Biology subjects

Kepceoglu, A.

Publications and source records attributed to Kepceoglu, A..

6 recordsLinked to original sources

bioRxiv2026

A novel sample delivery method for powder X-ray diffraction at Turkish Light Source

Powder X-ray diffraction (PXRD) measurements performed on platforms originally designed for single-crystal diffraction are strongly affected by how the powder sample is presented to the X-ray beam, including the delivery configuration and support geometry. Here, we developed a modified Terasaki-plate-based sample-delivery method for PXRD using a laboratory single-crystal diffractometer implemented with the XtalCheck-S plate-reader operational mode at Turkish Light Source. The method was regarded under comparable measurement conditions relative to a standard loop/pin-based and a grease-based Terasaki setup using 5-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-4-ethyl-4H-1,2,4-triazole-3-thiol as a model analyte. The loop-based method allowed only limited powder sampling, whereas the grease-based Terasaki setup enabled multi-well sample delivery but produced higher background and weaker diffraction profiles. Conversely, Kapton-sealed Terasaki ensured secure retention of small amounts powder while providing lower background and clearer diffraction patterns. Within short total data collection times of only 1-2 min, the Kapton-Terasaki method delivered the best overall PXRD performance among the tested methods. Search-match and profile-fitting analyses showed that all three approaches sampled the same crystalline material, while the Kapton-based method gave the lowest profile residual (Rp = 9.6%) and the most reliable whole-pattern profile. These results demonstrate that optimizing sample delivery, rather than modifying the core instrument hardware, can substantially extend PXRD capability on an existing in situ crystallography platform for rapid, laboratory-based screening and comparative multi-sample measurements.

biochemistry↗

bioRxiv2025

Solubility, Hydration, and Sulphate Coordination in Cubic Insulin Crystals from Esrapid™ Monomers Stabilized in Divalent Anionic Form of Citric Acid

Insulins structural adaptations have been extensively studied at neutral and basic pH; however, the effects of water and anion coordination on allosteric regions under various acidic conditions remain unexplored. Given the critical role of polar interactions in allosteric modulation, investigating structured water movements across extended pH intervals is essential for understanding solvent-mediated stabilization mechanisms in the monomer form of insulin. Structures of acid-stable cubic insulin crystals were determined in the divalent anionic form of citric acid solutions over a pH range of 2 to 6 to investigate the effects of water and anion coordination, along with charge distribution, on protein conformation. Synchrotron X-ray diffraction data were collected at resolutions ranging from 1.4 to 1.76 [A], with refined models exhibiting R-factors between 0.19 and 0.21. While the spatial arrangement of most proteins is highly conserved, [~]90% of the water coordination network and polar interactions alter local residue motion and intrinsic dynamics of the structures as the pH is changed. This is in line with previously determined structures at pH 7-11, allowing for a comprehensive structural analysis in the pH range of pH 2-11. Three key observations emerged: (i) water coordinations undergo a prominent shift toward the isoelectric point of insulin (pH 5-7), (ii) water molecules and anions function as allosteric modulators to stabilize the T-state of insulin at the pH range 2 to 6, and (iii) at extreme pH values 2 and 11, increased solubility correlates with the structural adoption of insulins most active form, wherein hydration within the allosteric pocket supports monomer stabilization in the T-state. Combined with the computational analyses, pH-dependent electrostatic redistributions primarily affect side-chain dynamics and local protein motion. This solvent-coupled allosteric regulation provides a mechanistic framework for solvent-mediated protein stabilization, offering a novel insight into the rational design of insulin formulations through controlled protonation and hydration strategies.

biophysics↗

bioRxiv2023

4D Crystallography Captures Transient IF1-Ribosome Dynamics in Translation Initiation

Initiation factor 1 (IF1) is one of multiple key ligands involved in the initiation of mRNA translation, a highly dynamic and carefully-orchestrated process. However, details surrounding IF1 transient interactions with the small 30S ribosomal subunit remain incompletely understood despite characterization of unbound and fully-bound 30S states. Improvements in X-ray light sources and crystallographic techniques are now enabling time-resolved structural studies at near-physiological temperature and near-atomic resolution and thus the structural investigation of such dynamic processes. Here, we employed time-resolved serial femtosecond X-ray crystallography (TR-SFX) to probe the binding of IF1 to the small 30S ribosomal subunit in real time. Our time-resolved structural data demonstrates transient cryptic short-, mid-, and long-range allostery among different regions of the small 30 ribosomal subunit during IF1 binding, revealing small- and large-scale protein-target interactions and dynamics within intermediate macromolecular states at unprecedented temporal and spatial resolution. These data represent one of the first such 4D crystallographic studies assessing protein-protein and protein-RNA interactions and could serve as the basis for subsequent studies of the ribosome and of the multitudinous dynamic processes which underpin biology, and therefore, of life.

biophysics↗

bioRxiv2022

Comparative Study of High-resolution LysB29(Nε-myristoyl) des(B30) Insulin Structures Display Novel Dynamic Causal Interrelations in Monomeric-Dimeric Motions

The treatment of insulin-dependent diabetes mellitus is characterized by artificial supplementation of pancreatic {beta}-cell ability to regulate sugar levels in the blood. Even though various insulin analogs are crucial for reasonable glycemic control, understanding the dynamic mechanism of the insulin analogs may help to improve the best-protracted insulin analog to assist people with Type 1 Diabetes (T1D) to live comfortably while maintaining tight glycemic control. Here we present the high-resolution crystal structure of NN304, known as insulin detemir, to 1.7 [A] resolution at cryogenic temperature. We computationally further investigated our crystal structures monomeric-dimeric conformation and dynamic profile by comparing it with a previously available detemir structure (PDB ID: 1XDA). Our structure (PDB ID: 8HGZ) obtained at elevated pH provides electrostatically triggered minor movements in the equilibrium between alternate conformational substates compared to the previous structure, suggesting it might induce an intermediate state in the dissociation pathway of the insulin detemirs hexamer:dihexamer equilibrium. Supplemented with orientational cross-correlation analysis by Gaussian Network Model (GNM), this alternate oligomeric conformation offers the distinct cooperative motions originated by loose coupling of distant conformational substates of a protracted insulin analog that has not been previously observed.

biophysics↗

bioRxiv2022

Rapid and High Resolution Ambient Temperature 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 mins with resolution extending to 2.39 [A] 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.

biophysics↗

bioRxiv2022

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 cryo-crystallography has never been performed in Turkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Turkiye 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.

biophysics↗