The effect of cosolutes on amyloid fibril formation in the case of β-lactoglobulin

Jaklin, Matej (author), Hribar Lee, Barbara (mentor)

Protein aggregation, particularly amyloid fibrils, poses challenges for biopharmaceutical production and contributes to incurable diseases such as neurodegeneration, transthyretin amyloidosis, and type II diabetes. Amyloid fibrils are highly stable aggregates composed of cross-β structures. They have vast polymorphism and complex formation pathways, involving intermediates like liquid-liquid separation (LLPS) droplets, oligomers, and protofilaments, that complicate structural characterization. A new nucleated confor-mational conversion (NCC) model proposes that fibrils arise from diverse intermediates along an energy funnel, akin to protein folding. Both intrinsic and extrinsic factors, such as temperature, pH, protein concentration, and co-solutes, influence aggregation. Amphiphilic protein β-lactoglobulin (BLG) is a widely available whey protein with high concentration of β-structures that serves as an excellent amyloid model. This study systematically varied process conditions and co-solutes to explore BLG aggregation across pH values using spectroscopic, microscopic, biochemical, and diffraction techniques. At pH 2 in glycine buffer and 80 °C, a novel fibrillization pathway was discovered involving LLPS, α-helix rearrangement, and reduced acid hydrolysis susceptibility. Hexameric oligomers aligned on LLPS surfaces and formed nanotubes and fibrils. Stirring promoted transitions from worm-like to rigid amyloid fibrils. At pH 7 and high temperatures (80 °C), BLG’s Cys121 initiates disulfide scrambling, preventing fibril formation. However, at low temperatures (50 °C), this scrambling was suppressed, leading to large colored microribbons and protein vesicles that formed from LLPS droplets. With added 2 M KSCN, unfolded BLG formed tapes, fibrils, and ring-like aggregates, showing NCC-driven kinetics and cross-α interactions. This work presents a new framework for amyloid fibrillization, linking LLPS, oligomerization, vesicle formation, and secondary structure transitions. It offers novel avenues for therapeutic development and the design of self-assembled biomaterials. BLG-derived structures also show promise in photonic applications, mimicking amelogenin-based enamel formation and enabling use in drug delivery, sensors, coatings, and optical devices.

• Language: English
• Keywords: β-lactoglobulin, amyloid fibrils, polymorphism, co-solutes, phase separation, amphiphilicity, protein vesicles, thiol groups, microscopy, aggregation, photonic crystals
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-174063
• COBISS.SI-ID: 254039811
• Publication date in RUL: 26.09.2025

Secondary language

• Language: Slovenian
• Title: Vpliv sotopljencev na tvorbo amiloidnih fibril na primeru β-laktoglobulina

Abstract

Agregacija proteinov v amiloidna vlakna predstavlja izziv za biotehnološke aplikacije in je vpletena v številne neozdravljive bolezni, kot so nevrodegenerativne bolezni, transtiretinska amiloidoza in sladkorna bolezen tipa II. Amiloidi so stabilni proteinski agregati s specifično zgradbo prepletenih β-plasti imenovano β-sterična zadrga. Njihovo nastajanje vključuje različne vmesne strukture, kot so kapljice fazne separacije tekoče-tekoče (LLPS), oligomeri in protofibrili. Na agregacijo vplivajo notranji in zunanji dejavniki, kot so pH, temperatura, strig, koncentracija in prisotnost sotopljencev. Z uporabo različnih sotopljencev lahko vplivamo na specifične interakcije v raztopini proteinov in na ta način odkrijemo nove potencialne spojine za zaviranje agregacije. Amfifilni protein kot je β-laktoglobulin (BLG), je široko dostopen protein mlečne sirotke in zaradi visokega deleža β-struktur predstavlja dober model za študij amiloidov. Pri pH 2 in uporabi glicina kot pufra sem pri 80 °C odkril nov mehanizem fibrilizacije BLG-ja, ki vključuje LLPS, prerazporeditev α-vijačnice in manšjo stopnjo razgradnje s kislinsko hidrolizo. Nastali so heksamerični oligomeri, nanotube in fibrili ter LLPS kaplje stabilizirane z mehkimi mikrogelnimi delci. Pri pH 7 in visoki temperaturi (80 °C) na novo izpostavljena skupina cisteina tvori inter-molekularne disulfidne mostičke, ki preprečijo nastanek amiloidnih fibril. Pri nižji temperaturi (50 °C) je bila reaktivnost prostega tiola nižja in BLG je namesto tega tvoril proteinske vezikle, mikrotrakove in barvne fotonske kristale, ki so nastali iz LLPS kapljic. Strukturna analiza nakazuje na urejeno povezovanje α-vijačnic in α-sterično zadrgo. To delo predstavlja drugačen pogled na amiloidno fibrilizacijo, saj skupaj poveže vse procese od LLPS, oligomerizacije, nastanka veziklov, fibril in vsebnost različnih tipov sekundarnih struktur. Zaradi nove teorije se ponudijo nove ideje za razvoj zdravil in boj proti nevrodegeneraciji ter načrtovanju novih biomaterialov. Strukture, pridobljene iz BLG-ja, obetajo tudi na področju fotonskih kristalov, posnemajo tvorbo zobne sklenine in imajo velik potencial za dostavo zdravil, na področju senzorjev, premazov ter za uporabo v optičnih napravah.

• Keywords: β-laktoglobulin, amiloidni fibrili, polimorfizem, so-topljenci, fazna separacija, amfifilnost, proteinski vezikli, tiolne skupine, mikroskopija, agregacija, fotonski kristali