Biology subjects

Kirmizialtin, S.

Publications and source records attributed to Kirmizialtin, S..

11 recordsLinked to original sources

bioRxiv2026

Connecting Cryo-EM and Crystallographic Views of RNA Folding through Ionic Conditions and Structural Flexibility

Discrepancies between biomolecular structures resolved by cryo-electron microscopy (cryo-EM) and X-ray crystallography (XRD) often arise from differences in ionic conditions and construct design, yet their mechanistic impact on RNA folding remains unclear. In the SARS-CoV-2 frameshifting stimulatory element, cryo-EM and XRD structures reveal distinct pseudoknot conformations--a bent and a coaxially stacked state--complicating its structure-function relationship. Here, combining all-atom explicit-solvent simulation results with a structure-based electrostatic model, we show that Mg{superscript 2} ions drive transitions between these states by stabilizing long-range tertiary interactions and modulating local dynamical coupling involving the slippery site and stem 3. Energy landscape analysis reveals distinct folding pathways, while deletion of the slippery segment in crystallographic constructs alters intermediates and produces pathways inconsistent with single-molecule optical tweezer experiments. This study demonstrates how condition-dependent experiments encode complementary interaction-level information and how physics-based computational approaches integrate these to yield a coherent, mechanistic picture of RNA folding. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/722415v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1a7c324org.highwire.dtl.DTLVardef@fcabceorg.highwire.dtl.DTLVardef@736704org.highwire.dtl.DTLVardef@7061e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Cat_Wiz: A stereochemistry-guided toolkit for locating, diagnosing and annotating Mg2+ ions in RNA structures.

Misassigned Mg2+ ions are pervasive in RNA structural databases, obscuring mechanistic interpretation, undermining comparative analyses and compromising machine-learning training sets. Here, we present Cat_Wiz, a Coot-integrated, stereochemistry guided toolkit that facilitates the localization, diagnosis, correction and annotation of Mg2+ binding sites. Cat_Wiz comprises three modules: MG_diagnosis which validates and regularizes existing assignments; MG_detect which identifies unmodelled ion binding sites; and MG_clamp which classifies recurrent Mg2+ clamp motifs. Cat_Wiz also includes a complete binding site annotation system. The stereochemical principles implemented in Cat_Wiz were derived from an earlier analysis of the 1.55 [A] resolution Escherichia coli ribosome and from surveys of the Cambridge Structural Database. These principles provide a robust experimental foundation for characterizing Mg2+ binding sites. Applications to ribosomes, hammerhead ribozymes, group I introns, and quaternary RNA assemblies demonstrate that Cat_Wiz rapidly locates overlooked ions, corrects misassignments, and improves stereochemical fidelity in hours rather than days. Beyond refinement, Cat_Wiz generates curated data that can seed diverse machine-learning and AI models. This transparent, cost-effective framework establishes reproducible standards for RNA-ion assignments and will drive progress in the design of RNA 3D architectures through the identification of unique Mg2+-dependent backbone folds. Cat_Wiz is also applicable to Mg2+ binding sites in proteins and all related biomolecular systems since it is based on universal stereochemical principles.

molecular biology↗

bioRxiv2025

Protein Crowders Remodel RNA Electrostatics, Hydration, and Dynamics: A Challenge to Steric Crowding Models

The intracellular environment is densely populated with macromolecules, creating crowded conditions. Whether in vitro environments or synthetic crowders like polyethylene glycol (PEG) accurately capture the complexity of RNA interactions in vivo remains unclear. Using extensive all-atom molecular dynamics simulations, we investigated the HIV-1 TAR RNA hairpin in dilute, PEG-crowded, and realistic protein-crowded solutions. We found that PEG primarily exerts excluded-volume effects, maintaining RNA hydration and Na+ ion condensation similar to dilute conditions. In contrast, protein crowders significantly altered RNA electrostatics, reducing Na+ condensation by nearly 60%, displacing surface hydration water, and forming chemically specific contacts dominated by positively charged residues, notably arginine and lysine. These interactions led to local RNA expansion and reshaped its conformational landscape without disrupting the global fold. Moreover, protein crowding dramatically slowed RNA translational and rotational dynamics, as well as local water and ion mobility, effects minimally observed with PEG. Our findings emphasize that crowder identity critically determines RNA behavior and challenge the use of PEG as a universal model for intracellular conditions, providing mechanistic predictions for RNA studies in biologically relevant environments.

biophysics↗

bioRxiv2024

Principles of ion binding to RNA inferred from the analysis of a 1.55 A resolution bacterial ribosome structure - Part I: Mg2+

The importance of Mg2+ ions for RNA structure and function can difficultly be overstated. Several attempts were made to establish a comprehensive Mg2+ binding site classification. However, such descriptions were hampered by poorly modelled ion binding sites. Recently, ribosome cryo-EM structures with resolutions < 2.0 [A] allowed better descriptions of site-bound ions. However, in a recent cryo-EM 1.55 [A] E. coli ribosome structure, incomplete ion assignments prevented a full understanding of their binding modes. We revisited this model to establish general binding principles applicable to any RNA of sufficient resolution. These principles rely on the 2.9 [A] distance separating two Mg2+-bound cis-water molecules. By applying these rules, we could assign all Mg2+ ions bound with 2 to 4 non-water oxygens. We also uncovered unanticipated motifs where up to five adjacent nucleotides wrap around a single ion. The formation of these complex motifs involves a hierarchical dehydration of the Mg2+ ions, a process that plays a significant role in ribosome biogenesis and in the folding of large RNAs. These binding principles enhance our understanding of the roles of ions in RNA structure and will help refining the solvation shell of different ribosomes and of RNAs with complex topologies.

molecular biology↗

bioRxiv2023

Sequence-dependent orientational coupling and electrostatic attraction in cation-mediated DNA-DNA interactions

Condensation of DNA is vital for its biological functions and controlled nucleic acid assemblies. However, the mechanisms of DNA condensation are not fully understood due to the inability of experiments to access cation distributions and the complex interplay of energetic and entropic forces during assembly. By constructing free energy surfaces using exhaustive sampling, and detailed analysis of cation distributions, we elucidate the mechanism of DNA condensation in different salt conditions and with different DNA sequences. We found that DNA condensation is facilitated by the correlated dynamics of localized cations at the grooves of DNA helices. These dynamics are strongly dependent on salt conditions and DNA sequences. In the presence of magnesium ions, major groove binding facilitates attraction. In contrast, in the presence of poly-valent cations, minor groove binding serves to create charge patterns leading to condensation. Our findings present a novel advancement to the field and have broad implications for understanding and controlling nucleic acid complexes in vivo and in vitro.

biophysics↗

bioRxiv2022

RNA triplex structures revealed by WAXS-driven MD simulations

RNA triple helices are commonly observed tertiary motifs that are increasingly associated with critical biological functions, including signal transduction. Because the recognition of their biological importance is relatively recent, their full range of structures and function has not yet been elucidated. The integration of solution wide-angle X-ray scattering (WAXS) with data-driven molecular dynamics (MD) simulations, described here, provides a new way to capture the structures of major-groove RNA triplexes that evade crystallographic characterization. This method yields excellent agreement between measured and computed WAXS profiles, and allows for an atomically detailed visualization of these motifs. Using correlation maps, the relationship between well-defined features in the scattering profiles and real space characteristics of RNA molecules is easily defined, including the subtle conformational variations in the double-stranded RNA upon the incorporation of a third strand by base-triples. This readily applicable approach provides unique insight into some of the interactions that stabilize RNA tertiary structure and enable function.

biophysics↗

bioRxiv2022

Differences in Ion-RNA binding modes due to charge density variations explain the stability of RNA in monovalent salts

The stability of RNA increases as the charge density of the alkali metal cations increases. The molecular mechanism for the stability does not exist. To fill this gap, we performed all-atom molecular dynamics (MD) pulling simulations to dissect the microscopic origin of this phenomenon. We first established that the free energy landscape obtained in the simulations is in excellent agreement with the single-molecule optical tweezer experiments. The origin of the stronger stability in Na+ compared to K+ is found to be due to the differences in the charge-density related binding modes. The smaller hydrated Na+ ion preferentially binds to the highly charged phosphates. In contrast, the larger K+ ions interact with the major grooves. As a result, the electrostatic repulsion between the phosphate groups is reduced more effectively by Na+ ions. Because the proposed mechanism is generic, we predict that the same conclusions are valid for divalent alkaline earth metal cations.

biophysics↗

bioRxiv2021

Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid

High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension (with the correct nucleotide), the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to greatly exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. We show that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, non-ideal base pairing, and a large increase in the distance from the 3-OH group of the primer strand to the -phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high fidelity DNA polymerases.

biochemistry↗

bioRxiv2020

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis

We examined the roles of Mg2+ ions in DNA polymerization by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase and by molecular dynamics simulation of Mg2+ binding. Binding of the Mg-nucleotide complex induces a conformational change of the enzyme from open to closed states in a process that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds weakly to the closed state of the enzyme-DNA-Mg.dNTP complex with an apparent Kd = 3.7 mM and facilitates the catalytic reaction. This weak binding of the catalytic Mg2+ is important to maintain fidelity in that the Mg2+ samples the correctly aligned substrate without perturbing the equilibrium at physiological Mg2+ concentrations. The binding of the catalytic Mg2+ increases nucleotide specificity (kcat/Km) by increasing the rate of the chemistry and decreasing the rate of enzyme opening allowing nucleotide release. Changing the free Mg2+ concentration from 0.25 to 10 mM increased nucleotide specificity (kcat/Km) by 12-fold. Mg2+ binds very weakly to the open state of the enzyme in the absence of nucleotide (Kd {approx} 34 mM) and competes with Mg.dNTP. Analysis based on publish crystal structures showed that HIV RT binds only two metal ions during incorporation of a correct base-pair. MD simulations support the kinetic studies suggesting weak binding of the catalytic Mg2+ in open and closed states. They also support the two-metal ion mechanism, although the polymerase may bind a third metal ion in the presence of a mismatched nucleotide.

biochemistry↗

bioRxiv2020

Dramatic shape changes occur as Cytochrome c folds

Extensive experimental studies on the folding of Cytochrome c (Cyt c) make this small protein an ideal target for atomic detailed simulations for the purposes of quantitatively characterizing the structural transitions and the associated time scales for folding to the native state from an ensemble of unfolded states. We use previously generated atomically detailed folding trajectories by the Stochastic Difference Equation in Length (SDEL) to calculate the time-dependent changes in the Small Angle X-ray scattering (SAXS) profiles. Excellent agreement is obtained between experiments and simulations for the time dependent SAXS spectra, allowing us to identify the structures of the folding intermediates, which shows that Cyt c reaches the native state by a sequential folding mechanism. Using the ensembles of structures along the folding pathways we show that compaction and the sphericity of Cyt c change dramatically from the prolate ellipsoid shape in the unfolded state to the spherical native state. Our data, which provides unprecedented quantitative agreement with all aspects of time-resolved SAXS experiments, shows that hydrophobic collapse and amide group protection coincide on the 100 microseconds time scale, which is in accord with ultrafast Hydrogen/Deuterium exchange studies. Based on these results we propose that compaction of polypeptide chains, accompanied by dramatic shape changes, is a universal characteristic of globular proteins, regardless of the underlying folding mechanism.

biophysics↗

bioRxiv2020

Structure-guided DNA-DNA attraction mediated by divalent cations

Probing the role of surface structure in electrostatic interactions, we report the first observation of sequence-dependent dsDNA condensation by divalent alkali cations. Disparate behaviors were found between two repeating sequences with 100% AT content, a poly(A)-poly(T) duplex (AA-TT) and a poly(AT)-poly(TA) duplex (AT-TA). While AT-TA exhibits non-distinguishable behaviors from random-sequence genomic DNA, AA-TT condenses in all divalent alkali ions (Mg2+, Ca2+, Sr2+, and Ba2+). We characterized these interactions experimentally and investigated the underlying principles using all-atom computer simulations. Both experiment and simulation demonstrate that AA-TT condensation is driven by nonspecific ion-DNA interactions, which depend on the structures of ions and DNA surface. Detailed analyses reveal sequence-enhanced major groove binding (SEGB) of point-charged alkali ions as the major difference between AA-TT and AT-TA, which originates from the continuous and close stacking of nucleobase partial charges in AA-TT but not in AT-TA. These SEGB cations elicit attraction via spatial correlations with the phosphate backbone of neighboring helices, reminiscent of the "DNA-zipper" model, which though assumes non-electrostatic cation groove binding a priori. Our study thus presents a distinct molecular mechanism of DNA-DNA interaction in which sequence-directed surface motifs act with abundant divalent alkali cations non-specifically to enact sequence-dependent behaviors. This physical insight allows a renewed understanding of the function of repeating DNA sequences in genome organization and regulation and offers a facile approach for DNA technology to control the assembly process of DNA nanostructures.

biophysics↗