The role of co-solutes in complexation of globular proteins with synthetic polyelectrolytes in aqueous solutions

Simončič, Matjaž (author), Lukšič, Miha (mentor)

In the present work, the influence of various co-solutes on the aggregation between globular proteins as well as on the complexation of globular proteins with synthetic polyelectrolytes (PEs) was investigated. Conclusions were drawn using an assortment of experimental techniques, molecular dynamics (MD) simulations, and other theoretical approaches. The modulating role of three types of co-solutes was studied: salts (NaCl, NaBr, NaI, NaNO3), polyethylene glycol (PEG) with different molecular weights, and two sugar analogs (sucrose, sucralose). The effect of co-solutes was investigated with respect to the colloidal stability of protein- and protein-PE-containing aqueous solutions as well as on the conformational stability of the protein in such solutions. It was found that protein aggregation and protein-PE complexation can be explained in light of the balance between attractive and repulsive electrostatic interactions (forces) depending on the pH of the solution with respect to the isoionic point of the protein (pI). The strength of the interactions and their modulation depend strongly on the heterogeneous charge distribution of the protein surface and the protein-PE system studied, however, some general conclusions can be drawn. The modulating role of salts was most pronounced considering protein-protein and protein-PE interactions, with the effect depending on the pH of the solution with respect to the pI as well as on the concentration of salt ions. Moreover, it was shown that the modulating effect of a salt depends on the chemical identity of the salt anion, as more chaotropic anions screen electrostatic forces more effectively. In addition, complexation of proteins with PEs for protein-PE pairs studied can be accompanied by conformational changes of the protein related to the extent of complex formation, which can in some cases be regulated by the presence of salt ions. Although the effect of neutral (non-ionic) co-solutes, such as PEG or sugars on protein aggregation and protein-PE complexation was less pronounced, we nevertheless showed that under certain conditions macromolecular association can be modulated. In particular, chemical modification of sucrose was demonstrated to change its water-structuring capability around proteins, which altered the stabilizing property of the sugar with respect to protein aggregation and protein-PE complexation. Protein surface anisotropy and the localization of protein binding sites (charge patches) were shown to be important in evaluating protein-PE complexation. A theoretical approach combining a machine-learning (ML) algorithm and target molecular docking for more efficient protein-ligand docking was developed and tested on a series of protein complexes with peptides as well as with small organic molecules. We demonstrated the advantages of implementing ML in classical docking protocols, which is particularly convenient when no experimental data on protein-ligand complexes are available, such as for protein complexes with synthetic PEs.

• Language: English
• Keywords: globular proteins, polyelectrolytes, complexation, co-solutes, aqueous solutions
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-145053
• COBISS.SI-ID: 148214275
• Publication date in RUL: 31.03.2023

Secondary language

• Language: Slovenian
• Title: Vloga sotopljencev pri kompleksaciji globularnih proteinov s sintetičnimi polielektroliti v vodnih raztopinah

Abstract

V pričujočem delu so prikazani rezultati vpliva različnih sotopljencev na agregacijo med globularnimi proteini ter na kompleksacijo globularnih proteinov s sintetičnimi polielektroliti (PE). Raziskave smo izvedli s pomočjo nabora različnih eksperimentalnih tehnik, simulacij molekulske dinamike (MD) in drugih teoretičnih pristopov. Osredotočili smo se na proučevanje modulacijske vloge treh vrst sotopljencev: soli (NaCl, NaBr, NaI, NaNO3), polietilen glikola (PEG) različnih molekulskih mas in dveh analogov sladkorja (saharoza, sukraloza). Vpliv sotopljencev smo proučevali z ozirom na koloidno stabilnost vodnih raztopin globularnih proteinov kot tudi vodnih mešanic le-teh s polielektroliti. Pri interakciji različnih zvrsti (sotopljenci, polielektroliti) s proteini smo spremljali tudi konformacijsko stabilnost obravnavanih proteinov v tovrstnih raztopinah. Ugotovili smo, da je agregacijo proteinov kot tudi kompleksacijo proteinov s polielektroliti moč pojasniti v luči ravnovesja med privlačnimi in odbojnimi elektrostatskimi interakcijami (silami), ki so v večji meri odvisne od pH-vrednosti raztopine z ozirom na izoionsko točko proteina (pI). Jakost interakcij in njihova modulacija zavisita od heterogene porazdelitve naboja na površini proteina kot tudi od proučevanega para protein-PE. Prisotnost ionov soli je na interakcije med samimi proteini kot tudi med proteini in polielektroliti imela največji učinek, pri čemer pa je bila modulacija odvisna pretežno od pH-vrednosti raztopine z ozirom na pI in od koncentracije same soli. Poleg tega smo pokazali, da je učinek soli na meddelčne interakcije odvisen tudi od vrste iona soli, saj bolj kaotropni anioni učinkoviteje senčijo elektrostatske sile med makromolekulami. Za proučevane sisteme smo pokazali, da lahko močne elektrostatske interakcije med proteini in polielektroliti povzročijo konformacijske spremembe proteina, ki so neposredna posledica tvorbe kompleksov, v določenih primerih pa jih lahko reguliramo z dodatkom soli. Čeprav je bil učinek nevtralnih (neionskih) sotopljencev, kot sta PEG ali sladkor, na agregacijo proteinov in kompleksacijo le-teh s polielektroliti manj izrazit, smo kljub temu pokazali, da je samozdruževanje makromolekul v tovrstnih raztopinah pri določenih pogojih vseeno moč modulirati z nevtralnimi sotopljenci. Na tej točki lahko omenimo kemijsko modifikacijo saharoze, ki bistveno spremeni njene sposobnosti strukturiranja molekul vode v okolici proteinov, kar posredno vpliva tako na interakcije med samimi proteini kot med proteini in polielektroliti v prisotnosti modificiranega sladkorja. S kombinacijo eksperimentalnega dela in teoretičnih pristopov smo pokazali, da sta anizotropija površine proteina in lokalizacija vezavnih mest drugih zvrsti na površini proteinov bistveni pri obravnavi asociacije proteinov z ligandi, denimo polielektroliti. Na podlagi tega smo razvili teoretični pristop, ki združuje napovedno moč algoritma strojnega učenja in tarčno molekulsko sidranje. Pristop, katerega namen je učinkovitejše tarčno molekulsko sidranje, je bil validiran na vrsti proteinskih kompleksov s peptidi kot tudi z majhnimi organskimi molekulami. S tovrstnim pristopom smo predstavili nekatere prednosti implementacije strojnega učenja v klasičnih protokolih sidranja, kot so denimo presejalni protokoli. Prav tako smo pokazali uporabnost takšnega pristopa pri oceni vezavne regije na proteinu, kadar eksperimentalni podatki o kompleksih protein-ligand niso na voljo (npr. kompleksi med proteini in sintetičnimi polielektroliti)

• Keywords: globularni proteini, polielektroliti, kompleksacija, sotopljenci, vodne raztopine