LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

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http://hdl.handle.net/11527/19401

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Yazar "Balta, Bülent" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans'a göz atma
  • Öge
    Computational investigation of reaction mechanism of FET3 protein in yeast
    (Graduate School, 2023-02-17) Ahıshalı, Büşra ; Balta, Bülent ; 521191104 ; Molecular Biology-Genetics and Biotechnology
    In Saccharomyces cerevisiae, a yeast species, iron uptake into the cell takes place with the Reducing Iron Uptake Model. Ferric chelates (Fe3C-L) are degraded on the cell surface by being reduced from Fe3+ to Fe2+ by the cell surface reductases Fre1p and Fre2p. The free reduced Fe2+ ions are taken up by Fet3p-Ftr1p, a high-affinity oxidase-permease complex, or by Fet4, another metal carrier. In this study, the reaction mechanism and the role of Fet3p in reducing iron uptake are examined. Fet3p is a membrane-bound protein and a member of the multicopper oxidase protein family. It metabolizes iron uptake with a high affinity for Fe2+ and plays a role in iron uptake together with iron-permease Ftr1p. Since Ftr1p can only transport the oxidized form of iron, Fe2+ needs to be oxidized before entering the cell. Fet3p couples the four-electron reduction of O2 to H2O with the one-electron oxidation of four Fe2+. The oxidized iron leaves the iron-binding site in Fet3p and is transferred to Ftr1p. Thus, Fe3+ ions are transported into the cytoplasm by a permease, Ftr1p. The understanding of the mechanism of Fet3p is of great importance to shed light on other multicopper oxidase members such as laccases and human ceruloplasmin, some having wide industrial applications. When the active site structure of Fet3p is examined, it has 4 copper as a cofactor in the active site. These coppers are divided into 3 types according to their characteristics: Type 1 (T1), Type 2 (T2), and binuclear Type 3 (T3a and T3b). T2 and T3 coppers form the trinuclear cluster (TNC). Iron as a substrate is not observed in any of the crystal structures of Fet3p. However, according to the information obtained from mutation studies and comparing them with the crystal structures of other MCOs, especially copper efflux oxidase (CueO), the amino acids in the iron-binding region of Fet3p and the location of iron were determined. Fet3p couples four one-electron oxidations of 4 Fe2+ as a substrate to the four-electron reduction of dioxygen to water by taking four protons from the environment. This process is mediated by oxidation-reduction reactions of copper ions as cofactors and consists of two stages. In the first stage, the O2 molecule, which will be reduced to H2O during the reaction, enters the TNC through the solvent channel and binds to the TNC. The O-O bond is cleaved by taking two electrons from two coppers (T1 and one of T3 coppers). Proton donation of E481 to one of the oxygens bridging T3 coppers facilitates this cleavage. Finally, all coppers are oxidized to Cu2+, and one O2- and two OH- ions are formed. In the second stage, the four reductions from Cu2+ to Cu+ with oxidation of four Fe2+ to Fe3+, and four protonations occur, and OH- and O2- ions are converted to two water molecules. In the literature, most of the first stage of the reaction mechanism of MCOs, especially dioxygen-cleavage and peroxy intermediate structure are known. However, the exact mechanism of the second stage, the order of electron and proton transfer reactions is not known because this part occurs fast. Due to the rapidity of these reactions, they have not been studied before and the order of the reaction is unknown due to the difficulty of following the protonation order experimentally. In addition to examining the reaction scheme, it is known that D283 plays an important role in iron binding to substrate-binding site, and electron transfer (ET) is enhanced by D283. However, in the crystal structure, the loop containing D283 is oriented away from the active site, suggesting that it closes only after the binding of Fe2+. Thus, to find out the role of D283 on ET and reaction pathways, the geometries are separately examined when the loop containing D283 is open and closed. In order to elucidate the unknown parts, computational methods were used in this present study, so the possible reaction mechanism will be determined. Thus, it is aimed to understand the mechanisms of other multicopper oxidase members through Fet3p. The calculations and geometry optimizations were carried out using the Quantum Mechanics/Molecular Mechanics QM/MM approach. The M06-2X method, a Density Functional Theory (DFT) method, was used for quantum mechanical (QM) calculations. B3LYP, TPSS, and M06 methods were also used to investigate whether M06-2X is the most suitable method for energy calculations and geometry optimizations of Fet3p containing copper and iron metals. Although M06-2X is not recommended to be used on metals in the literature, all necessary electronic states and spin densities could be obtained only with M06-2X in this study. For this reason, the results were interpreted over the energies obtained with M06-2X. The determination of the QM region to be calculated during the QM/MM calculations is of great importance for the calculations to obtain more accurate results. While choosing the most ideal QM region, residues that have the potential to affect the reaction, electron transfer, and proton exchange, especially close to the region where the reaction took place, were determined. The proximity of amino acids that will contribute to electron transfer around copper and Fe was investigated; therefore, calculations were made accordingly by choosing different QM regions. Considering the computational costs, the most ideal QM region was determined. In the structure where the loop containing D283 is closed, the first Fe2+ oxidation occurs exothermically without protonation while T1 is reduced. Protonation of OH- or O2- ions are not needed due to the cost of protonation. It is examined whether the first electron transfers from T1 to TNC before the second iron binds; nevertheless, the structure could not be obtained without protonation. With the protonation of the TNC region, electron transfer to the TNC has yielded a stable structure. After the oxidized iron leaves, the second Fe2+ binds. Meanwhile, the electron already transferred from the first iron remains in the protonated TNC. Considering the necessity of a second proton transfer before oxidation, the proton taken from D94 returns back to D94 during the optimization, thus the second proton transfer is not necessary. The electron from Fe2+ transfers to T1 copper, and oxidation of the second iron takes place. The second oxidation, which was endothermic when D283 was open, is exothermic in the structure where the loop is closed. The results draw attention to the importance of the loop containing D283. After the second oxidation, the oxidized Fe3+ is replaced with the third Fe2+. For the third iron, structures with two protons, three protons, and four protons are examined. For the third and fourth Fe2+ the geometries when the loop with D283 is open are also examined. According to the results, even three protonations are not enough for third oxidation, and a fourth protonation is needed. When the loop containing D283 is open, the oxidation of the fourth Fe2+ is endothermic even in the presence of four protons in TNC, which is the maximum number of protons TNC can take. In the oxidation reactions protonation of TNC-O2- decrease the negativity of TNC; thus, electron transfer to TNC is more favorable. The protonation of TNC is important to reduce coppers at TNC (T2 and T3 coppers) and for transferring an electron from the substrate to TNC. Similarly, the transfer of an electron from the substrate to TNC and the reduction of TNC coppers force the TNC-O2- or T3-OH- to take proton.
  • Öge
    Investigation of protonation state dependent conformational dynamics of the nucleotide binding domain of Hsp70 protein homolog DnaK via computational methods
    (Graduate School, 2022) Uçar, Umut Çağan ; Balta, Bülent ; 771654 ; Moleküler Biyoloji-Genetik ve Biyoteknoloji Bilim Dalı
    70 kDa heat shock proteins (Hsp70) are a ubiquitious and well-conserved protein family with chaperone functions. They supervise protein folding process and assist in proper protein folding and renaturation by preventing partially folded or misfolded proteins from forming amorphous aggregates and amyloid fibrils. In terms of structure, Hsp70s are composed of approximately 45 kDa N-terminal nucleotide binding domain (NBD) with ATPase activity, 25 kDa C-terminal Substrate Binding Domain (SBD) with substrate peptide binding pocket. Also, there is a conserved hydrophobic linker segment connecting the 2 domains. Hsp70 proteins do not work alone. Rather, they need aid of some other proteins called "cochaperones" to work optimally. The two auxillary cochaperones required by the Hsp70s are the 40 kDa Hsp40s(J domain proteins) and nucleotide exchange factors (NEFs). Together with the cochaperones, Hsp70 proteins form 3-membered Hsp70 chaperone machinery. For Hsp70 chaperone system to carry out all of its functions, the NBD and SBD domains must communicate with each other during the functional cycle. This mutual signal transfer and crosstalk between the domains is an intricate example of "allosteric regulation". Basically, nucleotide status of the NBD exerts an effect on substrate affinity. Reciprocally, binding of a substrate protein to the SBD stimulates ATPase activity of the NBD. In ATP bound state of the NBD, the two domains are in docked conformation. On the other hand, in ADP bound or nucleotide free state of the NBD, these two domains are undocked and become independent of each other. In this undocked conformation, the two domains are connected to each other with the hydrophobic linker. The ATP-bound state of the NBD with docked NBD-SBD conformation binds and releases substrates at much higher rates than nucleotide free or ADP-bound state; thus, ATP-bound state of the Hsp70 is known as "low-affinity state" state with substrate binding pocket open. In contrast, when the NBD lacks any nucleotide or is bound by ADP, rate of substrate binding and release occur at way slower rates. This state of the Hsp70 proteins is called as "high-affinity state" with closed substrate binding pocket. Hence, Hsp70s shuttle between open and closed states. The hydrophobic linker is an undispensable component of the Hsp70 chaperone system. By functioning as a dynamic signal switch, the linker conveys messages in either direction, from NBD to SBD and from SBD to NBD, keeping both domains in touch. In literature, the linker, particularly the hydrophobic 388 VLLL 392 sequence, have been demostrated to be vital for both interdomain communication and dynamics of the ATPase domain. Likewise, there are many residues of the NBD playing key roles in the Hsp70 cycle, particularly in the mechanism of ATP hydrolysis. Especially, D8, K70, E171, D194, T199, and D201 residues of E.coli Hsp70 homolog DnaK and correspondants of these residues in other Hsp70 homologs have been given attention. In this study it was aimed to elucidate the conformational dynamics of the nucleotide binding ATPase domain (NBD) of E.coli DnaK in nucleotide free state by means of computational simulation techniques. First of all, so as to understand if protonation states of critical residues of interest have any effect on the opening-closure dynamics of the NBD in nucleotide free state, initially open and closed structures with different protonation states were prepared. By changing the protonation states of residues D194, D201, and H226 in distinct combinations, charge states +12, +13, and +14 were attained. Four protonation states, namely D194 protonated (194pr), D201 protonated (201pr), both D194 and D201 protonated (2pr), and both D194 and D201 unprotonated (2dep) were tested for both initially open and initially closed conformations. In each case, another residue of interest H226 was taken in protonated form. To broaden conformational space scanned and explore contribution of linker position to the dynamics of the NBD, positions of the linker were changed manually multiple times for each protonation state. According to our standard molecular dynamics (MD) simulation results (each 500 ns) of DnaK 1-392 construct of initially open conformations with these 4 protonation states, it was seen that protonation state can dramatically influence the tendency of initially open conformation towards closure. The cases in which D194 were unprotonated (2dep and 201pr) exhibited a tendency to close. Especially, 2dep became totally closed after 200 ns simulation period. On the other hand, 194pr had no tendency towards closure at all. On the other hand, all of the initially closed conformations for each of the 4 protonation states of the DnaK 1-392 retained their closed forms. Based on these simulation results, it can be deduced that the energy barrier between open and closed conformations was highest for 194pr and lowest for 2dep. In order to both decide on relative abundance of open - closed structures and circumvent sampling problems encountered during classical MD simulations, we carried out temperature replica exchange molecular dynamics (T-REMD) simulations for each protonation state. In addition to the 4 protonation states with protonated H226 residue investigated during MD simulations, 2 additional states, namely D194 protonated-H226 deprotonated (194prHID226) and D201 protonated-H226 deprotonated (201prHID226), were prepared by only removing the epsilon H atom of H226 from 194pr and 201pr cases, respectively. To elucidate whether the linker favors closed conformations, apart from these 6 different protonation cases with residues 1-392 of NBD, 6 further constructs were prepared by stripping the last 4 residues 389VLLL392 of the linker away from each of 1-392 construct. In total, 12 T-REMD simulations, 6 with 1-392 and 6 with 1-388 residues, and each with 300 ns-long were performed. Based on our T-REMD results, the most essential point to be understood is that closed conformations are much more favorable compared to open ones. Considering the ratios of open conformations, even the highest fractions of open conformations obtained in the case of 194pr388 did not exceed 25%. Looking at other cases, the percentage of open conformations can be as low as 2-3% for 201pr cases. If we look at 194pr cases, irrespective of the presence of last 4 residues of linker and protonation state of H226, open structures are most abundant in the cases of 194pr. Among 194pr and thereby all other protonation states-structures, 194pr388 (226 protonated) promotes, by far, the open structures most. Additionally, the role of the length of linker in favoring closed structures over open ones becomes more prominent when H226 is protonated with nearly 7% higher closed frames in 194pr392 than 194pr388. As opposed to 194pr, regardless of linker or protonation state of H226, 201pr cases are out and away the protonation states that exhibit lowest tendency towards opening with fraction of open structures no more than 5%. Unlike 194pr, there is no evident impact of linker on opening-closure behavior. Protonation state of H226 seemed not that important in either protonation state. In agreement with the standard MD simulations, 2dep was monitored to tend to have high number of closed frames. Indeed, 2dep comes after 201pr in terms of closed structure fractions. The contribution of the linker to the closure of the NBD was minor and seen only the last 200ns. As to 2pr states, the 2nd highest fraction of open frames, both in the presence and absence of 4 terminal residues of the linker, after 194pr were obtained. Another point of interest in this study was to scrutinize the pH dependence of DnaK ATPase domain. The active site of the ATPase domain comprises multiple charged amino acid residues; therefore, it can be expected from Hsp70 proteins to be susceptible to changes in pH conditions. To the best of our knowledge, only two experimental studies in early 2000's underlining pH dependent behavior of isolated DnaK NBD have been conducted thus far. On the other hand, no computational study has paid attention to change in the dynamics and activity of either full-length protein or NBD alone with respect to pH. Hence, which of the candidate residues in the active site are in protonated or deprotonated form around physiological pH remained elusive. To solve this mystery, thermodynamic integration (TI) simulations were performed, again for both DnaK 1-392 and DnaK 1-388. For TI simulations, for each of 12 protonation states used in the T-REMD, 2 initially closed structures alongside one open structure were picked from cluster analysis performed on the T-REMD trajectories. Additionally, pKa values of E171 and epsilon position of H226 were calculated in various protonation/deprotonation scenarios of D194 and D201. Regarding pKa values of epsilon H226, we inferred that pKa values were more or less the same around 9, indicating the fact that neither protonation states of active site aspartate and glutamate residues nor the lenght of the linker altered pKa values of H226. Protonation state of E171 is likely to depend on the protonation states of D194 and D201. When these aspartates were both deprotonated, E171 was protonated. D194, D201, and H226, on the other hand, must be in protonated states around physiological pH conditions. Consequently, according to our pKa values, we got +12 charge state, not +13 or +14. All in all, combining all atom MD simulations with REMD simulations and free energy TI simulations, the following questions were tried to be clarified in this thesis study: 1) Effect of protonation state on opening-closure abundance and dynamics of NBD 2)Whether the linker induces a change in the ratio of open to closed conformations. 3) pH dependence of the NBD through demonstration of the possible protonation/deprotonation status of critical residues 4) Whether linker promotes change in pKa of any of critical active site residues. At the end, we came to these conclusions. Dynamics of the DnaK NBD opening-closure were protonation state dependent. Intriguingly, protonation of 2 active site aspartate residues have opposite effect in such a way that protonation of D194 increases the abundance of open conformations, whereas D201 promotes more closed form of the NBD. In addition, at different charge states, there must be multiple protonation states in equilibrium. Regarding the linker, there was no direct indication of the linker being involved in opening/closure or pH dependence of the isolated DnaK NBD. Likewise, no apparent role of the linker in shifting the pKa of any critical residue in the active site.
  • Öge
    The computational study of the interaction of POT1 with SSDNA and TPP1
    (Graduate School, 2022) Gürbüz Önder ; Balta, Bülent ; 740109 ; Molecular Biology-Genetics and Biotechnology Programme
    Linear DNA sequences in eukaryotes have telomere sequences at the chromosome ends. In humans, telomeres consists of repeating TTAGGG sequences. Shelterin complex mainly interacts with telomere sequences. It interacts with both telomeric dsDNA and ssDNA. It protects it from DNA repair mechanism. POT1 is one of the members of shelterin complex. It interacts with telomeric ssDNA. It controls the length of the telomere sequence by forming complex with TPP1. The N-terminal domain of POT1, POT1N, binds to ssDNA while C terminal domain of POT1, POT1C, is known to interact with TPP1. The interaction between POT1 and ssDNA depends on many factors. In the shorter sequences, POT1 prefers to bind 3' end of the sequence by 10 fold. The preference changes as TPP1 binds to POT1. POT1 slides on ssDNA after forming complex with TPP1. There is no information about the mechanism of action in the literature. TPP1 interacts with POT1 and increases its affinity to ssDNA. The affinity of POT1 on ssDNA increases after interacting with POT1 and POT1-TPP1 interaction is regulated by cell cycle as well. It is likely that the POT1-TPP1 complex has at least 2 different conformations and these conformations have different interactions, conformations and geometries. There is no information about these structures in the literature. The preference of POT1 on ssDNA, potential initial binding and sliding mechanisms, POT1-TPP1 complex and the allosteric effect of TPP1 were investigated by standard Molecular Dynamics (MD) simulations and Replica Exchange Molecular Dynamics (REMD) simulations. Free energy of the system was calculated by Molecular Mechanic/Poisson-Boltzmann Surface Area (MM/PBSA) method. Another candidate for the mechanism was investigated by Potemtial of Mean Force (PMF) method. According to our results, the binding preference of POT1 depends on the length of ssDNA. In a longer DNA sequence (22 nucleotides), POT1 has the highest abundance in the middle of sequence, ~27%, followed by 5'end, ~ 17% and 3' end ~ 16%. In a shorter DNA sequence (16 nucleotides), POT1 prefers binding to the 3' end over 5' end (~32% and ~22%). This results shows that the preference of POT1 is influenced by ssDNA length. Our results indicate that extra nucleotide in the longer ssDNA simulations form secondary structure with each other and this might explain 5' end preference over 3' end. In the shorter ssDNA simulations, 3' end is loosely bound to POT1 ~23% of the time and ~8% in the solvent, while 5' end has ~8% protein bound and ~14% in the solvent and this explains 3' preference in the shorter ssDNA simulations. Our results show that there are multiple potential initial binding and sliding mechanisms. Most of the time, POT1 slides only 1 OB fold at a time while other OB fold conserves its position. The sliding happens per nucleotide in this mechanism. The relative abundance of these OB shifted structures were ~ 21%. Here, OB2 fold shifted structures had higher abundance than OB1 fold shifted ones which confirms the literature. Another potential mechanism involves the repetetive nature of telomere sequence. POT1 might prefer to skip a telomere repeat and OB fold can bind to the next telomere sequence. When OB1 fold binds to telomere sequence, OB2 fold moves 6 nucleotides and then OB1 folds shall slide on ssDNA. Furthermore, when ssDNA was interacted with only OB1 fold or OB2 fold, it clearly showed that OB1 fold prefers to bind ssDNA. The relative abundance of these structures were ~4.5% and ~1.5%, respectively. When whole sequence was shifted by 1 nucleotide, which is considered a potential mechanism, the structure has the lowest abundance. In order to test this for 2, 3, 4 and 5 nucleotides, 5 new initial structure was created. In these structures, ssDNA was 11 nucleotide long. The crystal structure and 6 new structures were simulated. G results show that all of these structures are not a candidate for a potential intermediate structures during the sliding. There are 2 different complexes for POT1-TPP1. In the first complex, it should slide easily on ssDNA and it should have less specific interactions. In second complex, it should bind it tightly and have specific interactions. Both structures were constructed based on O. nova. In the simulations, they survived more than 1 µs.