Biology subjects

Ahlawat, V.

Publications and source records attributed to Ahlawat, V..

6 recordsLinked to original sources

bioRxiv2026

Counteraction of HMGB1 at ss-dsDNA junctions maintains liquidity of protamine-DNA co-condensates

In the sperm nucleus, protamine replaces histones to mediate extreme DNA compaction. The histone-to-protamine transition involves the occurrence of double-strand breaks, and is facilitated by transition proteins including those containing high-mobility-group (HMG) boxes. Here we used optical tweezers and microscopy to study the actions of HMGB1 and protamine on DNA. Confocal scans of GFP-HMGB1 on overstretched {lambda}-DNA show 2-3 foci that spread on the DNA upon retraction. Spreading of foci coincides with reannealing of ssDNA tracks, confirming their localization at ss-dsDNA junctions. Whereas the force-extension curves of protamine-bound {lambda}-DNA show tangles that withstand forces > 60 pN, premixing protamine with HMGB1 produces only bends and bridges ([~] 20 pN). The counteraction of HMGB1 involves its acidic C-terminal tail, as HMGB1-{Delta}C fails to prevent tangle formation. In line with these single-molecule results, brightfield and confocal imaging shows that HMGB1 converts protamine-dsDNA aggregates into liquid droplets whereas HMGB1-{Delta}C fails to do so. Together, these observations support our hypothesis that chromatin-associated proteins like HMGB1 help maintain early protamine-mediated DNA condensates in a liquid state, enabling the recruitment of the repair machinery to restore the duplex structure.

biophysics↗

bioRxiv2025

Impact of Long-Term Integrated Nutrient Management Practices on Soil Bacterial Diversity: A 16S rRNA-Based Metagenomic Approach

The growing worlds population, expected to surpass 9 billion by 2050, demands a 70-100% increase in farm productivity, posing significant challenges for sustainable agriculture. Conventional farming, reliant on chemical fertilizers and pesticides, negatively impacts environmental sustainability and food quality. Metagenomics, a culture-independent technique, has revolutionized the study of microbial communities by enabling comprehensive analysis of microbial diversity and function. This study evaluated the effects of combined nutrient management practices--including farmyard manure (FYM), wheat straw, green manure, and NPK fertilizers--on soil bacterial diversity using 16S rRNA-based metagenomic analysis. Key findings revealed the dominance of bacterial phyla such as Proteobacteria, Actinobacteria, and Firmicutes, which play critical roles in nutrient cycling, organic matter decomposition, and plant growth promotion. Distinct taxonomic distributions were observed across the samples, with Proteobacteria and Firmicutes dominating in control (T1), NPK (T2), and NPK_Green Manure (T5) treatments, while a shift in phyla composition was noted in NPK_FYM (T3) and NPK_WheatStraw (T4). Genus-level analysis revealed that Pseudomonas and Bacillus species were abundant in control (T1) and NPK (T2), while Sphingomonas species were dominant in NPK_FYM (T3). Further diversity analyses revealed significant variation across the samples, with T3 showing the highest richness and control (T1) showing the lowest richness. The rarefaction curve confirmed sufficient sampling for capturing microbial diversity, while beta diversity analysis, including Principal Coordinate Analysis (PCA) and Bray-Curtis clustering, indicated distinct microbial community structures across different treatments. The combined use of organic amendments with NPK fertilizer enhanced microbial diversity and population, indicating their potential to support resilient and productive soil ecosystems.

bioinformatics↗

bioRxiv2025

Measuring bridging forces in protein-DNA condensates

Protein-DNA condensates mediate transcription and regulate gene expression and DNA replication and repair. The intermolecular bridging forces stabilizing condensates have direct roles in these processes. Here we use optical tweezers to measure bridging forces. In the presence of protamine, a single condensate is observed on a 20.5-knt single-stranded DNA (ssDNA) tethered between two microbeads. Stretching produces force curves with a sawtooth pattern, suggesting that the condensate is dissembled by the sequential rupture of individual protamine-ssDNA bridges. The bridging forces are 11.3 {+/-} 4.6 pN, with unfolding lengths of 1.3 {+/-} 0.8 {micro}m for single bridges. In contrast, double-stranded DNA (dsDNA) forms protamine-bridged tangles that can withstand forces high enough ([~]55 pN) for strand separation. ssDNA tracks unpeeled at nicks on dsDNA by overstretching seed tangle formation upon retraction, but the initial condensates have a sufficient ssDNA-to-dsDNA ratio to appear liquid-like, as indicated by a sawtooth pattern in the subsequent stretching. The presence of dsDNA raises bridging forces to 34 {+/-} 8 pN, which revert to [~]10 pN upon adding external ssDNA. In line with these single-molecule results, protamine-dsDNA mixtures form solid-like aggregates and require the addition of ssDNA to become liquid droplets. Conversely, adding dsDNA slows the fusion of protamine-ssDNA droplets. This work demonstrates the first measurements of bridging forces and shows that the ssDNA-to-dsDNA ratio can tune their magnitude in protein-DNA condensates.

biophysics↗

bioRxiv2023

Multiple modes of DNA compaction by protamine

In sperm cells, protamine replaces histones to compact DNA 10-20 times more than in somatic cells. To characterize the extreme compaction, we employed confocal microscopy and optical tweezers to determine the conformations and stability of protamine-bound {lambda}-DNA. Confocal images show increasing compaction of {lambda}-DNA at increasing protamine concentration. In the presence of protamine, single {lambda}-DNA molecules form bends and loops that unravel at 10-40 pN forces as well as coils that shorten the contour length by up to 40% and withstand forces strong enough ([~]55 pN) for strand separation. Strand separation nucleates coils, indicating protamine insertion into DNA bases. Protamine may participate in both local and higher-order chromatin organization, leading to extreme compaction and global transcription silencing. One-Sentence SummaryProtamine compacts sperm DNA in multiple modes, producing bends and loops but also coils that may block transcription.

biophysics↗

bioRxiv2020

Viscoelasticity of single macromolecules using Atomic Force Microscopy

We measured viscoelasticity of single protein molecules using two types of Atomic Force Microscopes (AFM) which employ different detection schemes to measure the cantilever response. We used a commonly available deflection detection scheme in commercial AFMs which measures cantilever bending and a fibre-interferometer based home-built AFM which measures cantilever displacement. For both methods, the dissipation coefficient of a single macromolecule is immeasurably low. The upper bound on the dissipation coefficient is 5 x 10-7 kg/s whereas the entropic stiffness of single unfolded domains of protein measured using both methods is in the range of 10 mN/m. We show that in a conventional deflection detection measurement, the phase of bending signal can be a primary source of artefacts in the dissipation estimates. It is recognized that the measurement of cantilever displacement, which does not have phase lag due to hydrodynamics of the cantilever, is better suited for ensuring artefact-free measurement of viscoelasticty compared to the measurement of the cantilever bending. We confirmed that the dissipation coefficient in single macromolecules is below the detection limit of AFM by measuring dissipation in water layers confined between the tip and the substrate using similar experimental parameters. Further, we experimentally determined the limits in which the simple point-mass approximation of the cantilever works in off-resonance operation. Significance StatementSingle Macromolecules, including unfolded proteins bear rubber-like entropic elasticity and internal friction characterized by finite dissipation coefficient. Direct measurement of this viscoelastic response is important since it plays a significant role, both in polymer physics as well as protein folding dynamics. The viscoelastic response of single polymer chain is difficult and prone to artefacts owing to the complications of hydrodynamics of macroscopic probe itself in the liquid environment. Using a special atomic force microscope, which allows quantitative estimate of viscoelasticity in liquid environments, we measured viscoelastic response of single molecule of Titin. We report here that the dissipation coefficient is below the detection limit of our experiments - with upper bound which is less than reported values in the literature.

biophysics↗

bioRxiv2018

Direct Measurement of Dissipation in a Single Protein using Small Amplitude Atomic Force Microscopy

In Krammers theory, stiffness and dissipation coefficient of a protein determine the rate of their conformational change. Using atomic force microscope, it is possible to measure viscoelasticity of a single protein, wherein its dissipative and elastic nature is directly and independently measured. Such measurements are performed, either by measuring the thermal fluctuations of the protein held under a constant force, or by providing small modulations to the protein by dithering the cantilever and measuring its response. In small amplitude approximation, where dither amplitude is comparable to persistence length of polymers, it is possible to measure the proteins viscoelastic response accurately. We measured dissipation in I27 at extremely low pulling speeds ([~] 50 nm/s) and low dither frequencies ([~]100 Hz). At these experimental parameters the dissipation is found to be [~]10-5 kg/s, well above the detection limit of conventional AFM and upper limit predicted by Benedetti et al. Our stiffness data clearly reveals unfolding intermediate of titins individual immunoglobulin units. The intermediate is elongation of folded domains by [~]8 [A], wherein two hydrogen bonds are broken between beta sheets. It was possible to measure this elongation in our experiments. The directly measured internal friction of unfolded polymer chain shows a scaling with tension on the chain. The measurements show that it is possible to measure internal friction in single molecules unambiguously using small amplitude AFM. It suggests that systematic experiments to unravel the relation between directly measured internal friction and folding rates of proteins are possible.

biophysics↗