Ispitivanje stabilnosti serin- I cistein-proteaza na niskim temperaturama

Abstract

Denaturacija globularnih proteina na niskim temperaturama predstavljauniverzalni fenomen. Narušavanje nativne strukture proteina usled izlaganja niskimtemperaturama 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 slabljenjahidrofobnih interakcija u unutrašnjosti nativnog proteina. Ispitivanje stabilnosti proteinana niskim temperaturama ima, pre svega, fundamentalni značaj koji se ogleda učinjenici da bi detaljno razumevanje mehanizma denaturacije proteina na niskimtemperaturama moglo značajno da doprinese razjašnjavanju jednog od najvažnijihproblema savremene biohemije, problema uvijanja proteina.Ispitivanje stabilnosti proteina na niskim temperaturama započeto je pre oko dvedecenije. Međutim, direktnu denaturaciju proteina, koja je posledica niske temperatureper se, teško je proučavati rutinski primenjivanim metodama, jer većina proteina imatačke denaturacije ispod temperature mržnjenja vode. Razvojem savremenih FT-IRinstrumenata, kao i primenom istih u određivanju promena sekundarnih strukturaproteina, došlo je do povećanog interesovanja za proučavanjem denaturacije proteinaizazvane niskim temperaturama.Praktični značaj proučavanja ove problematike ogleda se u tome štorazumevanje mehanizama denaturacije može pomoći u pronalaženju optimalnih uslovaza skladištenje proteina pri kojima će se produžiti njihov vek trajanja. Poznavanjestabilnosti 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 predmetistraživanja u okviru ove doktorske disertacije.Najvažniji cilj koji je postavljen u ovoj studiji jeste pronalaženje objašnjenja zagubitak aktivnosti izazvan niskom temperaturom ispitivanih serin- i cistein-proteaza nanivou detaljnih strukturnih promena proteina, odnosno, pokazati da su, zapravo,denaturacija proteaza i strukturni rearanţmani koji prate denaturaciju odgovorni zagubitak aktivnosti u mnogo većoj meri nego autoproteoliza. Dodatni cilj bilo jeoptimizovanje uslova za skladištenje komercijalno vaţnih proteaza na niskimtemperaturama.Praćenje stabilnosti proteina na niskim temperaturama nailazi na metodološkaograničenja kada je reč o primeni uobičajeno korišćenih metoda za ispitivanjestabilnosti proteina, kao što su spektroskopske metode i diferencijalna skenirajućakalorimetrija. Stoga je u ovoj studiji pribegnuto pristupu izlaganja proteaza uzastopnimciklusima zamrzavanja/odmrzavanja u cilju praćenja uticaja temperatura ispod nule nastrukturu proteaza. Rezultati praćenja promena primarne strukture model proteaza kojisu dobijeni kao deo ove disertacije, ukazuju da autoproteoliza ne može biti odgovornaza visoke procente gubitka aktivnosti, (čak do 75% u slučaju papaina, preko 40% uslučaju ficina i oko 60% u slučaju tripsina u kiselim uslovima) nakon šest ciklusazamrzavanja/odmrzavanja. Praćenje strukturnih perturbacija na nivou sekundarne itercijarne strukture pokazalo je da nakon šest do sedam uzastopnih ciklusazamrzavanja/odmrzavanja proteaze gube elemente nativne sekundarne strukture (α-heliks i neuređene strukture) u korist β-pločica (intramolekulskih u slučaju tripsina ukiselim uslovima). Naročito je izražen porast sadržaja intermolekulskih β-pločica (uslučaju papaina i ficina) koje predstavljaju strukturne elemente neophodne zaagregiranje. Agregiranje je potvrđeno gel-filtracijom. Opisani trendovi promenasekundarnih struktura detektovani su u literaturi i za druge proteine denaturisane niskimtemperaturama, 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 značajau ovoj studiji, predložen je alternativni način skladištenja na niskoj temperaturi urastvornom obliku kojim se izbegava denaturacija i ograničava autoproteoliza. Blagoalkalna pH vrednost (u blizini optimalne vrednosti za aktivnost tripsina) uz dodatakkrioprotektivnih agenasa (glicerola i lizina) za koje je poznato da stabilizuju proteine(mehanizmom preferencijalne potisnutosti sa površine proteina favorizujući nativnukonformaciju), dovela je do efikasnog očuvanja nativne strukture tripsina. Takođe,inhibicijom autoproteolitičke aktivnosti u prisustvu lizina koji okupira vezujuće mestotripsina, ograničena je i autoproteoliza. Tripsin skladišten na niskoj temperaturi na pHvrednosti od 8,0 uz dodatak glicerola ili lizina, pokazao je efikasnost identičnunetretiranom nativnom tripsinu u metodi identifikovanja BSA peptidnim mapiranjem,što sugeriše da skladištenje tripsina u blago alkalnim uslovima uz dodatakkrioprotektanata 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 consequenceof the collapse of the hydrophobic effect, an entropy parameter which represents a maindriving force for protein folding. Secondarily, protein denaturation at low temperaturesis a consequence of the weakening of hydrophobic interactions in the interior of aprotein's three dimensional structure. Investigation of the cold denaturation of proteinshas fundamental importance, because detailed understanding of the mechanism of colddenaturation could contribute to a great extent toward elucidationg one of the mostchallenging problems of contemporary structural biochemistry: the protein foldingproblem. Investigation of the cold stability of proteins began two decades ago. Directcold denaturation, which is a consequence of low temperature per se, is difficult toinvestigate using routine methods since the majority of proteins have cold denaturationpoints well below 0 °C. However, the development of modern FT-IR instruments andtheir application in secondary structure determination has lead to increased interest inthe investigation of the cold stability of proteins.The practical importance of this topic reflects the fact that understandingstructural rearrangements induced by cold denaturation could help define optimalconditions for protein storage in order to prolong their shelf lives. The cold stability ofproteins is especially important for enzymes used in biotechnology, medicine orresearch: 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 andcysteine proteases activity induced by low temperature, by showing that colddenaturation and subsequent massive structural rearrangements are responsible for thisloss, rather than autoproteolysis. In addition, optimal cold storage conditions weredefined for commercially important proteases.In this work, repeated freeze-thaw cycles were used to analyse the influence ofsub-zero temperatures on protease structures, due to methodological limitations inmonitoring protein stability at sub-zero temperatures by commonly used techniquessuch as spectroscopy and differential scanning calorimetry. Results from monitoring theprimary structure stability of model proteases obtained as a part of this dissertationsuggest that autoproteolysis cannot be the cause for the dramatic activity losses (as largeas 75% in the case of papain, above 40% in the case of ficin and around 60% in the caseof trypsin in acidic conditions) observed after six freeze-thaw cycles. Secondary/tertiarystructure perturbations after six-to-seven freeze-thaw cycles suggest that these modelproteases lose part of their native secondary structure elements (mainly α-helices andrandom coils) in favor of a β-sheet conformation (intramolecular in the case of trypsinin acidic conditions). An especially large increase was detected for intermolecular β-sheets (in the case of papain and ficin) which represents a structural element necessaryfor aggregation. Aggregation of cold denatured cysteine proteases was shown by sizeexclusionchromatography. Similar trends in secondary structure changes were detectedin 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 aconsequence of cold denaturation.Sequencing grade trypsin, being the most important commercial protease of thisstudy, was chosen for optimization of cold storage conditions. An alternative protocolfor cold storage of trypsin in solution is proposed which circumvents cold denaturationand limits autoproteolysis. Slightly alkaline conditions (close to the optimum pH valueof trypsin, pH around 8) with the addition of cryoprotectants (glycerol or lysine whichare known to stabilise proteins in solution by preferential exclusion from the proteinsurface favoring the native state) led to complete preservation of native structure. In thecase of lysine as a cryoprotectant, autoproteolysis was inhibited as well. After sevencycles of cold storage at pH around 8 with the addition of cryoprotectants, trypsin wasas efficient as untreated trypsin in trypsin mass fingerprinting of BSA, suggesting thatproposed conditions could prolong its shelf life.