Azotobacter vinelandii CA6 FeSI proteininin klonlanması, ifadesi ve karakterizasyonu

The [2Fe-2S] protein from Azotobacter vinelandii CA6, also known as Shethna protein I or FeSI protein, was cloned and overexpressed in E. coli and purified. SDS-PAGE analysis showed a band at ~11 kDa, the monomeric size of the protein, at each stage of the purification. Gel filtration profile of FeSI indicates it forms a dimer in its native state. The UV-visible spectrum showed absorbances at signature wavelengths, 344, 418 and 464 nm, due to the iron-sulfur cluster. The sequence of A. vinelandii CA6 FeSI protein are similar to the sequences of [2Fe-2S] ferredoxins from nitrogen-fixing Clostridium pasteurianum and Aquifex aeolicus, which is not a nitrogen fixer, including conserved cysteine residues. These suggest that FeSI may or may not be involved in nitrogen fixation as there is no evidence although the FeSI gene is present in the major nif gene cluster in Azotobacter vinelandii CA6. This study will be beneficial for understanding the function of FeSI in nitrogen fixation and the relations with other [2Fe-2S] proteins.Bu çalışmada, Azotobacter vinelandii CA6'dan Shethna protein I veya FeSI proteini olarak da bilinen [2Fe-2S] proteini izole edilmiş, E. coli'de aşırı ifade edilmiş ve saflaştırılmıştır. SDS-PAGE analizi, saflaştırmanın her aşamasında proteinin monomer boyutu olan ~11 kDa'da bir bant göstermiştir. FeSI jel filtrasyon profili, doğal halde dimer olduğunu işaret etmektedir. UV-görünür spektrumunda, demir-kükürt kümesine özgü 344, 418 ve 464 nm dalga boylarında absorbanslar ölçülmüştür. A. vinelandii CA6 FeSI proteininin dizisi, korunmuş sistein rezidüleri dahil olmak üzere, nitrojen sabitleyici Clostridium pasteurianum ve bir nitrojen sabitleyici olmayan Aquifex aeolicus'tan elde edilen [2Fe-2S] ferredoksinlerin dizilerine benzemektedir. Bu bulgular, FeSI geninin Azotobacter vinelandii CA6'daki majör nif gen kümesinde bulunmasına rağmen hiçbir kanıt olmadığı için FeSI'in nitrojen fiksasyonuna dahil olabileceğini veya olmayabileceğini düşündürmektedir. Bu çalışma, FeSI'in nitrojen fiksasyonundaki işlevini ve diğer [2Fe-2S] proteinleri ile olan ilişkilerini anlamak için faydalı olacaktır

Structural studies of proteins involved in carbon fixation

In this thesis, the structures of proteins involved in carbon fixation by enzymes of the 3 hydroxypropionate (3 HP) cycle of Chloroflexus aurantiacus, and the acetone carboxylase from Xanthobacter autrophicus were investigated. The crystal structures of C-terminal and N-terminal domains of malonyl CoA reductase, and mesaconyl C1-CoA hydratase were solved. Preliminary structural information was obtained for mesaconyl-CoA transferase and mesaconyl-C4-CoA hydratase, the right-hand side 3 HP cycle enzymes. The mechanism of 3 HP formation from malonyl CoA, catalysed by malonyl CoA reductase was studied using ligand bound structures, site-directed mutagenesis, and kinetics. The ATP dependent acetone carboxylation mechanism was examined structurally. Acetone carboxylase was natively purified from Xanthobacter autotrophicus and the AMP and acetate bound structure was determined at 1.9 Å. This thesis has applications in the biotechnology of carbon fixation.Open Acces

Rapid and efficient ambient temperature X-ray crystal structure determination at Turkish Light Source

High-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish light source (Turkish DeLight). The complete dataset was collected in 18.5 min with resolution extending to 2.39 Å and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature structure provides invaluable information about the structural dynamics of the lysozyme. Turkish DeLight provides robust and rapid ambient temperature biomacromolecular structure determination with limited radiation damage

Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish Light Source “Turkish DeLight”

X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g., proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryocrystallography has never been performed in Türkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, İstanbul, Türkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis