Biology subjects

Rangadurai, A. K.

Publications and source records attributed to Rangadurai, A. K..

3 recordsLinked to original sources

bioRxiv2024

Quantitative and systematic NMR measurements of sequence-dependent A-T Hoogsteen dynamics uncovers unique conformational specificity in the DNA double helix

The propensities to form lowly-populated short-lived conformations of DNA could vary with sequence, providing an important source of sequence-specificity in biochemical reactions. However, comprehensively measuring how these dynamics vary with sequence is challenging. Using 1H CEST and 13C R1{rho} NMR, we measured Watson-Crick to Hoogsteen dynamics for an A-T base pair in thirteen trinucleotide sequence contexts. The Hoogsteen population and exchange rate varied 4-fold and 16-fold, respectively, and were dependent on both the 3- and 5-neighbors but only weakly dependent on monovalent ion concentration (25 versus 100 mM NaCl) and pH (6.8 versus 8.0). Flexible TA and CA dinucleotide steps exhibited the highest Hoogsteen populations, and their kinetics rates strongly depended on the 3-neighbor. In contrast, the stiffer AA and GA steps had the lowest Hoogsteen population, and their kinetics were weakly dependent on the 3-neighbor. The Hoogsteen lifetime was especially short when G-C neighbors flanked the A-T base pair. The Hoogsteen dynamics had a distinct sequence-dependence compared to duplex stability and minor groove width. Thus, our results uncover a unique source of sequence-specificity hidden within the DNA double helix in the form of A-T Hoogsteen dynamics and establish the utility of 1H CEST to quantitively measure sequence-dependent DNA dynamics.

biochemistry↗

bioRxiv2024

Atomic resolution map of the solvent interactions driving SOD1 unfolding in CAPRIN1 condensates

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution Nuclear Magnetic Resonance (NMR) spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1, focused on both unfolded and folded SOD1 states in mixed phase and de-mixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the folding equilibrium of SOD1 towards the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates. Significance StatementBiomolecular condensates concentrate proteins and nucleic acids to regulate and perform key biological functions. Although the material properties of these condensates are well-studied, much less is understood about how the structure and dynamics of proteins within them are affected by the high concentration of biomolecules. In this study we have used NMR spectroscopy to study how the folding equilibrium and structural dynamics of the ALS protein SOD1 are modulated inside a condensate formed by CAPRIN1. Our study reveals that the CAPRIN1 condensed phase biases an immature form of SOD1 towards unfolded states that are susceptible to aggregation and provides insights into why this is the case, while a more mature form of the protein is much less affected.

biophysics↗

bioRxiv2023

NMR measurements of transient low-populated tautomeric and anionic Watson-Crick-like G·T/U in RNA:DNA hybrids: Implications for the fidelity of transcription and CRISPR/Cas9 gene editing

Many biochemical processes use the Watson-Crick geometry to distinguish correct from incorrect base pairing. However, on rare occasions, mismatches such as G*T/U can transiently adopt Watson-Crick-like conformations through tautomerization or ionization of the bases, giving rise to replicative and translational errors. The propensities to form Watson-Crick-like mismatches in RNA:DNA hybrids remain unknown, making it unclear whether they can also contribute to errors during processes such as transcription and CRISPR/Cas editing. Here, using NMR R1{rho} experiments, we show that dG*rU and dT*rG mismatches in two RNA:DNA hybrids transiently form tautomeric (Genol*T/U {rightleftarrows}G*Tenol/Uenol) and anionic (G*T-/U-) Watson-Crick-like conformations. The tautomerization dynamics were like those measured in A-RNA and B-DNA duplexes. However, anionic dG*rU- formed with a ten-fold higher propensity relative to dT-*rG and dG*dT- and this could be attributed to the lower pKa ({Delta}pKa [~]0.4-0.9) of U versus T. Our findings suggest plausible roles for Watson-Crick-like G*T/U mismatches in transcriptional errors and CRISPR/Cas9 off-target gene editing, uncover a crucial difference between the chemical dynamics of G*U versus G*T, and indicate that anionic Watson-Crick-like G*U- could play a significant role evading Watson-Crick fidelity checkpoints in RNA:DNA hybrids and RNA duplexes.

biophysics↗