Study of the mechanism of heat and chemical denaturation of model proteins using FTIR spectroscopy

Infrardeča spektroskopija sodi med manj pogoste metode za preučevanje konformacijskih sprememb proteinov. Kljub temu metoda nudi nekatere prednosti, kot so meritve majhnih količin vzorcev v različnih agregatnih stanjih. Pridobimo lahko informacije o medmolekulskih interakcijah, še posebej pa je metoda občutljiva pri karakterizaciji vodikovih vezi, ki sodijo med nekatere izmed glavnih sil, ki stabilizirajo strukturo proteinov. Proces denaturacije proteinov je pogosto sklopljen še z njihovo agregacijo. V magistrskem delu smo spremljali spremembe v sekundarni strukturi treh modelnih proteinov pri njihovi temperaturni in kemični denaturaciji z natrijevim dodecilsulfatom. Konformacijske spremembe smo zaznali z uporabo diferenčne spektroskopije. Z integriranjem vrhov v diferenčnih spektrih smo določili temperaturo denaturacije proteinov ter spremembe deležev elementov sekundarnih struktur. Pokazali smo, da lahko s FTIR zaznamo spremembo v temperaturi denaturacijskega prehoda ob prisotnosti majhne količine denaturanta. Z metodo dekompozicije in prileganja modelnih spektralnih trakov smo uspešno napovedali deleže sekundarnih struktur v nativnem stanju in iz površin modelnih trakov izračunali temperaturno odvisnost deleža α-vijačnic in β-agregatov za hemoglobin. Agregacija je pri hemoglobinu in konalbuminu nastopila v celotnem koncentracijskem območju preiskanim s FTIR spektroskopijo. Prisotnost dodecilsulfata je vodila do počasne agregacije proteinov. Pri visokih koncentracijah denaturanta je bila karakterizacija spektrov nemogoča zaradi prenasičenja signalov v spektru. Inzulin pri nizkih vrednostih pH raztopin ob temperaturnih meritvah ni kazal bistvenih sprememb v strukturi poleg slabenja vodikovih vezi. Ta podatek se sklada s predhodnimi rezultati, ki pokažejo rast fibril šele po začetni lag fazi.Infrared spectroscopy is an unusual method for studying conformational changes of proteins. Nevertheless, the method offers some advantages, such as the possibility to measure small amounts of samples in different states. We can obtain information about intermolecular interactions, and it is particularly sensitive in characterizing hydrogen bonds, which are among the leading forces that stabilize proteins. The process of denaturation is very often coupled with protein aggregation. In this work, we followed the changes in the secondary structure of three model proteins under temperature and chemical denaturation conditions with sodium dodecyl sulfate. We used difference spectroscopy to detect conformational changes of the proteins. By integrating peaks in difference spectra, we predicted denaturation temperatures of proteins and changes in the amount of secondary structures. We showed that we can detect changes in transition temperatures with FTIR when small amounts of denaturant are present. Using decomposition and peak fitting, we successfully predicted the amount of secondary structures in the native form and calculated the temperature dependence of the secondary structure content of α-helices and β-aggregates for hemoglobin. Hemoglobin and conalbumin experienced aggregation throughout the working concentration range of FTIR spectroscopy. The presence of dodecyl sulfate resulted in slow aggregation of the proteins, which we detected with differential spectroscopy. Characterization of spectra with high concentrations of dodecyl sulfate was not possible due to saturation of the protein spectra. Insulin at pH 3 showed no significant changes in secondary structure besides the weakening of hydrogen bonds at higher temperature measurements. These data are consistent with previous studies of insulin showing that fibrillation begins only after an initial lag phase