Konformacijska dinamika adenilacijske domene tirocidin-sintetaze 1 praćena metodom fluorescencijske sprektroskopije

doctoral thesis

• Title (english): Conformational dynamic af the adenylation domain in tyrocidine synthetase 1 followed by fluorescence spectroscopy
• Author: Matilda Šprung; MBZ: 289663
• Mentor: Maja Pavela-Vrančić (mentor); MBZ: 35251
• Committee member: Ita Gruić Sovulj (predsjednik povjerenstva)
• Committee member: Maja Pavela-Vrančić (član povjerenstva); MBZ: 35251
• Committee member: Tihana Žanić Grubišić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2014-12-11, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry
• Universal decimal classification (UDC): 54 - Chemistry. Crystallography. Mineralogy

Abstract

Neribosomske peptid-sintetaze (NRPS) su veliki multienzimski kompleksi koji nizom transpeptidacijskih reakcija kataliziraju sintezu biološki aktivnih peptidnih produkata. Proces biosinteze započinje na adenilacijskoj (A) domeni koja u prvom koraku katalizira nastanak aminoacil-adenilata, a u drugom prijenos aminokiseline na susjednu tiolacijsku (T) domenu. Kristalne strukture A-domena ukazuju na velike strukturne preinake tijekom katalitičkog ciklusa, a dovode se u vezu s usklađivanjem katalitičkih koraka i interakcijom sa susjednom T-domenom. U ovom radu, konformacijske promijene A-domene tirocidin-sintetaze 1 (TycA-A) ispitane su u otopini metodom fluorescencijske spektroskopije. TycA-A sadrži pet triptofana u primarnoj sekvenci: W227, W301, W323, W376 i W406. Procjena dostupnosti njihovih bočnih ogranaka izrađena je na temelju homolognih modela TycA-A u dvije konformacije. Gašenje fluorescencije akrilamidom provedeno na divljem tipu i mutantama TycA-A s pojedinačno zamjenjenim triptofanima, ukazuje na dvije skupine fluorofora različite dostupnosti okolnom otapalu. Prisustvo supstrata ima utjecaja jedino na W227 koji može poslužiti kao intrinzična fluorescencijska proba. Uloga nepripadajućih aminokiselina na konformaciju TycA-A, ispitana je na mutanti sa samo jednim fluorescirajućim triptofanom te ukazuje na suboptimalnu konformaciju i slabiju zaštitu reaktivnog međuprodukta od hidrolize. Promjena konformacije kao odraz vezanja supstrata također je ispitana pokusima ograničene triptičke digestije i diferencijalne fluorimetrije.

Abstract (english)

Nonribosomal peptide synthetases (NRPS) are large multienzyme complexes that catalyze the synthesis of biologically active peptides in an array of transpeptidation reactions. The process of biosynthesis starts with the adenylation (A) domain, which in the first half reaction catalyzes the synthesis of the aminoacil-adenylate, and in the second amino acid transfer to the adjacent tiolation (T) domain. Crystal structures of various A-domains show large structural rearrangements accompanying the catalytic process that are in correlation with the coordination of catalytic steps and interaction with the adjacent T-domain. Here we report the investigation of conformational changes in the A-domain of tyrocidine synthetase 1 (TycA-A) probed by fluorescence spectroscopy. TycA-A comprises five tryptophan residues in the primary sequence: W227, W301, W323, W376 and W406. Trp side chain accessibility was probed using TycA-A homology models in two distinctive protein conformations. Fluorescent quenching with acrylamide, carried out on the wild type and mutant TycA-A proteins caring individual Trp substitutions, revealed two independent groups of fluorophores. The Results show that the presence of reaction substrates has affected only the W227 residue which can serve as an intrinsic fluorescent probe. The effect of noncognate amino acid substrates on the TycA-A conformation was probed on mutant TycA-A bearing only one fluorescent Trp residue. The Results show a suboptimal protein conformation and reduced protection of the reaction intermediate from hydrolysis. Conformational changes induced by substrate binding were probed by limited proteolysis and differential scanning fluorimetry methods.

• Keywords: adenilacijska domena; neribosomske peptid-sintetaze; konformacijska dinamika; tirocidin-sintetaza 1
• Keywords (english): adenylation domain; nonribosomal peptide synthetases; conformational dynamics; tyrocidine synthetase 1
• Language: croatian
• URN:NBN: urn:nbn:hr:217:615100
• Study programme: Title: Doctoral study in chemistry; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje kemija (doktor/doktorica znanosti, područje prirodnih znanosti, polje kemija)
• Type of resource: Text
• Extent: 117 str. ; 30 cm
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-02-16 13:50:46