Eksperimentalna karakterizacija polialaninskih regij v fazni separaciji tekoče-tekoče

Struna, Gašper (author), Hadži, San (mentor)

Fazna separacija tekoče–tekoče (LLPS) predstavlja pomemben mehanizem organizacije biomolekul v celicah in sodeluje pri nastanku nemembranskih biomolekularnih kondenzatov. Poleg številnih fizioloških procesov je povezana tudi z razvojem različnih nevrodegenerativnih bolezni, zato je razumevanje molekulskih dejavnikov, ki pomembno vplivajo na njen nastanek, pomembno. Med zaporedji, ki lahko pomembno vplivajo na fizikalno-kemijske lastnosti peptidov, so tudi polialaninske regije, katerih podaljšanje je povezano z nepravilnim zvijanjem proteinov in agregacijo. Namen magistrskega dela je bil sintetizirati polialaninske peptide z različnim številom alaninskih ostankov ter raziskati vpliv dolžine polialaninskega odseka in robnih regij na njihove strukturne in biofizikalne lastnosti. Peptidom smo določili topnost, preverili vpliv dodatka soli na topnost, določili porazdelitveni koeficient med organsko fazo in vodo ter s spektroskopijo krožnega dikroizma spremljali vpliv dolžine peptida, koncentracije, temperature in drugih eksperimentalnih pogojev na sekundarno strukturo. Sposobnost tvorbe LLPS smo preverili preko primerjave med modelnima peptidoma PR-15 in WGR-1 ter s sintetiziranimi polialaninskimi peptidi. Ugotovili smo, da so temperatura in nekateri drugi eksperimentalni pogoji pri daljših peptidih povzročili izrazite spremembe CD-spektrov, medtem ko v preučevanem koncentracijskem območju vpliva koncentracije na sekundarno strukturo nismo zaznali. Medtem ko sta modelna peptida potrdila ustreznost uporabljenega eksperimentalnega pristopa za spremljanje LLPS, sintetizirani polialaninski peptidi v uporabljenih pogojih niso izkazovali primerljive sposobnosti tvorbe biomolekularnih kondenzatov, saj so pretežno tvorili agregate ali gelu podobne strukture. Dobljeni rezultati prispevajo k boljšemu razumevanju povezave med aminokislinskim zaporedjem polialaninskih regij, njihovimi strukturnimi lastnostmi in faznim obnašanjem ter predstavljajo izhodišče za nadaljnje raziskave vpliva zaporedja peptidov na nastanek biomolekularnih kondenzatov.

• Language: Slovenian
• Keywords: polialaninski peptidi, fazna separacija tekoče-tekoče, krožni dikroizem
• Work type: Master's thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-187769
• Publication date in RUL: 14.09.2026

Secondary language

• Language: English
• Title: Experimental characterization of polyalanine regions in liquid-liquid phase separation

Abstract

Liquid–liquid phase separation (LLPS) is an important mechanism of biomolecular organization in cells and contributes to the formation of membraneless biomolecular condensates. In addition to its involvement in numerous physiological processes, LLPS has also been associated with the development of various neurodegenerative diseases, making it important to understand the molecular factors that influence its formation. Polyalanine regions are among the sequences that can significantly affect the physicochemical properties of peptides, and their expansion has been associated with protein misfolding and aggregation. The aim of this master's thesis was to synthesize polyalanine peptides containing different numbers of alanine residues and to investigate the effects of polyalanine tract length and flanking regions on their structural and biophysical properties. Peptide solubility was determined, the effect of salt addition on solubility was examined, the partition coefficient between the organic and aqueous phases was determined, and circular dichroism spectroscopy was used to investigate the effects of peptide length, concentration, temperature, and other experimental conditions on secondary structure. The ability to undergo LLPS was assessed by comparing the model peptides PR-15 and WGR-1 with the synthesized polyalanine peptides. We found that temperature and some other experimental conditions caused pronounced changes in the CD spectra of the longer peptides, whereas no effect of concentration on secondary structure was detected within the concentration range investigated. While the model peptides confirmed the suitability of the experimental approach for monitoring LLPS, the synthesized polyalanine peptides did not exhibit a comparable ability to form biomolecular condensates under the conditions tested, as they predominantly formed aggregates or gels. The results contribute to a better understanding of the relationship between the amino acid sequence of polyalanine regions, their structural properties, and phase behavior, and provide a basis for further studies investigating the influence of peptide sequence on the formation of biomolecular condensates.

• Keywords: polyalanine peptides, liquid-liquid phase separation, circular dichroism