Interparticle interactions in aqueous solutions of globular proteins : doctoral dissertation
ID Brudar, Sandi (Author), ID Hribar-Lee, Barbara (Mentor) More about this mentor... This link opens in a new window, ID Pavšič, Miha (Thesis defence commission member), ID Spohr, Eckhard (Thesis defence commission member), ID Vlachy, Vojko (Thesis defence commission member)

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In the present dissertation we investigated the influence of interparticle interactions on the phase stability of some globular proteins, with our main focus on hen egg-white lysozyme (HEWL). Our research was performed as an interplay of diverse experimental techniques, theoretical approach and molecular dynamics computer simulations. Our work is divided into four parts: Phase stability and crystallization of HEWL, fibrillization of HEWL, expression and investigation of T4 lysozyme variants and molecular dynamics simulations of aqueous solutions of globular proteins. Firstly, we briefly studied the crystallization of HEWL by employing different crystallization techniques under various solution conditions. Results have shown that liquid-liquid phase separation (LLPS) enhances the crystallization of HEWL and produces more HEWL crystals with less impurities than via conventional solvent evaporation method. Impurities, which are initially present in purchased HEWL samples were shown to have insignificant impact on the phase stability of HEWL solutions. We proceeded by studying the phase stability of 10 different HEWL–buffer solutions. We found that the phase stability of HEWL–buffer solutions strongly depends on solution conditions and also on the choice of buffer. The results of phase stability of HEWL in multiple buffers at pH=7.0 have undoubtedly indicated towards the presence of buffer-specific effects. By using a simple model within the framework of Wertheim’s perturbation theory, we could predict the coexistence curves of HEWL in multiple buffers at pH=7.0, which were in good agreement with our own measurements of cloud point temperatures (Tcloud) of these solutions. In addition, we made use of this model to ascertain the influence of buffers at pH=7.0 on protein-protein interactions, as well as on model parameters. Our findings suggest to a competition between buffer and salt anions for binding to the positively charged surface of HEWL. By measuring the dynamic light scattering (DLS) of HEWL–buffer solutions, we determined the second virial coefficient and interaction diffusion coefficient (B22 and kD), which allowed us to gain knowledge on actual protein-protein interactions in investigated HEWL solutions. Both B22 and kD were shown to be in reasonable correlation with measured Tcloud values. In the second part we investigated how different solution conditions affect the fibrillization of HEWL. The formation of HEWL amyloid fibrils was detected by means of highly amyloidophilic dyes thioflavin T (ThT) and Congo red (CR). HEWL fibrillized only under agitated conditions at pH=2.0, where the conditions enabled partial protein unfolding and at the same time allowed the procedure to overcome the electrostatic repulsion barrier due to highly positively charged HEWL molecules. These conditions were only met by 0.5 M glycine buffer and KCl-HCl buffer at 0.25 M and 0.5 M ionic strength. At identical, favourable, solution conditions HEWL fibrils were not observed in phosphate buffer, suggesting to buffer-specific effects. Additional buffers were also found to have a stabilizing role as they too prevented the fibrillization of HEWL. Electrostatic charge screening was proved to play an important, but not decisive role for the fibrillization process. By adding a sufficient amount of inert polymer, PEG12000, we managed to arrest the fibrillization of HEWL in a previously highly amyloidogenic solution. In the third part of this dissertation we carried out the expression of a pseudo-wild type (WT*) T4 lysozyme and its respective single point mutant L99I*. The protein expression procedure was optimized to the extent that we harvested up to 100 mg of both proteins from 3.2 L of growth medium. Recorded circular dichroism (CD) spectra of both proteins have shown the replacement of leucine with isoleucine does not meaningfully affect the secondary structure content with respect to T4 WT* lysozyme. In addition, both T4 lysozyme variants exhibited almost 30 % higher α-helix content than HEWL atidentical solution conditions. Measurements of phase stability of T4 WT* lysozyme have demonstrated that T4 WT* lysozyme is less prone to phase separation than HEWL, since despite cooling the T4 WT* solutions to -6°C, no phase separation was achieved at identical solution conditions. Both CD spectroscopy and differential scanning calorimetry (DSC) have indicated that T4 WT* lysozyme is conformationally more stable than T4 L99I* lysozyme, since the latter unfolded at ≈4.3°C lower temperature. Lastly, we carried out multiple atomistic molecular dynamics simulations of aqueous solutions of HEWL, T4 WT* lysozyme and γ-D crystallin. The calculated density fluctuations have shown distinct protein-rich and protein-poor regions for all solutions with high protein content. The nature of protein self-association was found to be partially reversible, which was especially pronounced in solutions that were close to experimentally observed phase separation conditions. The observed self-association of proteins was reflected in their respective diffusion coefficients, which decreased with rising protein concentration and protein molecular weight, as well as with decreasing temperature. Hydration of proteins was assessed by calculating the water-oxygen – protein centre-of-mass (COM) pair distribution functions, as well as with the solvent accessible surface area (SASA). The latter results have shown the water-accessible surface area depends not only on the extent of self-association, but also on the degree of its reversibility. We have monitored the time-dependence of radius of gyration and demonstrated that in spite of forming protein clusters all proteins remained in their compact (native) state. The calculated protein-protein centre-of-mass (COM) pair distribution functions exhibited several interaction peaks, corresponding to most frequent contacts between proteins. A thorough analysis of residue-residue contacts between all pairs of proteins enabled us to determine the most important residue-residue pairs, as well as individual amino acids responsible for the formation of initial contacts. In solutions of both HEWL and γ-D crystallin, arginine was the predominating amino acid residue in both initiating first contacts, as well as forming overall residue pairs, with its partners being mostly residues capable of forming salt bridges and hydrogen bonds. Meanwhile, for solutions of T4 WT* lysozyme, the title of the prevailing amino acid belonged to lysine, which was most often seen to be paired with the oppositely charged aspartic acid.

Keywords:lysozyme, globular proteins, protein-protein interactions, cloud point temperature, phase stability, amyloid fibrils, buffer-specific effects, molecular dynamics, expression of proteins
Work type:Doctoral dissertation
Typology:2.08 - Doctoral Dissertation
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Place of publishing:Ljubljana
Publisher:[S. Brudar]
Number of pages:XXIV, 232 str.
PID:20.500.12556/RUL-122021 This link opens in a new window
COBISS.SI-ID:32433411 This link opens in a new window
Publication date in RUL:16.11.2020
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Secondary language

Title:Meddelčne interakcije v vodnih raztopinah globularnih proteinov
V pričujoči disertaciji smo proučili vpliv meddelčnih interakcij na fazno stabilnost nekaterih globularnih proteinov, s poudarkom na kokošjem lizocimu iz jajčnega beljaka (jajčni lizocim). Raziskavo smo izvedli v okviru prepleta raznolikih eksperimentalnih tehnik, teoretičnega pristopa in računalniških simulacij molekulske dinamike. Delo je razdeljeno na štiri dele: Fazna stabilnost in kristalizacija jajčnega lizocima, fibrilizacija jajčnega lizocima, ekspresija in proučevanje različic lizocima T4 ter simulacije vodnih raztopin globularnih proteinov z molekulsko dinamiko. Sprva smo na kratko, z uporabo različnih kristalizacijskih pristopov, raziskali kristalizacijo jajčnega lizocima pri različnih pogojih v raztopini. Rezultati so pokazali, da fazna separacija kapljevina-kapljevina (LLPS) spodbudi kristalizacijo jajčnega lizocima in pridela več kristalov zmanjšo prisotnostjo nečistoč kot pri konvencionalnem izhlapevanju topila. Za nečistoče, ki so prisotne v kupljenem jajčnem lizocimu je bilo prikazano, da imajo zanemarljiv vpliv na fazno stabilnost njegovih raztopin. Nadaljevali smo s proučevanjem fazne stabilnosti 10 različnih pufrskih raztopin jajčnega lizocima. Ugotovili smo, da je fazna stabilnost pufrskih raztopin jajčnega lizocima močno odvisna od pogojev v raztopini in prav tako od izbire pufra. Rezultati fazne stabilnosti jajčnega lizocima v številnih pufrih pri pH=7,0 so nedvomno pokazali na prisotnost pufer specifičnih efektov. Z uporabo preprostega modela v okviru Wertheimove perturbacijske teorije smo lahko napovedali koeksistenčne krivulje jajčnega lizocima v številnih pufrih pri pH=7,0, ki so se dobro ujemale z našimi lastnimi meritvami temperature zmotnitve (Tzmotnitve) teh raztopin. Poleg tega, smo z uporabo tega modela ovrednotili vpliv pufrov pri pH=7,0 na interakcije med proteini, kakor tudi na parametre modela. Naše ugotovitve predpostavljajo kompeticijo med anioni pufra in soli za vezavo na pozitivno nabito površino jajčnega lizocima. Z merjenjem dinamičnega sipanja svetlobe (DLS) pufrskih raztopin jajčnega lizocima, smo določili drugi virialni koeficient in interakcijski difuzijski koeficient (B22 in kD), ki sta nam podala informacije o dejanskih medproteinskih interakcijah v proučevanih raztopinah jajčnega lizocima. Za tako B22 kot kD je bilo prikazano, da sta v smiselni korelaciji z izmerjenimi vrednostmi Tzmotnitve. V drugem delu smo proučili, kako različni pogoji v raztopini vplivajo na fibrilizacijo jajčnega lizocima. Nastanek amiloidnih fibril jajčnega lizocima smo zaznali z uporabo visoko amiloidofilnih barvil tioflavina T (ThT) in Conga rdeče (CR). Jajčni lizocim je fibriliziral zgolj pod vzbujenimi pogoji pri pH=2,0, kjer so pogoji omogočali delno razvitje proteina in hkrati omogočili, da je proces uspel premostiti elektrostatsko odbojno bariero zaradi visoko pozitivno nabitih molekul jajčnega lizocima. Omenjeni pogoji so bili izpolnjeni zgolj v 0,5 M glicinskem pufru ter v KCl-HCl pufru z 0,25 in 0,5 M ionsko jakostjo. Pri enako ugodnih pogojih pa fibrile jajčnega lizocima niso bile opažene v fosfatnem pufru, kar nakazuje na pufer specifične efekte. Tudi za nekatere dodatne pufre je bila ugotovljena stabilizirajoča vloga, saj so prav tako preprečili fibrilizacijo jajčnega lizocima. Ugotovljeno je bilo, da senčenje elektrostatskih odbojev igra pomembno, a ne ključno vlogo pri procesu fibrilizacije. Z dodatkom zadostne količine inertnega polimera, PEG12000, smo uspeli preprečiti fibrilizacijo jajčnega lizocima v predhodno visoko amiloidogeni raztopini. V tretjem delu te disertacije smo izvedli ekspresijo psevdo-divjega tipa (WT*) lizocima T4 in njegove točkovne mutante L99I*. Ekspresijo proteinov smo optimizirali do te mere, da smo pridobili do 100 mg obeh proteinov iz 3,2 L medija za gojenje. Posneti spektri cirkularnega dikroizma (CD) obeh proteinov so pokazali, da nadomestitev levcina z izolevcinom nima pomenljivega vpliva na sestavo sekundarne strukture glede na lizocim T4 WT*. Poleg tega sta obe različici lizocima T4 prikazali skoraj 30 % višjo vsebnost α-vijačnic kot jajčni lizocim pri identičnih pogojih v raztopini. Meritve fazne stabilnostilizocima T4 WT* so dokazale, da je lizocim T4 WT* manj dovzeten za fazno separacijo kot jajčni lizocim, saj fazna separacija, navkljub ohlajanju lizocima T4 WT* pri enakih pogojih v raztopini vse do -6°C, ni bila dosežena. Tako CD spektroskopija kot diferenčna dinamična kalorimetrija (DSC) sta pokazali, da je lizocim T4 WT* konformacijsko bolj stabilen od lizocima T4 L99I*, saj se je slednji razvil pri ≈4,3°C nižji temperaturi. Na koncu predstavimo še izvedbo številnih atomističnih simulacij vodnih raztopin jajčnega lizocima, lizocima T4 WT* in γ-D kristalina z uporabo molekulske dinamike. Izračunane fluktuacije v gostoti so pokazale jasno razvidne, s proteini obogatene in s proteini osiromašene regije, za vse raztopine z visoko vsebnostjo proteinov. Narava samozdruževanja proteinov je bila delno reverzibilna, kar je bilo še posebej izraženo v raztopinah, ki so bile blizu eksperimentalnim pogojem fazne separacije. Omenjeno samozdruževanje proteinov se je odrazilo v njihovih difuzijskih koeficientih, ki so se znižali z rastočo koncentracijo proteinov in njihovo molekulsko maso, kakor tudi s padajočo temperaturo. Hidratacijo proteinov smo ocenili z izračunom parskih porazdelitvenih funkcij za center mase kisikovega atoma vode in proteina, kakor tudi s topilu dostopno površino (SASA). Rezultati slednje so pokazali, da je vodi dostopna površina odvisna ne le od obsega samozdruževanja, pač pa tudi odstopnje reverzibilnosti tega procesa. Spremljali smo tudi časovno odvisnost radija giracije in dokazali,da so navkljub tvorbi proteinskih klastrov vsi proteini ostali v svojem kompaktnem (nativnem) stanju. Izračunane parske porazdelitvene funkcije za medproteinske centre mas (COM) so pokazale številne vrhove interakcij, ki ustrezajo najbolj pogostim kontaktom med proteini. Celovita analiza kontaktov aminokislina-aminokislina med vsemi pari proteinov nam je omogočila določitev najbolj pomembnih parov aminokislina–aminokislina, kakor tudi posameznih aminokislin, odgovornih zatvorbo začetnih kontaktov. V raztopinah tako jajčnega lizocima kot γ-D kristalina je bil arginin prevladujoči aminokislinski ostanek v tako sprožitvi prvih kontaktov, kakor tudi v celokupni tvorbi parov aminokislin, pri čemer so bili njegovi partnerji večinoma ostanki, ki so sposobni tvoriti solne mostičke in vodikove vezi. V raztopinah lizocima T4 WT* pa je naziv prevladujoče aminokisline pripadel lizinu, ki je bil najpogosteje opažen v paru z nasprotno nabito asparaginsko kislino

Keywords:lizocim, globularni proteini, medproteiske interakcije, temperatura zmotnitve, fazna stabilnost, amiloidne fibrile, pufer specifični efekti, molekulska dinamika, ekspresija proteinov, doktorske disertacije

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