Biology subjects

Khare, S.

Publications and source records attributed to Khare, S..

22 records · Page 2Linked to original sources

bioRxiv2019

Mathematical models of protease-based enzymatic biosensors

An important goal of synthetic biology is to build biosensors and circuits with well-defined input-output relationships that operate at speeds found in natural biological systems. However, for molecular computation, most commonly used genetic circuit elements typically involve several steps from input detection to output signal production: transcription, translation, and post-translational modifications. These multiple steps together require up to several hours to respond to a single stimulus, and this limits the overall speed and complexity of genetic circuits. To address this gap, molecular frame-works that rely exclusively on post-translational steps to realize reaction networks that can process inputs at a timescale of seconds to minutes have been proposed. Here, we build mathematical models of fast biosensors capable of producing Boolean logic functionality. We employ protease-based chemical and light-induced switches, investigate their operation, and provide selection guidelines for their use as on-off switches. We then use these switches as elementary blocks, developing models for biosensors that can perform OR and XOR Boolean logic computation while using reaction conditions as tuning parameters. We use sensitivity analysis to determine the time-dependent sensitivity of the output to proteolytic and protein-protein binding reaction parameters. These fast protease-based biosensors can be used to implement complex molecular circuits with a capability of processing multiple inputs controllably and algorithmically. Our framework for evaluating and optimizing circuit performance can be applied to other molecular logic circuits.

synthetic biology↗

bioRxiv2019

Loss of function from widely distributed, synonymous mutations at single codons

Mutational tolerance inferred from laboratory-based mutational studies is typically much higher than observed natural sequence variation. Using saturation mutagenesis, we show that the ccdA antitoxin component of the ccdAB toxin-antitoxin system is unusually sensitive to mutation with over 60% of mutations leading to loss of function. Multi-base synonymous mutations at a codon display enhanced propensity to show altered phenotypes, relative to single-base ones. Such mutations modulate RNA structure, leading to altered relative translation efficiencies of the two genes in the operon, and a CcdA:CcdB protein ratio below one. These insights were used to predict and experimentally validate synonymous mutations that lead to loss of function in the unrelated relBE operon as well as the lacZ gene. Thus, synonymous mutations can have significant phenotypic effects, in the absence of overexpression or extraneous reporters. More generally, proteins are likely more sensitive to mutation than inferred from previous saturation mutagenesis studies.

molecular biology↗

bioRxiv2018

Dissection of protonation sites for antibacterial recognition and transport in QacA, a multi-drug efflux transporter.

QacA is a drug:H+ antiporter (DHA2) with 14 transmembrane helices, that renders antibacterial resistance to methicillin-resistant Staphylococcus aureus (MRSA) strains, with homologues in other pathogenic organisms. It is a highly promiscuous antiporter, capable of H+- driven efflux of a wide array of cationic antibacterial compounds and dyes. Our study, using a homology model of QacA, reveals a group of six protonatable residues in its vestibule. Systematic mutagenesis resulted in identification of D34 (TM1), and a cluster of acidic residues in TM13 including E407 and D411 and D323 in TM10, as being crucial for substrate recognition and transport of monovalent and divalent cationic antibacterial compounds. The transport and binding properties of QacA and its mutants were explored using whole cells, inside-out vesicles, substrate-induced H+ release and microscale thermophoresis. We identify two sites, D34 and D411 as vital players in substrate recognition while E407 facilitates substrate efflux as a protonation site. We also observe that E407 plays a moonlighting role as a substrate recognition site for dequalinium transport. These observations rationalize the promiscuity of QacA for diverse substrates. The study unravels the role of acidic residues in QacA with implications for substrate recognition, promiscuity and processive transport in multidrug efflux transporters, related to QacA.\n\nHighlightsO_LIA homology model of QacA with 14 TM helices was used to test the importance of acidic residues within the vestibule.\nC_LIO_LIMono and Divalent cationic substrate recognition requires two sites D34 (TM1) and D411 (TM13).\nC_LIO_LIE407 (TM13) is important for protonation driven efflux.\nC_LIO_LISubstrate recognition of a divalent substrate, dequalinium occurs at E407 instead of D411 providing glimpses into the promiscuity of substrate recognition in QacA.\nC_LI

biochemistry↗

bioRxiv2018

Stimulus-responsive self-assembly of enzymatic fractal structures by computational design

Fractal topologies, which are statistically self-similar over multiple length scales, are pervasive in nature. The recurrence of patterns at increasing length scales in fractal-shaped branched objects, e.g., trees, lungs, and sponges, results in high effective surface areas, and provides key functional advantages, e.g., for molecular trapping and exchange. Mimicking these topologies in designed protein-based assemblies will provide access to novel classes of functional biomaterials for wide ranging applications. Here, we describe a modular, multi-scale computational design method for the reversible self-assembly of proteins into tunable supramolecular fractal-like topologies in response to phosphorylation. Computationally-guided atomic-resolution modeling of fusions of symmetric, oligomeric proteins with Src homology 2 (SH2) binding domain and its phosphorylatable ligand peptide was used to design iterative branching leading to fractal-like assembly formation by enzymes of the atrazine degradation pathway. Structural characterization using various microscopy techniques and Cryo-electron tomography revealed a variety of dendritic, hyperbranched, and sponge-like topologies which are self-similar over three decades ([~]10nm-10m) of length scale, in agreement with models from multi-scale computational simulations. We demonstrate control over mesoscale topology (by linker design), formation dynamics, and functional enhancements due to dynamic multi-component assemblies constructed with three atrazine degradation pathway enzymes. The described design method should enable the construction of a variety of novel, spatiotemporally responsive catalytic biomaterials featuring fractal topologies.

biophysics↗