para-ethynyl-(L)-phenylalanine) were synthesized and a non-cytotoxic protocol was established. The triazole ring formed during this reaction may contribute as a moderate σ-donor/π-acceptor ligand to the metal binding site. Since the cell limits the incorporation of boronic acids, an aqueous protocol for Miyaura borylation using a highly active palladacycle catalyst was established and can be transferred to a selective borylation of proteins. It allows subsequent Suzuki cross coupling and therefore broadens the possibilities for chemical modifications and the establishment of new metalloenzymes. Different metal chelating amino acids were investigated, such as Hydrochinolin-Alanine, Bipyridyl-Alanine, Dipyridine-Lysines and phosphorous containing amino acids.en
dc.language.isoenen
dc.subjectaqueous catalysisen
dc.subjectpalladium catalysisen
dc.subjectcross couplingen
dc.subjectunnatural amino acidsen
dc.subjectprotein modificationsen
dc.titleArtificial Metalloenzymes through Chemical Modification of Engineered Host Proteinsen
dc.typeDissertationen
dc.contributor.departmentPhysical Sciences and Engineering (PSE) Divisionen
thesis.degree.grantorKing Abdullah University of Science and Technologyen_GB
dc.contributor.committeememberkhashab, niveenen
dc.contributor.committeememberHamdan, Samiren
dc.contributor.committeememberHadjichristidis, Nikolaosen
thesis.degree.disciplineChemical Sciencesen
thesis.degree.nameDoctor of Philosophyen
dc.person.id114519en
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