Biology subjects

Tatli, O.

Publications and source records attributed to Tatli, O..

3 recordsLinked to original sources

bioRxiv2024

X-ray Crystallographic and Hydrogen Deuterium Exchange Studies Confirm Alternate Kinetic Models for Homolog Insulin Monomers

Despite the crucial role of various insulin analogs in achieving satisfactory glycemic control, a comprehensive understanding of their in-solution dynamic mechanisms still holds the potential to further optimize rapid insulin analogs, thus significantly improving the well-being of individuals with Type 1 Diabetes. Here, we employed hydrogen-deuterium exchange mass spectrometry to decipher the molecular dynamics of newly modified and functional insulin analog. A comparative analysis of H/D dynamics demonstrated that the modified insulin exchanges deuterium atoms faster and more extensively than the intact insulin aspart. Additionally, we present new insights derived from our 2.5 [A] resolution X-ray crystal structure of modified hexamer insulin analog at ambient temperature. Furthermore, we obtained a distinctive side-chain conformation of the Asn3 residue on the B chain (AsnB3) by operating a comparative analysis with a previously available cryogenic rapid-acting insulin structure (PDB_ID: 4GBN). The experimental conclusions have demonstrated compatibility with modified insulins distinct cellular activity, comparably to aspart. Additionally, the hybrid structural approach combined with computational analysis employed in this study provides novel insight into the structural dynamics of newly modified and functional insulin vs insulin aspart monomeric entities. It allows further molecular understanding of intermolecular interrelations driving dissociation kinetics and, therefore, a fast action mechanism.

biophysics↗

bioRxiv2024

Understanding the characteristic behavior of the wild-type and mutant protein structure of FLT3 protein by computational methods

FLT3 emerges as a commonly mutated protein with significant prognostic implications in acute myeloid leukemia (AML). Point mutations or deletions in the tyrosine kinase domain (TKD) at the activation loop and internal tandem duplications (ITD) in the juxtamembrane (JM) region (and less commonly in the TKD) are the primary mutations that occur in the FLT3 protein. Besides, AML treatment with tyrosine kinase inhibitor drugs may result in the acquisition of TKD mutations in the FLT3-ITD structure. All these mutations will induce activation of the kinase activity of FLT3 protein leading to activation of downstream signaling pathways. Therefore, finding better therapeutics against each of these mutant FLT3 proteins is crucial in the treatment of AML. This study aims to comprehend the characteristic behavior of TKD mutants (C and F in Y842), ITD mutants, and the combination of ITD with TKD mutations (C and F in Y842) in the FLT3 protein through computational approaches, including Molecular Dynamic (MD) simulation, cluster analysis, and machine learning techniques. The MD simulation studies revealed the alterations in the optimized state, flexibility, and compactness nature between FLT3-WT and mutated FLT3 proteins and identified significant changes in the point mutants, ITD, and the combined ITD and TKD mutated FLT3 protein structures. Cluster analysis also confirmed that these mutations significantly impact the overall flexibility of the protein structures, especially in the point-mutated structures of FLT3-Y842C and FLT3-ITD-Y842F. These findings emphasize the diverse protein conformations of mutated structures of the FLT3 protein, contributing to the deregulation of FLT3 protein function, and identified these mutated proteins as promising therapeutic targets in the treatment of AML.

bioinformatics↗

bioRxiv2020

Lentiviral vector-based SARS-CoV-2 pseudovirus enables analysis of neutralizing activity in COVID-19 convalescent plasma

As the COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread around the globe, effective vaccination protocols are under deployment. Alternatively, the use of convalescent plasma (CP) therapy relies on the transfer of the immunoglobulin repertoire of a donor that has recovered from the disease as a means of passive vaccination. While the lack of an effective antiviral treatment inadvertently increases the interest in CP products, initial clinical evaluation on COVID-19 patients revealed that critical factors determining the outcome of CP therapy need to be defined clearly if clinical efficacy is to be expected. Measurement of neutralizing activity against SARS-CoV-2 using wildtype virus presents a reliable functional assay but the availability of suitable BSL3 facilities for virus culture restricts its applicability. Instead, the use of pseudovirus particles containing elements from the SARS-CoV-2 virus is widely applied to determine the activity of CP or other neutralizing agents such as monoclonal antibodies. In this study, we present our approach to optimize GFP-encoding lentiviral particles pseudotyped with the SARS-CoV-2 Spike and Membrane proteins for use in neutralization assays. Our results show the feasibility of pseudovirus production using a C-terminal truncated Spike protein which is greatly enhanced by the incorporation of the D614G mutation. Moreover, we report that the use of Sodium Butyrate during lentiviral vector production dramatically increases pseudovirus titers. Analysis of CP neutralizing activity against particles pseudotyped with wildtype or D614G mutant Spike protein in the presence or absence the M protein revealed differential activity in CP samples that did not necessarily correlate with the amount of anti-SARS-CoV-2 antibodies. Our results indicate that the extent of neutralizing activity in CP samples depends on the quality rather than the quantity of the humoral immune responses and varies greatly between donors. Functional screening of neutralizing activity using pseudovirus-based neutralization assays must be accepted as a critical tool for choosing CP donors if clinical efficacy is to be maximized.

immunology↗