Protein function critically depends on the protein ability to adopt a specific structure. Remarkably, a protein can efficiently fold to the native state from the unfolded state(s) on physiological time scale. Understanding how unfolded polypeptides self-assemble into a correctly folded structure represents one of the great challenges of the 21st century’s science. Proteins can be induced to collapse into partially folded states, under equilibrium or non-equilibrium conditions. A variety of partially folded proteins were shown to represent suitable models for intermediates forming during protein folding; therefore, determining the structural properties of these partially folded states becomes crucial for a deeper understanding of the protein folding mechanism. Partially folded proteins also play a role for important cell functions and are thought to have a central role in protein stability. However, due to the cooperative nature of folding a minimal amount of intermediates is observed. Fast-kinetics is widely employed for the characterization of transiently populated intermediates; of these, the most important is the molten globule, a compact, partially folded state possessing native-like secondary structure but lacking the extensive, specific side-chain packing interactions of the native structure. As demonstrated by structural studies, in the molten globule the side-chains adopt a greater variety of conformations with respect to the native protein, and the protein is characterized by high flexibility (if compared to the native state). In order to understand what changes take place in the protein during the final stages of folding, spectroscopic techniques, as circular dichroism, Fourier transform infrared, Raman and NMR have have been successfully employed; these techniques provide precious information on changes occurring in distinct regions of the macromolecule. Cytochrome c is a single polypeptide chain organized in five α-helices and three omega loops. Ω-loops are protein segments devoided of secondary structure, localized mostly on protein surface, characterized by the presence of polar aminoacids and enhanced flexibility. Also, cytochrome c contains heme as prosthetic group, which is linked to the polypeptide chain by two tioether bonds formed by the cysteins in position 14 and 17. The heme-iron is also axially coordinated with the two residues His 18 and Met 80. The molten globule (A state) of ferric cytochrome c has been extensively characterized. At pH 2.2, ferric cytochrome c is substantially unfolded at low ionic strength; upon addition of salts, the protein cooperatively folds to a compact structure, the A state, stabilized by the binding of anions to the positively charged groups on the protein surface. The A state possesses α-helix structure comparable to that of the native form, but a fluctuating tertiary conformation. In particular the hydrophobic core containing the two major helices (the N and C-terminal helices) and the heme group is preserved in the A state, stabilized by non bonded interactions, while the loop regions are fluctuating and partially disordered. Of the two native axial ligands of the heme-iron, only His 18 is thought to remain coordinated to the heme iron while the Met80 axial bond is lost in the presence of large anions, and is recovered by the presence of small anions. With this in mind, in this thesis our intent is to define wich contribution is provided by the hydrogen bond linking the histidine in position 26 (Ω loop 20s) to the carbonyl group of the residue in position 44 (Ω-loop 40s), to stability, folding, and functional properties of cytochrome c. At the same time, we want to explore the possibility to generate a molten globule state of cytochrome c at neutral pH. The interest on His 26 lies in the fact that this residue, it is an invariant residue in vertebrate and higher plant cytochromes c . In yeast cytochrome the lack of the H26-E44 H-bond, due to mutation of H26, is expected to free the two loops with a consequent increase of the protein flexibility and the likely displacement of M80 from the axial coordination to the heme-iron; thus, such a mutation shoud stabilize a compact intermediate state acting as a suitable model for a kinetic folding compact intermediate of cyt c. Yeast iso-1-cytochrome c represents an ideal model-system for c-class cytochromes; a high-resolution X-ray structure of the protein is available and a system to generate mutants by direct mutagenesis has been developed. Our study highlights the crucial role played by the His26-Glu 44 Hbond, wich confers great stability to native iso-1-cit c by keeping the 20’s and the 40’s Ω-loops joined and sterically close, thereby enhancing the overall rigidity of the macromolecule. The absence of the such H-bond induces changes in the tertiary structure of the macromolecule associated to a dramatic decrease of protein stability. The flexibility of the H26Y variant weakens the native M80-Fe(III) axial bond strength, therefore altering the protein behaviour. As a result, the alkaline conformational transition occurs at lower pH (pKa = 7.5 rather than 8.4) and, contrary to the native protein, at neutral pH the H26Y variant results to be a mixture of (at least) two species with different axial coordination to the metal. These observations indicate that this H-bond is particularly important for protein stabilization; its rupture induces in the protein the formation of a “molten globule” state under physiological-like conditions. This state is an equilibrium mixture of two forms, one characterized by a native-like coordination to the heme iron, the other possessing a misligated endogeneous ligand axially coordinated to the metal, as shown by the following scheme: His18-Fe(III)-Met80 ↔ His18-Fe(III)-X where X is an endogenous ligand. Resonance Raman measurements indicate probably a lysine as the most likely nonnative ligand. In order to determine which role is played by the other two histidines located in the aminoacidic sequence of the protein (H33 and H39), we have produced and characterized the double mutants H26YH33Y, H26YH39K, H33YH39K, which retain only one His in the amino acidic sequence. In particular, the H39K mutation inserts a lysine at position 39, as in the sequence of horse cyt c. Data obtained indicate that neither H33 or His39 induce significant alteration in the protein structure and stability; this highlights the critical role played H 26 for protein stability. The properties of the H26Y mutant of equine cyt c were also investigated. Although showing a close structural similarity with respect to yeast iso-1-cyt c (as revealed by X-ray cristallography), this protein exhibits a higher stability we wish to underline. Recombinant horse ferricyt c produced in our laboratory shows spectral properties and stability practically identical to the native protein, as shown by CD, electronic absorption and high frequency RR data. Interestingly, also the H26Y mutant does not exhibit significant changes with respect to the wt protein, at neutral pH. However, the H26Y mutant undergoes alkaline isomerization at a pH lower than the wt form (pKa: 8.3 vs 9.2), but higher than that of the yeast variant. The observed ∆pKa between the mutant and wt form (∆ (∆Go) = 5.1 KJ/mol) measures the stability decrease induced by the H26 mutation; the same ∆pKa was determined for horse and yeast cyt c, which suggests that the H26 mutation decreases the stability of the two proteins to a similar extent, despite the fact that equine cyt c displays a higher stability. Therefore, the properties of the ionizing group controlling the alkaline transition (i.e. the trigger) are equally affected in both proteins. Hence, the groups responsible for the higher stability of horse cyt c at neutral pH can be excluded from any involvement in the conformational changes associated with the alkaline isomerization of cyt c.
L’attività funzionale biologica di una proteina dipende essenzialmente dalla sua abilità ad adottare una struttura specifica. In condizioni fisiologiche, la macromolecola si ripiega nel lo stato nativo (biologicamente attia) partendo da uno stato non ripiegato. Capire il meccanismo attraverso cui la catena polipeptidica si ripiega ad assumere la sua struttura tridimensionale nativa (folding) è certamente una delle sfide più importanti della scienza del XXI secolo.Le proteine possono “collassare” in stati parzialmente ripiegati in condizioni di equilibrio o di non equilibrio.Tali stati possono potenzialmente rappresentare degli ottimi modelli di intermedi del processo di folding; studiarne le proprietà strutturali, è determinante per capire più a fondo il meccanismo di folding.Tali intermedi giocano un ruolo cruciale sulle funzioni cellulari e sulla stabilità ; ciò indica che il processo di ripiegamento proteico è di tipo cooperativo.Studi di cinetica rapida sono spesso utilizzati per caratterizzare intermedi transienti,dei quali il più importante è certamente il molten globule, uno stato compatto,caratterizzato da regioni a struttura secondaria simili allo stato nativo, ma con struttura terziaria fluttuante; esso manca infatti di molte delle interazioni terziarie specifiche presenti nella struttura nativa. Dagli studi strutturali, si osserva che nel molten globule la catena laterale adotta una grande varietà di conformazioni rispetto alla proteina nativa, in accordo con una sua aumentata flessibilità (rispetto alla proteina nativa).Lo studio del folding del citocromo c è stato effettuato utilizzando una vasta gamma di metodiche spettroscopiche; tra queste, il dicroismo circolare , la trasformata di Fourier , la spettroscopia Raman e NMR, che hanno permesso di osservare cambiamenti strutturali di alcune regioni distinte della macromolecola.Il citocromo c è costituito da una singola catena polipeptidica di 104 aminoacidi, organizzata in cinque regioni a-elica e tre regioni ad ansa(Ω loops). Gli Ω-loosp sono segmenti proteici privi di struttura secondaria, localizzati sulla superficie della proteina, costituiti principalmente da amminoacidi polari, e caratterizzati da elevata flessibilità. Il citocromo c contiene il gruppo prostatico (l’eme) legato covalentemente alla matrice proteica tramite due legami tioetere con le cisteine Cys14 e Cys17. Il Fe-eme è inoltre coordinato in posizione prossimale all’His18 e in posizione distale alla Met 80. Lo stato A (i.e. il molten globule ) del citocromo c di cavallo, è stato studiato approfonditamente. A pH 2.2 e a bassa forza ionica, il ferri-citocromo è sostanzialmente non ripiegato (“unfolded”); tuttavia, l’aggiunta di sali, induce la proteina a ripiegarsi in una struttura compatta, lo stato A, stabilizzato dal legame degli anioni ai gruppi carichi positivi situati sulla superficie della proteina. Lo stato A ha struttura α-elicaoidale comparabile a quella dello stato nativo, ma la sua conformazione terziaria è fluttuante. In particolare, nello stato A rimane conservato il “core idrofobico”, costituito dalle eliche N- e C terminali e dal gruppo eme, è stabilizzato da interazioni non covalenti; tuttavia le regioni a cappio ( loops) sono fluttuanti e parzialmente disordinate. Dei due ligandi assiali del ferro-eme, solo l’istidina 18 rimane coordinata al metallo, mentre il legame assiale con la metionina 80 risulta modulato dal tipo e dalle dimensioni degli anioni presenti in soluzione..Scopo di questa tesi è definire il ruolo del legame idrogeno formato nella proteina nativa, tra l’anello imidazolico dell’istidina in posizione 26 (nell’Ω-loop 20s) e il gruppo carbonilico del residuo in posizione 44 (nell’Ω-loop 40s), per la stabilità e le proprietà strutturali e funzionali del citocromo c. Allo stesso tempo, la sostituzione della H26 per mutagenesi, permetterà di stabilire le cause per cui tale residuo è invariante nella sequenza aminoacidica della proteina ( sia nei vertebrati che nelle piante superiori). L’ipotesi più accreditata è che la rottura del legame idrogeno, dovuta alla mutazione di H26, liberi i due loops determinando così un aumento della flessibilità della proteina e il probabile spiazzamento della Met80 dalla sesta posizione di coordinazione al ferro eme. Questa mutazione, se dovesse stabilizzare uno stato intermedio compatto, potrebbe produrre un valido modello di intermedio di folding del citocromo c. Il citocromo del iso-1 di lievito rappresenta sistema ideale nello studio dei citocromi c di classe c; la sua struttura determinata a Raggi X è disponibile ed è stato sviluppato un sistema di mutagenesi capace di produrre mutanti stabili. Il nostro studio ha evidenziato il ruolo chiave del legame-H tra His26-Glu 44 nel conferire stabilità della proteina nativa, bloccando i Ω-loops 20s e il 40s , con conseguente aumento della rigidità della macromolecola. L’assenza di tale legame induce cambiamenti conformazionali nella struttura terziaria della macromolecola che riducono sensibilmente la stabilità dalla proteina. L’alta flessibilità mostrata dal mutante H26Y, indebolisce il legame assiale Fe(III)-Met80, e altera il comportamento della proteina stessa. Come risultato, la transizione conformazionale alcalina avviene a pH più basso (pKa = 7.5 anzichè 8.4) e, contrariamente a quanto si osserva nella proteina nativa, a pH neutro il mutante H26Y resulta essere composto da almeno due specie con diversa coordinazione assiale al metallo. I dati ottenuti indicano che di questo legame-H la rottura induce nella proteina la formazione di uno stato di “molten globule” a pH fisiologico, caratterizzato da due forme in equilibrio,secondo il seguente schema: His18-Fe(III)-Met80↔His18-Fe(III)-X dove X è un ligando endogeno,probabilmente una lisina secondo quanto suggerito dalle misure di Raman effettuate.Con lo scopo di determinare il ruolo svolto delle altre due istidine presenti nella sequenza aminoacida della proteina di lievito (H33 and H39), sono stati prodotti e caratterizzati i doppi mutanti H26YH33Y, H26YH39K, H33YH39K, che conservano ciascuno una sola istidina nella sequenza aminoacidica. In particolare, la H39 è stata mutata con una lisina presente nella sequenza del citocromo c equino. I dati ottenuti indicano che H33 e His39 non alterano le proprietà della proteina, evidenziando così il ruolo critico svolto dal residuo H26.Sono state studiate anche le proprietà del mutante H26Y del cyt c equino, dal momento che malgrado mostri una struttura simile a quella dell’iso-1-cyt c. del lievito (come rilevato dalla cristallografia a raggi X), ha però maggiore stabilità. Il citocromo c equino prodotto nel nostro laboratorio mostra proprietà spettroscopiche ed una stabilità praticamente identiche alla proteina nativa, (evidenziate da misure CD, assorbimento elettronico e Raman). A pH neutro il mutante H26Y equino non mostra cambiamenti significativi rispetto alla proteina wt (nativa) ma è caratterizzato da un’isomerizzazione alcalina con pk più basso rispetto alla forma wt (pKa: 8.3 anzichè 9.2), ma comunque più alto di quello del mutante di lievito. La variazione di pKa osservata fra il mutante e la forma wt ( ∆ (∆Go ) = 5.1 KJ/mol) indica l’effetto l’effetto della mutazione in posizione 26 sulla stabilità della proteina .E’ interessante notare che una una stessa variazione di ∆pKa viene osservata per il citocromo c di lievito; ciò suggerisce che la mutazione H26Y influisce sulla stabilità delle due proteine in modo simile, nonostante la maggiore stabilità del citocromo c equino a pH 7.0, ciò suggerisce che le proprietà del gruppo ionizzante, che controlla la transazione alcalina (i.e. “ trigger ”) sono ugualmente influenzate in entrambe le proteine; dunque, i gruppi responsabili per la maggiore stabilità del citocromo c equino a pH neutro non sono coinvolti nei cambiamenti conformazionali associati all’isomerizzazione alcalina del citocromo c.
Caroppi, P.a. (2005). Ruolo delle istidine nella struttura e nella stabilità del citocromo C.
Ruolo delle istidine nella struttura e nella stabilità del citocromo C
CAROPPI, PAOLA ANNAMARIA
2005-01-01
Abstract
Protein function critically depends on the protein ability to adopt a specific structure. Remarkably, a protein can efficiently fold to the native state from the unfolded state(s) on physiological time scale. Understanding how unfolded polypeptides self-assemble into a correctly folded structure represents one of the great challenges of the 21st century’s science. Proteins can be induced to collapse into partially folded states, under equilibrium or non-equilibrium conditions. A variety of partially folded proteins were shown to represent suitable models for intermediates forming during protein folding; therefore, determining the structural properties of these partially folded states becomes crucial for a deeper understanding of the protein folding mechanism. Partially folded proteins also play a role for important cell functions and are thought to have a central role in protein stability. However, due to the cooperative nature of folding a minimal amount of intermediates is observed. Fast-kinetics is widely employed for the characterization of transiently populated intermediates; of these, the most important is the molten globule, a compact, partially folded state possessing native-like secondary structure but lacking the extensive, specific side-chain packing interactions of the native structure. As demonstrated by structural studies, in the molten globule the side-chains adopt a greater variety of conformations with respect to the native protein, and the protein is characterized by high flexibility (if compared to the native state). In order to understand what changes take place in the protein during the final stages of folding, spectroscopic techniques, as circular dichroism, Fourier transform infrared, Raman and NMR have have been successfully employed; these techniques provide precious information on changes occurring in distinct regions of the macromolecule. Cytochrome c is a single polypeptide chain organized in five α-helices and three omega loops. Ω-loops are protein segments devoided of secondary structure, localized mostly on protein surface, characterized by the presence of polar aminoacids and enhanced flexibility. Also, cytochrome c contains heme as prosthetic group, which is linked to the polypeptide chain by two tioether bonds formed by the cysteins in position 14 and 17. The heme-iron is also axially coordinated with the two residues His 18 and Met 80. The molten globule (A state) of ferric cytochrome c has been extensively characterized. At pH 2.2, ferric cytochrome c is substantially unfolded at low ionic strength; upon addition of salts, the protein cooperatively folds to a compact structure, the A state, stabilized by the binding of anions to the positively charged groups on the protein surface. The A state possesses α-helix structure comparable to that of the native form, but a fluctuating tertiary conformation. In particular the hydrophobic core containing the two major helices (the N and C-terminal helices) and the heme group is preserved in the A state, stabilized by non bonded interactions, while the loop regions are fluctuating and partially disordered. Of the two native axial ligands of the heme-iron, only His 18 is thought to remain coordinated to the heme iron while the Met80 axial bond is lost in the presence of large anions, and is recovered by the presence of small anions. With this in mind, in this thesis our intent is to define wich contribution is provided by the hydrogen bond linking the histidine in position 26 (Ω loop 20s) to the carbonyl group of the residue in position 44 (Ω-loop 40s), to stability, folding, and functional properties of cytochrome c. At the same time, we want to explore the possibility to generate a molten globule state of cytochrome c at neutral pH. The interest on His 26 lies in the fact that this residue, it is an invariant residue in vertebrate and higher plant cytochromes c . In yeast cytochrome the lack of the H26-E44 H-bond, due to mutation of H26, is expected to free the two loops with a consequent increase of the protein flexibility and the likely displacement of M80 from the axial coordination to the heme-iron; thus, such a mutation shoud stabilize a compact intermediate state acting as a suitable model for a kinetic folding compact intermediate of cyt c. Yeast iso-1-cytochrome c represents an ideal model-system for c-class cytochromes; a high-resolution X-ray structure of the protein is available and a system to generate mutants by direct mutagenesis has been developed. Our study highlights the crucial role played by the His26-Glu 44 Hbond, wich confers great stability to native iso-1-cit c by keeping the 20’s and the 40’s Ω-loops joined and sterically close, thereby enhancing the overall rigidity of the macromolecule. The absence of the such H-bond induces changes in the tertiary structure of the macromolecule associated to a dramatic decrease of protein stability. The flexibility of the H26Y variant weakens the native M80-Fe(III) axial bond strength, therefore altering the protein behaviour. As a result, the alkaline conformational transition occurs at lower pH (pKa = 7.5 rather than 8.4) and, contrary to the native protein, at neutral pH the H26Y variant results to be a mixture of (at least) two species with different axial coordination to the metal. These observations indicate that this H-bond is particularly important for protein stabilization; its rupture induces in the protein the formation of a “molten globule” state under physiological-like conditions. This state is an equilibrium mixture of two forms, one characterized by a native-like coordination to the heme iron, the other possessing a misligated endogeneous ligand axially coordinated to the metal, as shown by the following scheme: His18-Fe(III)-Met80 ↔ His18-Fe(III)-X where X is an endogenous ligand. Resonance Raman measurements indicate probably a lysine as the most likely nonnative ligand. In order to determine which role is played by the other two histidines located in the aminoacidic sequence of the protein (H33 and H39), we have produced and characterized the double mutants H26YH33Y, H26YH39K, H33YH39K, which retain only one His in the amino acidic sequence. In particular, the H39K mutation inserts a lysine at position 39, as in the sequence of horse cyt c. Data obtained indicate that neither H33 or His39 induce significant alteration in the protein structure and stability; this highlights the critical role played H 26 for protein stability. The properties of the H26Y mutant of equine cyt c were also investigated. Although showing a close structural similarity with respect to yeast iso-1-cyt c (as revealed by X-ray cristallography), this protein exhibits a higher stability we wish to underline. Recombinant horse ferricyt c produced in our laboratory shows spectral properties and stability practically identical to the native protein, as shown by CD, electronic absorption and high frequency RR data. Interestingly, also the H26Y mutant does not exhibit significant changes with respect to the wt protein, at neutral pH. However, the H26Y mutant undergoes alkaline isomerization at a pH lower than the wt form (pKa: 8.3 vs 9.2), but higher than that of the yeast variant. The observed ∆pKa between the mutant and wt form (∆ (∆Go) = 5.1 KJ/mol) measures the stability decrease induced by the H26 mutation; the same ∆pKa was determined for horse and yeast cyt c, which suggests that the H26 mutation decreases the stability of the two proteins to a similar extent, despite the fact that equine cyt c displays a higher stability. Therefore, the properties of the ionizing group controlling the alkaline transition (i.e. the trigger) are equally affected in both proteins. Hence, the groups responsible for the higher stability of horse cyt c at neutral pH can be excluded from any involvement in the conformational changes associated with the alkaline isomerization of cyt c.File | Dimensione | Formato | |
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