Prepoznavanje proteinov s pomočjo strukturne prilagodljivosti

primerjava vezanja intrinzično nestrukturiranih proteinov in kameloidnih protiteles

Samo Purič (Avtor), San Hadži (Mentor), Miha Pavšič (Član komisije za zagovor), Jurij Lah (Član komisije za zagovor)

Povzetek

V magistrskem delu smo s strukturnega in termodinamskega vidika preučili dva primera vezanja proteinov, pri katerih pride do prepoznavanja s strukturno prilagoditvijo enega od partnerjev. V prvem delu smo se osredotočili na interakcije med intrinzično nestrukturiranimi proteini (IDP-ji), ki ob vezavi na globularno tarčo tvorijo izključno α-vijačnico. S pomočjo literaturnih podatkov in naših lastnih meritev smo sestavili reprezentativen vzorec α-heličnih IDP-jev. Ugotovili smo, da je delež α-vijačnice v nevezani obliki znaten, IDP-ji so torej predzviti, ob vezavi pa se delež α-vijačnice poveča. Prisotnost takšne prehodne helične strukture v prostem stanju je iz termodinamskega vidika ugodna, saj zmanjša delež za katerega se morajo IDP-ji ob vezavi na tarčo še dodatno zviti. Izkaže se, da je razlog za opažene visoke deleže urejene strukture v nevezanem stanju prisotnost hidrofobnega aminokislinskega ostanka levcina in nekaterih vezavnih motivov, ki so sestavni del intrinzično neurejenih trans-aktivacijskih domen (še posebej pogost je levcinski motiv LXXLL). Levcin namreč zaradi svojih lastnosti pomembno vpliva na stabilnost predzvite strukture, prav tako pa tvori pomembne interakcije, ki stabilizirajo kompleks α-heličnih IDP-jev s tarčo. V drugem delu naloge pa smo s pomočjo izotermne titracijske kalorimetrije (ITC) analizirali interakcije med fragmenti kameljih protiteles in njihovimi tarčami (antigena HigB2 in MazF). Termodinamske prispevke vezanja smo poskušali pojasniti s pomočjo lastnosti aminokislinskega zaporedja zank CDR. Ugotovili smo, da je strukturna prilagodljivost hipervariabilnih regij sicer prisotna, vendar pa ne predstavlja poglavitnega prispevka k visoki afiniteti vezave. Razmeroma visoka vsebnost hidrofobnih ak-ostankov v regijah CDR3 in povezava števila le-teh z afiniteto vezave kažeta na to, da imajo interakcije nanoteles s pripadajočimi antigeni najverjetneje izrazit hidrofoben značaj.

Ključne besede

vezanje proteinov;strukturna prilagodljivost;intrinzično nestrukturirani proteini;nanotelesa;kameloidna protitelesa;magistrska dela;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2022
Tipologija:	2.09 - Magistrsko delo
Organizacija:	UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik:	[S. Purič]
UDK:	577.112(043.2)
COBISS:	98002435
Št. ogledov:	227
Št. prenosov:	23
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	Protein recognition by conformational plasticity: a comparison of binding by intrinsically disordered proteins and with camelid antibodies
Sekundarni povzetek:	The presented thesis examines two cases of protein binding from a structural and a thermodynamic point of view in which protein recognition occurs through the structural adaptation of one of the binding partners. First, we focused on the interactions of intrinsically disordered proteins (IDPs) which consist of sequences that fold into α-helices upon binding to their globular protein targets. Using data from literature and from our own measurements, we compiled a representative dataset of α-helical IDPs. We found that IDPs in general possess significant residual helicity in their unbound state (pre-folded structure) and gain helicity upon binding their targets. The presence of such a transient helical structure in the unbound state is advantageous from a thermodynamic point of view, as it reduces the entropic penalty of folding and thereby increases interaction affinity. It turns out that the reason for the observed ordered structure in the unbound state is, among other things, the presence of leucine – a hydrophobic amino acid residue – and some typical binding motifs that are an integral part of intrinsically disordered transactivation domains (the LXXLL leucine motif is especially common). Due to its properties, leucine significantly affects the stability of the pre-folded structure and forms important stabilizing interactions between an IDP and its target. In the second part of this thesis, the interactions of camelid antibody fragments (nanobodies) and their targets (HigB2 and MazF antigens) were analyzed using isothermal titration calorimetry (ITC). The goal was to explain the thermodynamic contributions of binding using the properties of the amino acid sequences of the hypervariable regions (CDR loops) of nanobodies. We observed that some conformational plasticity of the hypervariable loops is present but found that it doesn’t represent a major contribution to the high binding affinity of nanobodies. A relatively high content of hydrophobic amino acid residues in the CDR3 regions and its apparent connection with binding affinity both suggest a pronounced hydrophobic character of nanobody-antigen interactions.
Sekundarne ključne besede:	protein binding;structural plasticity;intrinsically disordered proteins;nanobodies;Beljakovine;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS):	Magistrsko delo/naloga
Študijski program:	1000377
Konec prepovedi (OpenAIRE):	1970-01-01
Komentar na gradivo:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Strani:	51 str.
ID:	14503988