Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because challenges producing proteins to spot on arrays. We generated by printing complementary DNAs onto glass slides and then translating target with mammalian reticulocyte lysate. Epitope tags fused allowed them be immobilized situ. This obviated need purify proteins, avoided stability problems during storage, captured sufficient functional studies. used technology map...

The human 3-methyladenine DNA glycosylase [alkyladenine (AAG)] catalyzes the first step of base excision repair by cleaving damaged bases from DNA. Unlike other glycosylases that are specific for a particular type base, AAG excises chemically diverse selection substrate alkylation or deamination. 2.1-Å crystal structure complexed to containing 1, N 6 -ethenoadenine suggests how modified can be distinguished normal in enzyme active site. Mutational analyses residues contacting alkylated...

Abstract The bacterial divisome is a macromolecular machine composed of more than 30 proteins that controls cell wall constriction during division. Here, we present model the structure and dynamics core complex E. coli divisome, supported by combination prediction, molecular simulation, single-molecule imaging, mutagenesis. We focus on septal synthase formed FtsW FtsI, its regulators FtsQ, FtsL, FtsB, FtsN. results indicate extensive interactions in four regions periplasmic domains complex....

Most cancer-associated BRCA1 mutations identified to date result in the premature translational termination of protein, highlighting a crucial role for C-terminal, BRCT repeat region mediating tumor suppressor function. However, molecular and genetic effects missense that map remain largely unknown. Using protease-based assay, we directly assessed sensitivity folding domain an extensive set truncation single amino acid substitutions derived from breast cancer screening programs. The protein...

Abstract Excitatory neurotransmission is principally mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype ionotropic glutamate receptors (AMPARs). Negative allosteric modulators are therapeutic candidates that inhibit AMPAR activation and can compete with positive to control function through unresolved mechanisms. Here we show inhibition pushes AMPARs into a distinct state prevents both modulation. We used cryo-electron microscopy capture bound glutamate, while...

Photoswitched tethered ligands (PTLs) can be used to remotely control protein function with light. We have studied the geometric and conformational factors that determine efficacy of PTL gating in ionotropic glutamate receptor iGluR6 using a family photoiosomerizable MAG (maleimide-azobenzene-glutamate) PTLs covalently attach clamshell ligand-binding domain. Experiments molecular dynamics simulations modified proteins show optical switching depends on 2 factors: (i) relative occupancy...

Abstract N -methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders diseases. However, complete elimination off-target effects small chemical compounds challenging thus, there is a need to explore alternative strategies targeting NMDAR subtypes. Here we report...

ABSTRACT In allosteric proteins, identifying the pathways that signals take from ligand‐binding sites to enzyme active or binding pockets and interfaces remains challenging. This avenue of research is motivated by goals understanding particular macromolecular systems interest creating general methods for their study. An especially important protein subject many investigations in allostery SARS‐CoV‐2 main protease (Mpro), which necessary coronaviral replication. It both an attractive drug...

Significance Glycine-activated ionotropic glutamate receptors (iGluRs) encoded in ctenophore genomes are evolutionary precursors to NMDA receptors, which play important roles synaptic plasticity. Ctenophore iGluRs feature a distinct interdomain salt bridge the ligand-binding domain, molecular lock, that thus far has not been found of other organisms. We use combination crystallographic, biochemical, electrophysiological, and computational approaches elucidate role this lock iGluR. find...

The human alkyladenine DNA glycosylase has a broad substrate specificity, excising structurally diverse group of damaged purines from DNA. To more clearly define the structural and mechanistic bases for specificity glycosylase, kinetics excision binding activities were measured several different undamaged within identical sequence contexts. We found that1,N 6-ethenoadenine (εA) hypoxanthine (Hx) excised relatively efficiently, whereas 7,8-dihydro-8-oxoguanine, O 6-methylguanine, adenine,...

K2PØ, the two-pore domain potassium background channel that determines cardiac rhythm in Drosophila melanogaster, and its homologues establish excitable membrane activity mammals are of unknown structure. K2P subunits have two pore domains flanked by transmembrane (TM) spans: TM1-P1-TM2-TM3-P2-TM4. To spatial relationships we identified pairs sites display electrostatic compensation. Channels silenced addition a charge loop 1 (P1) or P2 were restored to function countercharges at specific...

Ionotropic glutamate receptors (iGluRs) mediate neurotransmission at the majority of excitatory synapses in brain. Little is known, however, about how reaches recessed binding pocket iGluR ligand-binding domains (LBDs). Here we report process to a prototypical iGluR, GluA2, atomistic detail using unbiased molecular simulations. Charged residues on LBD surface form pathways that facilitate by effectively reducing three-dimensional diffusion spatially constrained, two-dimensional one. Free...

Abstract Inflammasomes are filamentous signaling platforms integral to innate immunity. Currently, little is known about how these structurally similar filaments recognize and distinguish one another. A cryo-EM structure of the AIM2 PYD filament reveals that architecture upstream essentially identical adaptor ASC filament. In silico simulations using Rosetta molecular dynamics followed by biochemical cellular experiments consistently demonstrate individual assemble bidirectionally. By...

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that responsible for the majority of excitatory transmission at synaptic cleft. Mechanically speaking, agonist binding to ligand domain (LBD) activates receptor by triggering a conformational change is transmitted transmembrane region, opening channel pore. We use fully atomistic molecular dynamics simulations investigate process in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, an iGluR subtype. The...