Metal içeren hekzahistidin zincirlerinin modellenmesi ve metalin amino asit etkileşimlerindeki rolünün incelenmesi
Modeling of metal-containing hexahistidine chains and examining the role of metal in amino acid interactions
Yazar: ALPARSLAN NUMAN YILDIZ
Danışman: PROF. DR. MİNE YURTSEVER
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Kimya Ana Bilim Dalı / Kimya Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Kimya = Chemistry

Özet:
Histidin oligomerleri, protein ve DNA saflaştırma adımlarında kullanılan çok yaygın bir etiketleme yöntemidir. Bu amaçla kullanılan histidin oligomerleri literatürde His-tag olarak adlandırılmaktadır. Çoklu histidin etiketleri veya misina doğada bulunan bir oligomer olduğu için deneysel moleküler biyoloji çalışmalarında sıklıkla kullanılmaktadır. Histidin etiketleri, insan vücudunda kolayca bulunan bakır (Cu+2), çinko (Zn+2), nikel (Ni+2) ve kobalt (Co+2) gibi iki değerlikte metal iyonlarını koordine ederek protein veya DNA bağlama kapasitelerini artırabilir. Metalin neden bağlanmayı arttırdığı, kompleks yapılarının metale göre nasıl ve neden değiştiği, metal tipinin bağlanma verimini arttırmada neden önemli olduğu vb. hala açık değildir. Bazı soruları cevaplamaya çalışan birkaç teorik çalışma olmasına rağmen, ayrıntılı mekanik problemler hala çözüme ulaşamamıştır. Bu çalışmada, YFT yöntemleri kullanılarak, bağlanma enerjisini kantitatif olarak açıklamak için histidin etiketleri taşıyan metalin kimyasal yapısı gerçekçi bir şekilde modellenmiştir. Modeller, imidazol halkalarının nitrojen atomları aracılığıyla Ni+2, Zn+2, Cu+2 ve Co+2 metal iyonlarına koordineli hekza histidin oligomerlerinden oluşturulmuştur. Modeller daha sonra N-terminalleri yoluyla amino asit dimerlerine (proteini temsil eden en küçük birimler) bağlanmıştır. Tüm model yapıları öncelikle B3LYP/6-31G(d,p) seviyesinde oksijen atomları tarafından metal katyonlarının eksik değerliklerinin tamamlanması ile optimize edilmiştir. Metal atomları için Stevens ve grubu tarafından geliştirilen yöntem olan gen anahtar kelimesi ile 6-31G(d,p) seviyesinde baz seti kullanılmıştır.  Tüm hesaplamalar, IEFPCM çözücü modeli kullanılarak örtülü su ortamında gerçekleştirilmiştir. Yapılan kuantum mekanik çalışmalarda hangi geçiş metalinin histidin etiketi yapısında daha kararlı bir yapı oluşumuna katkıda bulunduğu sonucuna varılmıştır. Elde ettiğimiz bu sonuç literatürdeki sonuçlarla yüksek oranda benzerlik göstermektedir. Bu çalışmaların ardından ise optimize edilen histidin etiketleri kullanılarak protein – ligand yerleştirme (docking) çalışması yapılmıştır. Bu yerleştirme çalışmalarında insan DNA'sına en çok benzeyen 1BNA dodokomer yapısı olan 12 dizilik bir yapı hedef makromolekül olarak kullanılmıştır. Kenetlenme sonucunda optimize edilen histidin etiket yapımızın DNA'ya daha çok küçük oluktan bağlandığı ve daha çok guanin bazı ile etkileşime girdiği görülmüştür. Elde edilen bu sonuçları desteklemek adına bir simülasyon gerçekleştirilmiştir. Bu simülasyonda ise protein ve ligandımızın gerçek bir çözücü ortamında nasıl davrandığı simüle edilmiştir. Elde edilen sonuçlar kuantum mekanik ve protein yerleştirme sonuçlarımızla benzerlik göstermektedir.

Abstract:
Amino acids (aa) are called substances containing amino and acid groups. Proteins are formed by combining basic units of amino acids. Therefore, during amino acid deficiency, errors occur in protein synthesis and diseases occur due to protein deficiency. In this regard, amino acids also play an important role in gene expression. Amino acids are used to assist in the translation of messenger RNAs called mRNAs. The amino acid histidine, which means tissue in Greek, is an amino acid that was first isolated from protamine by Albrecht Kossel in 1896. Histidine, a basic amino acid, has an imidazole ring in its structure and its pK value is 6.0. The amino acid histidine can exist in both neutral form and protanated form. There are three different histidine structures depending on the presence of hydrogen on the nitrogen atoms in the imidazole ring. The HID structure is the presence of hydrogen at the delta nitrogen atom in the imidazole ring. The HIE structure is the presence of hydrogen at the epsilon nitrogen atom found in imidazole. The HIP structure is the presence of a hydrogen atom in both nitrogen atoms. As time progressed and research increased, efforts were made to understand the role of metals in our lives. It has begun to be used in medicine and pharmaceutical treatments, especially thanks to many fields and technologies that allow the use of metal cations as central components. For this reason, metals will be used widely in the future with these aspects. Metals have such positive aspects as well as negative aspects. Storage problems arise with metals that are used very frequently. Since a regular storage area cannot be created for metals, their leakage into running water poses a danger. With this leakage, the amount of metal found in plants and animals increases. This increase indirectly affects humans as well, as humans need both animals and plants to survive. Metal ions that pass into humans in this way can enter the bloodstream and potentially cause cell damage, neurological diseases, oncogenesis and other diseases. In addition to these effects, once metals enter the bloodstream, they can bind to amino acids in proteins in the blood, thanks to the amine and carboxyl ends they have, as well as the reactive potential of their R groups. This binding may lead to the inhibition of ongoing cellular processes, the increase of free radicals due to the increase in redox reactions, or structural deterioration. Metal atoms can coordinate with the oxygen atoms of the water molecules around them in order to complete the six-way coordination. Or coordination around the metal atom can be achieved with resins, one of the newly developed methods. Although resins are a commercially developed product, they have different combinations. With these combinations, metal binding affinities were tried to be increased.  
Quadruple coordination occurs between the oxygen atoms in its structure and the metal. The remaining two coordinations occur with the nitrogen atoms in the histidine molecule. NTA chelates are a common method used in chromatography. Histidine oligomers are a very common labeling method used in protein and DNA purification steps. Histidine oligomers used for this purpose are called His-tags in the literature. Multiple histidine tags, or fishhooks, are frequently used in experimental molecular biology studies because they are a naturally occurring oligomer. IMAC chromatography method is an important method used in the protein purification stage. The working principle of this chromatographic method and the main event that provides adsorption is the coordination between immobilized metal ions and a donor. IMAC method is divided into two: normal and reverse. In the normal method, metal ions are chelated on a support. The protein given for purification binds to metal ions. In the reverse IMAC method, the metal ion immobilized on the protein surface is bonded to the donor group attached to the support via a coordination bond. The reverse IMAC method is generally limited to metalloproteins. In addition, this method is not only used in the purification process. Enzyme reactors can be created by adsorbing enzymes to metal chelate supports. Recombinant proteins are protein sequences cloned into an expression vector that support gene expression and can be encoded by recombinant DNA. Histidine tags consist of six histidine amino acids. After these histidine residues are transferred to a vector by genetic engineering methods, a recombinant protein is obtained. This recombinant protein is subjected to the IMAC method and purified with the help of chelators and transition metals. Histidine tags can be purified under natural or denaturing conditions. Histidine tags can enhance protein or DNA binding capacities by coordinating divalent metal ions such as copper (Cu2+), zinc (Zn2+), nickel (Ni2+), and cobalt (Co2+), which are readily found in the human body. Why metal increases bonding, how and why complex structures change depending on the metal, why metal type is important in increasing bonding efficiency, etc. It still hasn't been explained. Although there are many theoretical studies trying to answer some questions, detailed mechanical problems are still unsolved. In this study, using DFT methods, the chemical structure of the metal bearing histidine tags was modeled realistically to describe the binding energy quantitatively. Quantum mechanics is a branch that describes the behavior of atoms, electrons, or particles at the molecular level. Software developed for structure optimization is used in quantum chemistry studies. The basic principle of these software is to read input and perform optimization operations according to these inputs. We carried out our quantum mechanics studies with the Gaussian 6.0 application, one of the most used applications. There are two different issues that are important in these studies: The first is the choice of method appropriate to the structure. The other is to choose the base depending on the structure situation. Appropriate methods and basis sets for the models were selected and the models were created. The models were constructed from hexa histidine oligomers coordinated to metal ions Ni2+, Zn2+, Cu2+, and Co2+ through the nitrogen atoms of the imidazole rings. The models were then linked to amino acid dimers (the smallest units representing the protein) via their N-termini. All model structures were first optimized by filling in the missing values of metal cations with oxygen atoms at the B3LYP/6-31G(d,p) level. For metal atoms, a basis set consisting of 6-31G(d,p) levels was used with the gen keyword, the method developed by Stevens and his group. All calculations were performed using the IEFPCM solver model in implicit water environment. Quantum mechanics studies have concluded which transition metal in the histidine tag structure contributes to the formation of a  
more stable structure. This result we obtained is very similar to the results in the literature. Following these studies, protein – ligand docking studies were carried out using optimized histidine tags. In these docking studies, a 12-sequence structure with a 1BNA dodocomere structure, which is most similar to human DNA, was used as the target macromolecule. The synthetic DNA dodocomere structure is little more than a full turn of the dextral double-stranded B form. The two ends of the spiral are interlocked at 190 angles. In this structure, small grooves overlap. At the center of the molecule where distortion is least, the structure has an average rotation of 36.90 per step, or 9.8 base pairs per rotation. The dihedral angle between the A and T base pairs at the center of the molecule is 17.30, and the angle between the G and C bases at the two ends is 11.50. While purine groups have high delta values, pyrimidine sugars have low delta values in the structure. All poses obtained as a result of docking were examined and the best poses were examined. As a result of this study, it was observed that our optimized histidine tag structure binds more to DNA in the minor groove and interacts more with the guanine base. The coupling distance to guanine bases is similar in length for three different metals. A simulation was performed to support these results. All conditions in the simulation were chosen the same for 3 different metals. In this simulation, how our protein and ligand behave in a real solvent environment is simulated. The simulation time was set to 50 ns. The results obtained are similar to our quantum mechanical and protein docking results.