Mapping of sufrace-exposed cysteines in ATPase ISWI

master's thesis

• Title (croatian): Mapiranje površinski izloženih cisteina u ATP-azi ISWI
• Author: Petra Vizjak
• Mentor: Felix Müller-Planitz (mentor)
• Committee member: Marko Močibob (predsjednik povjerenstva)
• Committee member: Đani Škalamera (član povjerenstva)
• Committee member: Branimir Bertoša (član povjerenstva)
• Committee member: Jasmina Rokov Plavec (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2017-09-29, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

In an eukaryotic cell, DNA molecule exists in the form of chromatin. The repetitive unit of
chromatin is a nucleosome, 147 bp of DNA wrapped around histone octamer. Nucleosome
position along DNA is important for transcription regulation. Chromatin remodeler ISWI
(Drosophila melanogaster) slides nucleosomes along DNA. The aim of this work is to map
surface-exposed cysteines in the protein ISWI. The surface-exposed cysteines are easily
avaliable for chemical reaction. To probe accessibility of cysteines for chemical reaction,
commonly used reagents for spectrophotometric detection of thiols were used: 5,5ʹ-dithiobis-
(2-nitrobenzoic acid) and 2-nitro-5-thiocyanatobenzoic acid. To unequivocally identify surfaceexposed
cysteines, ISWI was treated with primary alkylating reagent (N-ethylmaleimide),
denaturated and treated with secondary alkylating reagent (iodoacetic acid). The two reactions
add to the cysteine different modification groups which can be discriminated by mass
spectrometry. Half of the total cysteines could be clasified either as buried or surface-exposed
in the protein structure.

Abstract (croatian)

U eukariotskoj stanici, molekula DNA nalazi se u obliku kromatina. Ponavljajuća jedinica
kromatina je nukleosom, 147 parova baza DNA namotano oko histonskog oktamera. Položaj
nukleosoma na DNA ima važnu ulogu u kontroli transkripcije. Remodelirajući protein ISWI
(Drosophila melanogaster) pomiče nukleosome duž DNA. Cilj ovog rada je mapirati
površinski izložene cisteine proteina ISWI. Rezultati će kasnije biti iskorišteni za analizu
postojećih strukturnih modela proteina ISWI. Cisteini koji se nalaze na površini proteina, brže
podliježu kemijskim reakcijama. Kako bi se potvrdilo da cisteini reagiraju različitom
kinetikom, korišteni su reagensi za spektrofotometrijsko utvrđivanje tiola: 5,5ʹ-ditiobis-(2-
nitrobenzojeva kiselina) i 2-nitro-5-tiocianatobenzojeva kiselina. Za točnu identifikaciju
površinski izloženih cisteina, ISWI je tretiran s primarnim alkilirajućim reagensom (Netilmaleimidom),
denaturiran i tretiran sa sekundarnim alkilirajućim reagensom (jodoctenom
kiselinom). Produkt tih dviju reakcija je dodatak različitih modifikacijskih skupina na cisteine,
koje se potom mogu razlučiti spektrometrijom masa. Polovica od ukupnih cisteina mogla se
klasificirati kao površinski izloženi ili zaklonjeni unutar strukture proteina.

• Keywords: cysteine mapping; cysteine alkylation; Ellman; ISWI; NTCB
• Keywords (croatian): alkiliranje cisteina; Ellman; ISWI; mapiranje cisteina; NTCB
• Language: english
• URN:NBN: urn:nbn:hr:217:949570
• Study programme: Title: Graduate university studies in chemistry; specializations in: Research programme; Course: Research programme; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-10-20 10:51:25