Biology subjects

Hazra, J. P.

Publications and source records attributed to Hazra, J. P..

6 recordsLinked to original sources

bioRxiv2025

Unraveling single-molecule reactions via multiplexed in-situ DNA sequencing

DNA sequence regulates complex reactions and protein-DNA interactions, yet sequence effects remain poorly understood due to the lack of direct, high-throughput approaches to study sequence-dependence at the single-molecule level. Here, we introduce Single-molecule Phenotyping and In-Situ Sequencing (SPIN-Seq), a DNA-based, protein-free method that links functional and structural properties of a single DNA molecule with the sequence of that same molecule. After performing functional assays on immobilized DNA molecules, SPIN-Seq uses sequencing-by-transient-hybridization on the same surface and instrument to read sequences in each immobilized DNA molecule, directly linking phenotype and genotype. Applying SPIN-Seq, we dissected the interaction of a transcription factor with its target sequence, and revealed the sequence-dependence of RNA polymerase pausing and reaction-path branching during initial transcription. Our method provides powerful, systematic ways to understand complex molecular mechanisms and their sequence dependence.

biophysics↗

bioRxiv2022

Tip-links serve as force-pass filter to fulfil the role of gating-springs

Tip-links as gating-spring in the mechanotransduction in hearing is still a debate. While the molecular elasticity of individual tip-link proteins warrants its candidature, the apparent rigidity from the heterotetrameric tip-links assembly refutes the claim. Using force-clamp experiments and simulations, we report that the heterotetrameric assembly is the natural selection for the gating-springs. Tip-links follow slip-ideal-slip bonds with increasing force. While in slip, the complex dissociates monotonously, ideal-bond interface responds indifferently to various auditory inputs. Insensitivity to forces renders tip-links as low-force pass filter, characteristic of gating-spring. Individual tip-links, however, forms slip-catch-slip bonds under tension. While catch bonds turn stronger with force from loud sound, our Langevin dynamics indicated the transition from slip-catch to slip-ideal bonds as cooperative effect of the dimers of individual protein complexes in tip-links. From molecular dynamics, we deciphered the molecular mechanism of catch bonds and its importance in deafness.

biophysics↗

bioRxiv2020

An Envelope Protein of a Prokaryotic Organelle Function towards Conserving Catalytic Activity of the Native Enzyme at Higher Temperatures

A classic example of an all-protein natural nano-bioreactor, the bacterial microcompartments are a special kind of prokaryotic organelles that confine enzymes within a small volume enveloped by an outer layer of shell proteins. This arrangement provides conditional metabolic aid to the bacteria. The outer shell allows selective diffusion of small molecules and sequesters toxic metabolites. In this work we use 1,2-propanediol utilization microcompartment as a model to study the effect of molecular confinement on the stability and catalytic activity of native enzymes in microcompartment. We observe a 50% decrease in the activity of free enzyme PduCDE at 45{degrees}C, while PduMCP retains its optimum activity till 50{degrees}C followed by more than 40% reduced activity at 55{degrees}C. PduBB, the major component of the outer shell contributes to the increased catalytic activity of PduCDE. PduBB also prevents the unfolding and aggregation of PduCDE under thermal stress. Using a combination of experimental and theoretical studies we probe the interactions of the shell proteins PduBB, N-terminal truncated PduB and single mutant PduBM38L with PduCDE. We observe that all the three variants of PduB* shell proteins interact with the enzyme in vitro, but only PduBB influences its activity and stability, underscoring the significance of the unique combination of PduB and PduB in PduMCP assembly.

molecular biology↗

bioRxiv2019

Variable Mutations at the p53-R273 Oncogeic Hotspot Position Leads to Altered Properties

Mutations in p53 protein, especially in the DNA binding domain is one of the major hallmarks of cancer. The R273 position is a DNA contact position and has several oncogenic variants. Surprisingly, cancer patients carrying different mutant-variants of R273 in p53 have different survival rates indicating that the DNA contact inhibition may not be the sole reason for reduced survival with R273 variants. Here, we probed the structural properties of three major oncogenic variants of the R273: ([R273L], [R273H], and [R273C])p53. Using a series of biophysical, biochemical and theoretical simulation studies, we observe that these oncogenic variants of the p53 not only suffer a loss in DNA binding, but also show distinct structural stabilty, aggregation and toxicity profiles. [R273C]p53 shows maximum amyloidogenicity while [R273L]p53 shows maximum aggregation. Further probe in the aggregation mechanism show that [R273C]p53 aggregation is disulphide mediated whereas hydrophobic interactions dominate self-assembly in [R273L]p53. MD simulation studies clearly show that -helical intermediates are observed in [R273C]p53 whereas {beta}-sheets are observed for [R273L]p53. Our study indicates that each of the R273 variant has its own distinct property of stability and self-assembly, the molecular basis of which, may lead to different types of cancer pathogenesis in vivo. These studies will aid the design of therapeutic strategies for cancer using residue specific or process specific protein aggregation as target.\n\nStatement of significanceThe present work stems from an interesting observation that genetic mutations that results in switching of one amino acid to different variants at the same codon show different cancer cell progression. We are trying to understand the molecular reason behind the different gain-of-function opted by these variants. With the help of biophysical and biochemical experiments, and computational studies we have observed that the different thermal stability, unique mechanism of unfolding and self-assembly might be one of the crucial parameters for their different oncogenic effect. These studies thus call for the need of developing therapeutic strategies that consider the resultant mutant-variant as a target rather than mutation position. This is an important lead towards the understanding of cancer.

biophysics↗

bioRxiv2019

Broken force dispersal network in tip-links by the mutations induces hearing-loss

Tip-link as force-sensor in the hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in inner-ear. This delicate balance between structure and function of tip-links is regulated by Ca2+-ions present in endolymph. Mutations at the Ca2+-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca2+ concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an AFM. AFM allowed us to mimic the high and wide range of force ramps (103 - 106 pN.s-1) as experienced in the inner ear. We performed in silico network analyses to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca2+ rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.\n\nSignificance StatementForce-sensors in inner ear are the key components in the hearing. Mutations in force-sensors often lead to congenital hearing loss. Loss of hearing has become a threat to humanity, with over 5% of world population suffering from deafness and 40% of which is congenital, primarily due to mutations in the sensory machinery in inner-ear. A better understanding of the molecular mechanism of the underlined hearing loss due to mutations is, therefore, necessary for better therapeutics to deaf. Here with a zoomed region of the force-sensors, we pointed out the differences in the force-propagation properties of the mutant and wild-type force-sensors. Our observation on restoring of functions of mutants in Ca2+-rich buffer indicates methods of developing low-cost therapeutic strategies against deafness.

biophysics↗

bioRxiv2017

Molecular mechanism of cell-cell adhesion mediated by cadherin-23

Adherin-junctions are traditionally described by the homophilic-interactions of classical cadherin-proteins at the extracellular region. However, the role of long-chain non-classical cadherins like cadherin-23(Cdh23) is not explored as yet even though it is implicated in tissue-morphogenesis, cancer, and force-sensing in neuronal tissues. Here, we identified a novel antiparallel-binding interface of Cdh23 homodimer in solution by combining biophysical and computational methods, in-vitro cell-binding, and mutational modifications. The dimer consists of two electrostatic-based interfaces extended up to two terminal domains, atypical to classical-cadherins known so far, and forms the strongest interactions in cadherin-family as measured using single-molecule force-spectroscopy. We further identified single point-mutation, E78K, that completely disrupts this binding. Interestingly, the mutation, S77L, found in skin cancers falls within the binding interface of the antiparallel-dimer. Overall, we provide the molecular architecture of Cdh23 at the cell-cell junctions which are likely to have far-reaching applications in the fields of mechanobiology and cancer.

biophysics↗