The Structural Transition Of Apocytochrome C And Cytochrome C Induced By Polyanion With The Different Hydrophobicity

The structural transition of protein, including protein folding and unfolding, is the important field of protein research. Proteins must fold into their correct three-dimensional conformation in order to attain their biological function. Protein folding is the most fundamental and universal example of biological self-assembly. How a polypeptide chain folds into a stable, native structure in vivo is dependent on amino acid sequence and native solution environment. Generally, a polypeptide chain with given amino acid sequence can spontaneously fold into a certain three-dimensional structure with unique biological function. But structural transition could take place when the environment of protein is changed. That is to say, the three-dimensional structure of protein is determined by not only its thermodynamic stability, but also the micro-environment of protein and kinetic process. The structural transition of protein is a general phenomenon in vivo. Recently, the research found that the structural transition and misfolding of protein could lead to serious diseases. On the other hand, the overexpression of recombinant proteins in Escherichia coli often results in the accumulation of the protein, producing inactive insoluble deposits inside the cells, called inclusion bodies. Therefore, the study on the mechanism of protein folding and the development of more efficient folding methods is very important in theory and practice.The correctly non-covalent assembly of nascent polypeptide chains in the crowded cellular environment requires the assistance of so-called molecular chaperone proteins. Molecular chaperones can bind with a number of different polypeptides. Chaperone binding may not only block intermolecular aggregation by shielding the interactive surfaces of non-native polypeptides but may also prevent or reverse intramolecular misfolding. Molecular chaperone releases their substrates in a regulated manner, resulting in productive folding of the protein. The mechanism of the molecular chaperone-assisted folding provides a strategy to study protein folding in vitro. Cleland et al found that protein folding could be assisted by poly(ethylene glycol) (PEG) in 1990. In recent years, some other polymers have been reported as the folding additives. With the development of macromolecular science, the various polymers i.e., homopolymer, random polymer, block polymer, draft polymer with the given structure and molecular weight can be designed and synthesized according tothe requirement. Especially, environment-response polymers can be designed and synthesized with the different hydrophility/hydrophobicity property. The structure and the hydrophilic/hydrophobic balance of these polymers can be adjusted in molecular level; and more, these polymers can form assembly with the controlled size. Therefore, the influence of the various interactions on protein folding can be investigated through the interaction of different polymers with protein to find polymers with the suitable structure as the folding additives. In this thesis, I investigated the influence of the electrostatic and hydrophobic interactions and hydrogen bonding on the structure of apo cytochrome c (apo cyt c) and cytochrome c (cyt c) with sulfonated polystyrene and the alternating copolymers of maleic acid and alkene. Cyt c is a basic protein, whose isoelectric point is 10.6. The heme-free apo cyt c is the precursor of the mitochondria protein cyt c and has random coil structure in solution.The first part of the thesis is the interaction of sulfonated polystyrene (SPS) with apo cyt c. The previous study by Jie Gong found that SPS could induce apo cyt c a conformational transition form random coil to α-helical structure. The α-helical content of apo cyt c was dependent on concentration, degree of sulfonation and chain length of SPS, as well as the pH and ionic strength of the solution. I further study the interaction of SPS particles and apo cyt c using a combination of techniques, i.e., dynamic light scattering, steady-state fluorescence, and atom force microscopy. We find that when SPS THF/H₂O (50/50, v/v) solution was added dropwise into an excessive amount of water, the stable colloidal particles formed because the hydrophobic backbone chains collapsed and the ionic groups transferred toward the particle surfaces. The size distribution of SPS particles is independent of SPS concentration and low concentration of NaCl but increases with the pH of the solution. In acidic and neutral pH, SPS interacts with apo cyt c forming complex particles, whose size depends on the relative amount of apo cyt c and sulfonated polystyrene. When SPS is in excess, apo cyt c interacts with SPS particles forming stable complexes and excessive SPS particles bind to the periphery of the complexes preventing them from further aggregation. When apo cyt c is in excess, apo cyt c links the complexes forming much larger particles. Fluorescence study demonstrates that apo cyt c not only can neutralize the negative charges on the surface of SPS particles, but may also insert into the cores disrupting the original structure of SPS particles. The interaction of SPS particles and apo cyt c is similar to the interaction of lipidmicelles and apo cyt c. Perhaps SPS particles can partially mimic a lipid membrane environment.The second part is the folding of apo cyt c induced by the alternating copolymers of maleic acid and alkene. The hydrophility/hydrophobicity of these copolymers is jointly determined by the degree of ionization and the length of alkyl chain, therefore, they offer a good model to investigate the influence of electrostatic and hydrophobic interactions and hydrogen bonding on the structural transition of protein. In this study, 1 investigate the structural transformation of apo cyt c induced by two alternating copolymers, poly(isobutylene-alt-maleic acid) (PIMA) andpoly(l-tetradecene-alt-maleic acid) (PTMA). At pH 2.1, 6.5, 10.5 and 11.8, the ionization degrees of PIMA are 11%, 53%, 87% and 100%, respectively; the ionization degrees of PTMA are 8%, 55%, 97% and 100%. In aqueous solution, PIMA is in an expanded state while PTMA forms particles that tend to dissociate by increasing pH and decreasing concentration. Circular dichroism, steady-state fluorescence and atomic force microscopy show that apo cyt c transforms from the U_A state, expanded random coil at pH 2.1 to the C state, compact conformation with no helical structure at higher pH. After adding the alternating copolymers of maleic acid and alkene, apo cyt c undergoes a conformational transition from random coil to α-helical structure. The α-helical content induced by copolymers is influenced by the concentration of the copolymers, the hydrophobic alkyl chain length of the copolymers and the pH of the solution. At pH 2.1, apo cyt c carries 24 positive charges and the decrease of the electrostatic repulsion within the polypeptide is a dominant factor for apo cyt c folding, so the electrostatic attraction between the copolymer and apo cyt c promotes the folding of apo cyt c. At pH 11.8, both the copolymers and protein carry negative charges. The hydrophobic attraction of PTMA and apo cyt c induces partial helical structure while the alkyl in PIMA is too short to compensate the electrostatic repulsion of PIMA and apo cyt c, so no helical structure is induced by PIMA at pH 11.8. These results suggest that the hydrophobic interaction is important for the folding of apo cyt c. Over the entire pH range, the electrostatic and hydrophobic attractions between the copolymer and apo cyt c exclude water molecules that originally bond with apo cyt c through hydrogen bonds, promoting the formation of hydrogen bonds within the helical structure. On the other hand, the hydrogen bonds formed between the ionized carboxyl of the copolymer and the amideof apo cyt c partly restrain the hydrogen bonds formed between the carbonyl and amide within the polypeptide backbone, making helical structural decrease. The results by dynamic light scattering show that at the pH lower than the isoelectric point of protein, precipitation occurs when the ratio of the positive to negative charges is close to the stoichiometric value and the copolymer-protein complex particles can be stabilized by the charges on the surface when the positive or negative charges are in excess. The interaction of the copolymer with apo cyt c at neutral and alkali pH destroys the hydrophobic aggregation of PTMA or apo cyt c and forms new complex particles. A competition exists between the interaction of the copolymer with apo cyt c and the self-aggregation of PTMA or apo cyt c alone. Compared with the protein conformational transition induced by small molecules, polymers have several advantages: very low concentration can promote the conformational transition of the protein; electrostatic and hydrophobic interactions and hydrogen bonds formed between polymer and protein can be manipulated by the pH of the solution and the component of the polymer.The third part is the reversibility of structural transition of cyt c on interacting with and releasing from alternating copolymers of maleic acid and alkene. The interactions of globular proteins with oppositely charged polymers can form soluble complexes, coagula, or precipitates, depending on the concentrations of protein and polymer, pH and ionic strength of aqueous solution. The interactions of proteins with polymers have been widely studied because of the practical applications of polymers in protein separation and purification, protein immobilization and stabilization, protein folding, and protein encapsulation and release. The electrostatic and hydrophobic interactions and hydrogen bonding may occur between proteins and polymers. And these non-covalent interactions are important for the stability and structural transition of protein. However, as far as we know, there have not been systematic reports on the structural transition of proteins induced by interacting with polymers and the recovery of the structure of the proteins released from the polymers. Hence, circular dichroism, absorption spectroscopy and atomic force microscopy were used to investigate the influence of the electrostatic and hydrophobic interactions on the structure of cyt c on interacting with and releasing from the copolymers. At the physiological pH of 7.4, the interaction of PIMA with cyt c can only partly disturb the integrity of the heme pocket and has no influence on the asymmetry environment of Trp59, while PTMAhas very intensive influence on the structure of cyt c, that is, the hydrophobic interaction between PTMA and cyt c completely destroy the hydrophobic core of cyt c. Both PIMA and PTMA have no significant influence on the secondary structure of cyt c. After adding 0.15 M NaCl, physiological ionic strength, PIMA-cyt c complexes dissociate and the released cyt c recovers its native structure, whereas NaCl has no significant influence on PTMA-cyt c complexes. 0.5 M GuHCl destroys PTMA-cyt c complexes, forming GuHCl-PTMA precipitates; the cyt c released from the complexes regenerates its native structure. Compared with electrostatic interaction, hydrophobic interaction leads to more stable polymer-cyt c complexes and more intensive influence on cyt c structure, but cyt c can recover its native state after release. The results obtained in this study may be useful in protein separation and purification, protein immobilization and stabilization, protein folding, and protein encapsulation and release.