Biology subjects

Shams, A.

Publications and source records attributed to Shams, A..

3 recordsLinked to original sources

bioRxiv2020

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

Proteins evolve through the modular rearrangement of elements known as domains. It is hypothesized that extant, multidomain proteins are the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) that comprehensively assays all possible deletions of a protein. Using MISER, we generated a deletion landscape for the CRISPR protein Cas9. We found that Cas9 can tolerate large single deletions to the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

molecular biology↗

bioRxiv2020

vCOMBAT: a Novel Tool to Create and Visualize a COmputational Model of Bacterial Antibiotic Target-binding

MotivationAs antibiotic resistance creates a significant global health threat, we need not only to accelerate the development of novel antibiotics but also to develop better treatment strategies using existing drugs to improve their efficacy and prevent the selection of further resistance. We require new tools to rationally design dosing regimens to from data collected in early phases of antibiotic and dosing development. Mathematical models such as mechanistic pharmacodynamic drug-target binding explain mechanistic details of how the given drug concentration affects its targeted bacteria. However, there are no available tools in the literature that allows non-quantitative scientists to develop computational models to simulate antibiotic-target binding and its effects on bacteria. ResultsIn this work, we have devised an extension of a mechanistic binding-kinetic model to incorporate clinical drug concentration data. Based on the extended model, we develop a novel and interactive web-based tool that allows non-quantitative scientists to create and visualize their own computational models of bacterial antibiotic target-binding based on their considered drugs and bacteria. We also demonstrate how Rifampicin affects bacterial populations of Tuberculosis (TB) bacteria using our vCOMBAT tool. AvailabilityvCOMBAT online tool is publicly available at https://combat-bacteria.org/.

bioinformatics↗

bioRxiv2020

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 deafness

Cochlear hair cells possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15A) traffics and delivers critical molecules required for stereocilia development and is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3. To understand the molecular mechanism of how MYO15A delivers proteins within stereocilia, we performed a kinetic study of the ATPase motor domain to characterize its mechano-chemical cycle. Using the baculovirus-Sf9 system, we purified a recombinant minimal motor domain (S1) by co-expressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B co-expression had similar ATPase activities (kcat = ~ 6 s-1 at 20{degrees}C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP and actin binding, hydrolysis and phosphate release. Actin-attached ADP release was the slowest measured transition (~ 12 s-1 at 20{degrees}C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (~ 0.5) and weak thermodynamic coupling between ADP and actin binding. This is consistent with MYO15 being adapted for strain sensing as a monomer, or processive motility if oligomerized into ensembles. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and ultimately disrupt stereocilia trafficking necessary for normal hearing.

biochemistry↗