Ispitivanje stabilnosti serin- I cistein-proteaza na niskim temperaturama

Abstract

Denaturacija globularnih proteina na niskim temperaturama predstavlja univerzalni fenomen. Narušavanje nativne strukture proteina usled izlaganja niskim temperaturama dešava se primarno kao posledica kolapsa hidrofobnog efekta, entropijskog faktora koji u najvećoj meri doprinosi stabilizaciji nativne strukture. Sekundarno, denaturacija proteina na niskim temperaturama posledica je slabljenja hidrofobnih interakcija u unutrašnjosti nativnog proteina. Ispitivanje stabilnosti proteina na niskim temperaturama ima, pre svega, fundamentalni značaj koji se ogleda u činjenici da bi detaljno razumevanje mehanizma denaturacije proteina na niskim temperaturama moglo značajno da doprinese razjašnjavanju jednog od najvažnijih problema savremene biohemije, problema uvijanja proteina. Ispitivanje stabilnosti proteina na niskim temperaturama započeto je pre oko dve decenije. Međutim, direktnu denaturaciju proteina, koja je posledica niske temperature per se, teško je proučavati rutinski primenjivanim metodama, jer većina proteina ima tačke denaturacije ispod temperature mržnjenja vode. Razvojem savremenih FT-IR instrumenata, kao i primenom istih u određivanju promena sekundarnih struktura proteina, došlo je do povećanog interesovanja za proučavanjem denaturacije proteina izazvane niskim temperaturama. Praktični značaj proučavanja ove problematike ogleda se u tome što razumevanje mehanizama denaturacije može pomoći u pronalaženju optimalnih uslova za skladištenje proteina pri kojima će se produžiti njihov vek trajanja. Poznavanje stabilnosti enzima naročito je važno u slučaju enzima koji se koriste u biotehnologiji, medicini ili nauci, kao što je slučaj sa sve četiri model proteaze koje su predmet istraživanja u okviru ove doktorske disertacije. Najvažniji cilj koji je postavljen u ovoj studiji jeste pronalaženje objašnjenja za gubitak aktivnosti izazvan niskom temperaturom ispitivanih serin- i cistein-proteaza na nivou detaljnih strukturnih promena proteina, odnosno, pokazati da su, zapravo, denaturacija proteaza i strukturni rearanţmani koji prate denaturaciju odgovorni za gubitak aktivnosti u mnogo većoj meri nego autoproteoliza. Dodatni cilj bilo je optimizovanje uslova za skladištenje komercijalno vaţnih proteaza na niskim temperaturama. Praćenje stabilnosti proteina na niskim temperaturama nailazi na metodološka ograničenja kada je reč o primeni uobičajeno korišćenih metoda za ispitivanje stabilnosti proteina, kao što su spektroskopske metode i diferencijalna skenirajuća kalorimetrija. Stoga je u ovoj studiji pribegnuto pristupu izlaganja proteaza uzastopnim ciklusima zamrzavanja/odmrzavanja u cilju praćenja uticaja temperatura ispod nule na strukturu proteaza. Rezultati praćenja promena primarne strukture model proteaza koji su dobijeni kao deo ove disertacije, ukazuju da autoproteoliza ne može biti odgovorna za visoke procente gubitka aktivnosti, (čak do 75% u slučaju papaina, preko 40% u slučaju ficina i oko 60% u slučaju tripsina u kiselim uslovima) nakon šest ciklusa zamrzavanja/odmrzavanja. Praćenje strukturnih perturbacija na nivou sekundarne i tercijarne strukture pokazalo je da nakon šest do sedam uzastopnih ciklusa zamrzavanja/odmrzavanja proteaze gube elemente nativne sekundarne strukture (α- heliks i neuređene strukture) u korist β-pločica (intramolekulskih u slučaju tripsina u kiselim uslovima). Naročito je izražen porast sadržaja intermolekulskih β-pločica (u slučaju papaina i ficina) koje predstavljaju strukturne elemente neophodne za agregiranje. Agregiranje je potvrđeno gel-filtracijom. Opisani trendovi promena sekundarnih struktura detektovani su u literaturi i za druge proteine denaturisane niskim temperaturama, odnosno niskom pH vrednošću, indicirajući da je inaktiviranje papaina, ficina i tripsina (u kiselim uslovima) na niskoj temperaturi posledica denaturacije. U slučaju tripsina za sekvenciranje, proteaze od najvećeg komercijalnog značaja u ovoj studiji, predložen je alternativni način skladištenja na niskoj temperaturi u rastvornom obliku kojim se izbegava denaturacija i ograničava autoproteoliza. Blago alkalna pH vrednost (u blizini optimalne vrednosti za aktivnost tripsina) uz dodatak krioprotektivnih agenasa (glicerola i lizina) za koje je poznato da stabilizuju proteine (mehanizmom preferencijalne potisnutosti sa površine proteina favorizujući nativnu konformaciju), dovela je do efikasnog očuvanja nativne strukture tripsina. Takođe, inhibicijom autoproteolitičke aktivnosti u prisustvu lizina koji okupira vezujuće mesto tripsina, ograničena je i autoproteoliza. Tripsin skladišten na niskoj temperaturi na pH vrednosti od 8,0 uz dodatak glicerola ili lizina, pokazao je efikasnost identičnu netretiranom nativnom tripsinu u metodi identifikovanja BSA peptidnim mapiranjem, što sugeriše da skladištenje tripsina u blago alkalnim uslovima uz dodatak krioprotektanata može da produži njegov vek trajanja.

Cold denaturation of globular proteins represents a universal phenomenon. Disruption of native structure at low temperatures primarily happens as a consequence of the collapse of the hydrophobic effect, an entropy parameter which represents a main driving force for protein folding. Secondarily, protein denaturation at low temperatures is a consequence of the weakening of hydrophobic interactions in the interior of a protein's three dimensional structure. Investigation of the cold denaturation of proteins has fundamental importance, because detailed understanding of the mechanism of cold denaturation could contribute to a great extent toward elucidationg one of the most challenging problems of contemporary structural biochemistry: the protein folding problem. Investigation of the cold stability of proteins began two decades ago. Direct cold denaturation, which is a consequence of low temperature per se, is difficult to investigate using routine methods since the majority of proteins have cold denaturation points well below 0 °C. However, the development of modern FT-IR instruments and their application in secondary structure determination has lead to increased interest in the investigation of the cold stability of proteins. The practical importance of this topic reflects the fact that understanding structural rearrangements induced by cold denaturation could help define optimal conditions for protein storage in order to prolong their shelf lives. The cold stability of proteins is especially important for enzymes used in biotechnology, medicine or research: which is the case for all four model proteases investigated in this dissertation. The overall goal of this study was to explain the dramatic loss of serine and cysteine proteases activity induced by low temperature, by showing that cold denaturation and subsequent massive structural rearrangements are responsible for this loss, rather than autoproteolysis. In addition, optimal cold storage conditions were defined for commercially important proteases. In this work, repeated freeze-thaw cycles were used to analyse the influence of sub-zero temperatures on protease structures, due to methodological limitations in monitoring protein stability at sub-zero temperatures by commonly used techniques such as spectroscopy and differential scanning calorimetry. Results from monitoring the primary structure stability of model proteases obtained as a part of this dissertation suggest that autoproteolysis cannot be the cause for the dramatic activity losses (as large as 75% in the case of papain, above 40% in the case of ficin and around 60% in the case of trypsin in acidic conditions) observed after six freeze-thaw cycles. Secondary/tertiary structure perturbations after six-to-seven freeze-thaw cycles suggest that these model proteases lose part of their native secondary structure elements (mainly α-helices and random coils) in favor of a β-sheet conformation (intramolecular in the case of trypsin in acidic conditions). An especially large increase was detected for intermolecular β- sheets (in the case of papain and ficin) which represents a structural element necessary for aggregation. Aggregation of cold denatured cysteine proteases was shown by sizeexclusion chromatography. Similar trends in secondary structure changes were detected in the literature for other proteins denatured by low temperature and/or low pH values, indicating that inactivation of papain, ficin and trypsin (in acidic conditions) was a consequence of cold denaturation. Sequencing grade trypsin, being the most important commercial protease of this study, was chosen for optimization of cold storage conditions. An alternative protocol for cold storage of trypsin in solution is proposed which circumvents cold denaturation and limits autoproteolysis. Slightly alkaline conditions (close to the optimum pH value of trypsin, pH around 8) with the addition of cryoprotectants (glycerol or lysine which are known to stabilise proteins in solution by preferential exclusion from the protein surface favoring the native state) led to complete preservation of native structure. In the case of lysine as a cryoprotectant, autoproteolysis was inhibited as well. After seven cycles of cold storage at pH around 8 with the addition of cryoprotectants, trypsin was as efficient as untreated trypsin in trypsin mass fingerprinting of BSA, suggesting that proposed conditions could prolong its shelf life.