University of Groningen Exploring and exploiting bacterial protein glycosylation systems Yakovlieva, Liubov
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(2) . Exploring and exploiting bacterial protein glycosylation systems Liubov Yakovlieva. .
(3) Liubov Yakovlieva PhD thesis Rijksuniversiteit Groningen The work described in this thesis was financially supported by the NWO Veni grant (722.016.006) and the Rosalind Franklin Fellowship (ERC COFUND 60021). Cover design: Liubov Yakovlieva Printed by: Ipskamp Printing B.V.. .
(4) Exploring and exploiting bacterial protein glycosylation systems. PhD thesis. to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. C. Wijmenga and in accordance with the decision by the College of Deans. This thesis will be defended in public on Tuesday 6 July 2021 at 16.15 hours. by. Liubov Yakovlieva born on 10 March 1994 in Kyiv, Ukraine.
(5) Supervisors Prof. M. T. C. Walvoort Prof. A. J. Minnaard. Assessment Committee Prof. J. G. Roelfes Prof. A. J. Wilson Prof. R. Gerardy-Schahn.
(6) . “I must not fear. Fear is the mind-killer. Fear is the little-death that brings total obliteration. I will face my fear. I will permit it to pass over me and through me. And when it has gone past I will turn the inner eye to see its path. Where the fear has gone there will be nothing. Only I will remain.” Litany against Fear Frank Herbert, “Dune” (1965). .
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(10) List of abbreviations……………………………..…………………………………..……….… 1 Chapter 1………………….………………………….…………………….……...……….......…5 General introduction Chapter 2………….……………………………..………..………………...…………...........…13 Processivity in bacterial glycosyltransferases Chapter 3………………………….………………...……………………………….…....….….45 Semiprocessive hyperglycosylation of adhesin by bacterial N-glycosyltransferases Chapter 4……………………………….…………………………………….……..…...............89 Generation of azidoglucose-labeled adhesin protein Chapter 5…………………………….………………………….…...………………….......….127 Insight into the oligomeric states of bacterial NGTs and the H. influenzae adhesin fragment Chapter 6…………………………….……………….……………...……………………...….143 Site-selective palladium-catalyzed oxidation of glucose in glycopeptides Chapter 7………………………………………….………………….…..………….......….….189 Substrate recognition studies of EarP rhamnosyltransferase Chapter 8……………………………….………..….………………..………….….….............223 Summary and outlook Nederlandse samenvatting..………………………………..….………………...….…....….229 Popular summaries …………………………………………………………...….….....…….237 List of publications………………………………………………………………..…..…...….239 Curriculum vitae………………..………………………………………………….…...............240 Acknowledgements…………………………………………………………..........................241. .
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(14) AA Ab Ac ABC ACN ADEM AE AIDA Ala APC APDC Ara Arg Asn Asp ATP AzGlc Boc BOP. BQ BSA Bz cat. CAZy CDP CeS CLIPS CMP CNS CPS Cryo-EM CV Cys d DBU DC-SIGN. . DCE. amino acid antibody acetyl ammonium bicarbonate acetonitrile acute disseminated encephalomyelitis anion exchange adhesin involved in diffused adherence alanine antigen-presenting cell ammonium pyrrolidine dithiocarbamate arabinose arginine asparagine aspartic acid adenosine triphosphate azidoglucose tert-butyloxycarbonyl (benzotriazol-1yloxy)tris(dimethylamino) phosphonium hexafluorophosphate benzoquinone bovine serum albumin benzoyl catalyst Carbohydrate-Active enZYmes cytidine diphosphate cellulose synthase complex Chemical Linkage of Peptides onto Scaffolds cytidine monophosphate central nervous system capsular polysaccharide cryogenic electron microscopy column volume cysteine doublet. 1,8-diazabicycloundec-7ene dendritic cell - specific intracellular adhesion molecule 3-grabbing nonintegrin 1,2-dichloroethane. DCM dd DIC DIPEA DMF DMSO DNA DOSY DP EDTA EF-P EG ELISA eq ESI ESKAPE. Et FA FF Fmoc FPLC Galf GalNAc Galp GDP Glc GlcA Gln GlcNAc Glu Gly GT h HATU HEPES Hex HFIP His HMW1ct HSQC Hz. dichloromethane doublet of doublets chemical shift, ppm N,N-diisopropylcarbodiimide N,N-diisopropylethylamine N,N-dimethylformamide dimethyl sulfoxide deoxyribonucleic acid diffusion ordered NMR spectroscopy degree of polymerization ethylenediaminetetraacetic acid elongation factor P early glycoforms enzyme-linked immunosorbent assay molar equivalents electrospray ionization Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. ethyl formic acid fast flow fluorenylmethyloxycarbonyl fast protein liquid chromatography galactofuranose N-acetylgalactosamine galactopyranose guanidine diphosphate glucose glucuronic acid glutamine N-acetylglucosamine glutamic acid glycine glycosyltransferase hours (1[bis(dimethylamino)methylene]-1H1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid hexose 1,1,1,3,3,3-hexafluoro-2-propanol histidine C-terminal fragment of high molecular weight adhesin heteronuclear single quantum correlation hertz. . . 1.
(15) I-TASSER IC50 Ile IPTG IR ITC J kcat KD Kdo Km koff. kon KOW LC-MS Leu LPS LS Lys m m/z MALDITOF MALLS Man ManNAc MBP MD Me MHC min MOE MOG MOPS MRSA. 2. Iterative Threading ASSEmbly Refinement inhibitor concentration that results in half-maximal enzyme activity isoleucine isopropyl βthiogalactopyranoside infrared spectroscopy isothermal titration calorimetry coupling constant enzyme turnover number dissociation constant 3-deoxy-D-manno-octolusonic acid Michaelis constant rate constant for the dissociation of the proteinligand complex rate constant for the association of the proteinligand complex Kyrpides-Woese-Ouzounis liquid chromatographycoupled mass spectrometry leucine lipopolysaccharide light scattering lysine multiplet mass over charge ratio matrix-assisted laser desorption ionization time of flight multi-angle light laser scattering mannose N-acetylmannose myelin basic protein molecular dynamics methyl major histocompatibility complex minutes metabolic oligosaccharide engineering myelin oligodendrocyte glycoprotein 3-(N-morpholino) propanesulfonic acid methicillin-resistant Staphylococcus aureus. MS MTBE MurNAc Mw MW-SPPS MWCO n.a. n.d. NADH NAG NAM NCAM NDP NGT NMR NOE NOESY NTA NTHi OD OGT ON OST Oxyma PAGE Pbf. Pd PDB PG Phe Pintr Pls Pn ppm Pro PST q qNMR Rha RI RIF RNA RP-LC rpm RT s SAAT. multiple sclerosis methyl tert-butyl ether N-acetylmuramic acid molecular weight microwave assisted solid-phase synthesis molecular weight cutoff not available not determined nicotinamide adenine dinucleotide N-acetylglucosamine N-acetylmuramic acid neural cell adhesion molecule nucleotide diphosphate N-glycosyltransferase nuclear magnetic resonance Nuclear Overhauser Effect Nuclear Overhauser Effect Spectroscopy nitrilotriacetic acid nontypeable Haemophilus influenzae optical density O-GlcNAc transferase overnight oligosaccharyltransferase ethyl cyanohydroxyiminoacetate polyacrylamide gel electrophoresis 2,2,4,6,7pentamethyldihydrobenzo furan5-sulfonyl palladium protein data bank peptidoglycan phenylalanine intrinsic processivity plasmin-sensitive protein processivity factor parts per million proline polysialyltransferase quartet quantitative NMR rhamnose refractive index relative ionization factor ribonucleic acid reverse-phase liquid chromatography rotations per minute room temperature singlet self-associating autotransporters.
(16) SDR SDS SEC SEDS Ser SH Sia SPE SPPS SPR SPS SRRP STDNMR t TB TBA TBAOH TBS tBu tBuOH TCEP TDP TEAA Tf TFA THF Thr TIS TLC TOCSY TOF TON TPR Tris TR-NOE Trp Trt Tyr UDP UMP Und UniDec UV V0 Val WTA Xyl. . serine-aspartate repeats sodium dodecyl sulphate size-exclusion chromatography Shape, Elongation, Division and Sporulation family of proteins serine SUMO-His6 sialic acid solid phase extraction solid-phase peptide synthesis surface plasmon resonance solvent purification system serine-rich repeat protein Saturation transfer difference Nuclear Magnetic Resonance triplet terrific broth tetrabutylammonium tetrabutylammonium hydroxide Tris buffer saline tert-butyl tert-butanol tris (2-carboxyethyl) phosphine thymidine diphosphate triethylammonium acetate triflate trifluoroacetic acid tetrahydrofuran threonine triisopropylsilane thin-layer chromatography total correlation spectroscopy time of flight turnover number tetratrico peptide repeat tris(hydroxymethyl)aminomethane transfer NOE tryptophan trityl tyrosine uridine diphosphate uridine monophosphate undecaprenol Universal deconvolution ultraviolet initial velocity valine wall teichoic acid xylose. 3.
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