Ca2+-calmodulin-dependent protein kinase II (CaM-kinase II) is a ubiquitous Ser/Thr-directed that expressed from family of four genes (α, β, γ, and δ) in mammalian cells. We have documented the three-dimensional structures biophysical enzymatic properties gene products. Biophysical analyses showed each isoform assembles into oligomeric forms their at 21–25 Å revealed all isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising association domain...

The calcium-calmodulin-dependent protein kinase II is a major component of brain synaptic junctions and has been proposed to play variety important roles in function. A complementary DNA representing portion the smaller 50-kilodalton subunit rat enzyme cloned sequenced. calmodulin-binding region identified synthetic analog prepared that binds calmodulin with high affinity presence calcium. Like polypeptide, concentration messenger RNA varies both neuroanatomically during postnatal...

Mitochondrial function is intimately linked to cellular survival, growth, and death. Mitochondria not only generate ATP from oxidative phosphorylation, but also mediate intracellular calcium buffering, generation of reactive oxygen species (ROS), apoptosis. Electron leakage the electron transport chain, especially damaged or depolarized mitochondria, can excess free radicals that damage proteins, DNA, lipids. Furthermore, mitochondrial releases pro-apoptotic factors initiate cell Previous...

Many important gram-negative bacterial pathogens use highly sophisticated type III protein secretion systems (T3SSs) to establish complex host-pathogen interactions. Bacterial-host cell contact triggers the activation of T3SS and subsequent insertion a translocon pore into target membrane, which serves as conduit for passage effector proteins. Therefore initial interaction between T3SS-bearing bacteria host cells is critical step in deployment machine, yet this process remains poorly...

Significance We have used cryogenic electron microscopy (cryo-EM) to achieve direct, probe-free imaging of lateral domains in biomimetic lipid membranes under native conditions and characterize differences their structures. First, measurements membrane thickness laterally uniform single-component show that cryo-EM is capable subangstrom resolution interleaflet thickness. All-atom simulations are predict the appearance submicron vesicles with coexisting liquid these quantitatively validated...

Neuronal synapses are the fundamental units of neural signal transduction and must maintain exquisite fidelity while also accommodating plasticity that underlies learning development. To achieve these goals, molecular composition spatial organization synaptic terminals be tightly regulated; however, little is known about regulation lipid in membranes. Here we quantify comprehensive lipidome rat membranes during postnatal development observe dramatic developmental lipidomic remodeling first...

Lipid membranes are ubiquitous biological organizers, required for structural and functional compartmentalization of the cell sub-cellular organelles. Membranes in living cells compositionally complex, comprising hundreds dynamically regulated, distinct lipid species. Cellular physiology requires tight regulation these lipidomic profiles to achieve proper membrane functionality. While some general features tissue- organelle-specific complements have been identified, less is known about...

Studies of the structural organization calcium/ calmodulin-dependent protein kinase IIα (CaM KIIα) and truncated CaM KIIα by three-dimensional electron microscopy engineering show that structures consist 12 subunits are organized in two stacked hexameric rings with 622 symmetry. The body is gear-shaped, consisting six slanted flanges, has foot-like processes attached narrow appendages to both ends flanges. Truncated lacks functional domains a structure very similar KIIα. Thus, reside...

The activity of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) plays an integral role in regulating synaptic development and plasticity. We designed a live-cell-imaging approach to monitor activity-dependent clustering green fluorescent (GFP)-CaMKII holoenzymes, termed self-association, process that we hypothesize contributes the translocation CaMKII nonsynaptic sites activated neurons. show GFP-CaMKII self-association human embryonic kidney 293 (HEK293) cells requires catalytic...

Activity-mediated gene expression is thought to play an important role in many forms of neuronal plasticities. We have used pentylenetetrazol-induced seizure that produces synchronous and sustained activity as a model examine the mechanism(s) activation. The transcription factor CREB (Ca2+/cAMP response element-binding protein) be necessary for long-term memory formation both invertebrates vertebrates. When phosphorylated on Ser133 either by cAMP-dependent protein kinase and/or...

Abstract: The activities of Ca 2+ /calmodulin (CaM)‐dependent, /phospholipid‐dependent, and cyclic AMP‐dependent protein kinases (CaM‐KII, PKC, PKA, respectively) were determined in rat brains after global ischemia. Both CaM‐KII PKC significantly depressed both hippocampal cerebral cortical regions ischemic animals, whereas no change was detected PKA activity. loss activity more dramatic sustained than the correlated with duration These decreases enzyme found supernatant pellet fractions...

A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca2+ binding. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive and accesses time scales tens microseconds. used in conjunction with multiphoton microscopy examine fast Ca2+-induced transitions acrylodan-labeled calmodulin. We find that kinetic rates two homologous globular domains differ more than an order magnitude. characteristic constants are...

Development of biologically relevant crowding solutions necessitates improved understanding how the relative size and density mobile obstacles affect probe diffusion. Both each co-solute in a mixture will contribute to measured microviscosity as assessed by altered translational mobility. Using multiphoton fluorescent correlation spectroscopy, this study addresses excluded volume dextran polymers from 10 500 kDa quantified measurements calmodulin labeled with green protein diffusing probe....

The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins cell. To investigate structure-function relationship CaM, we combined methods computer simulation and experiments based on circular dichroism (CD) structural characteristics CaM that influence recognition crowded cell-like conditions. We developed a unique multiscale solution charges computed from quantum chemistry, together with protein reconstruction, coarse-grained molecular simulations,...

Self-assembly of plasma membrane-associated Ras GTPases has major implications to the regulation cell signaling. However, structural basis homo-oligomerization and fractional distribution oligomeric states remained undetermined. We have addressed these issues by deciphering dimers higher-order oligomers K-Ras4B, most frequently mutated isoform in human cancers. focused on constitutively active G12V K-Ras two its variants, K101E K101C/E107C, which respectively destabilize stabilize oligomers....

Deep learning excels at cryo-tomographic image restoration and segmentation tasks but is hindered by a lack of training data. Here we introduce cryo-TomoSim (CTS), MATLAB-based software package that builds coarse-grained models macromolecular complexes embedded in vitreous ice then simulates transmitted electron tilt series for tomographic reconstruction. We demonstrate the effectiveness these simulated datasets different deep use on real cryotomographic reconstructions. Computer-generated...

We have examined the interactions of p85 regulatory subunit phosphatidylinositol 3-kinase with endothelium-specific Flt-1 receptor tyrosine kinase using yeast two-hybrid system. find that both amino- and carboxyl-terminal SH2 domains bind to Flt-1. performed site-directed mutagenesis on tail in order identify site(s) is responsible for interactions. A single phenylalanine change at position 1213 inhibits binding domains. Phosphopeptide mapping wild type mutant protein expressed insect cells...

The availability and interactions of signaling proteins are tightly regulated in time space to produce specific localized effects. For calmodulin (CaM), a key transducer intracellular Ca 2+ signaling, binding its variety targets initiates cascades regulates subcellular localization, thereby making it unavailable for subsequent interactions. Among CaM's numerous targets, /CaM-dependent protein kinase II is one the most striking due unique ability increase affinity CaM by autophosphorylation...