Mehdi Sadeghi, Bijan Ranjbar, Mohammad Reza Ganjalikhany,
Volume 7, Issue 3 (Special Issue 2016)
Abstract
The Cu dependent restriction deoxyribozyme is the unique example of known deoxyribozymes. The uniqueness of this deoxyribozyme is originated from specific cleaving of single stranded DNA and formation of triple helix DNA structure which is necessary for substrate recognition and binding. The most established method for measuring the kinetic parameters of deoxyribozyme is based on use of radiolabeled substrates which have several difficulties. In this study we present accurate, fast and inexpensive methods for kinetic study of the deoxyribozyme which is based on extrinsic fluorescence and UV-visible spectroscopy techniques. As mentioned above, DNA triple helix formation is necessary for substrate identification and also enzyme activity. Circular dichroism spetropolarimetery is used for structural study of enzyme. Analysis of spectrum results from this technique indicates structural changes which is a direct evidence for the triple helix formation in enzyme-substrate complex. Extrinsic fluorescence experiment is based on high affinity of SYBR gold to double stranded DNA compared to single stranded DNA. Enzyme activity can be measured by SYBR gold fluorescence emission upon addition of cofactor to the enzyme-substrate complex. Continuous hyperchromicity assay method which is based on UV-visible spectroscopy was used for measuring of enzyme activity by hyperchromicity of the enzyme-substrate complex after addition of cofactor. Comparison of the results show that the continuous hyperchromicity assay is more accurate than the extrinsic fluorescence method, because of this method is based on intrinsic physicochemical properties of DNA without interference of external factors.
Shahrbanoo Jafari, Rahman Emamzadeh, Mahboobeh Nazari, Mohamad Reza Ganjalikhany,
Volume 14, Issue 4 (9-2023)
Abstract
Aim: Follistatin-like protein 1 (FSTL1) is a secreted glycoprotein that plays an important role in regulating cell survival, proliferation, differentiation, migration, inflammation, and modulating the immune system. The FK domain in FSTL1 has 10 conserved cysteine residues that form 5 disulfide bonds. Despite extensive studies on the function of FSTL1, limited structural information is available about this biologically important molecule.
Materials and Methods:Using the SWISS-MODEL server and using the crystal structure of the FK domain of the mouse FSTL1 protein with the code (PDB: 6jzw) as a template, structural models of the FK domain of the human FSTL1 protein were prepared. In the next step, the resulting structures were checked using Swiss-PDB Viewer 4.10, Chimera 1.12 software, Ramachandaran diagram and PDBSUM server, in terms of the distance between two cysteine residues, the modeling error range, and the formation of disulfide bonds. Molecular dynamics simulations were performed using the AMBER software package with the ff14SB force field.
Results: The results showed that the FK domain without disulfide bond has root mean square deviations (RMSD) and root mean square fluctuations (RMSF), higher than the native FK domain. In addition, the radius of gyration in domain without disulfide bonds is significantly lower than that of native FK domain. The results show that the disulfide bonds of the FK domain play a role in the stability of the structural folding of the FK domain and the removal of these bonds increases the structural flexibility of this domain.
