A key issue in neuronal circuit regulation is how synapse formation is initiated. Synapse formation could start when one or more synaptic scaffold proteins that can initiate synapse formation reach certain threshold concentrations in the dendritic shaft, which might lead to their oligomerization or even liquid-liquid phase separation (LLPS). By combining in vitro reconstitution of purified proteins with live-cell single-molecule and confocal imaging, we demonstrated that SynGAP alone forms assemblies of nanoscale clusters containing several to several tens of molecules at 10-nM order concentrations and micron-scale LLPS hydrogel-like condensates at submicromolar concentrations. The trimers of SynGAP’s intrinsically disordered region (IDR) induced by its coiled-coil domain are responsible for SynGAP condensation. CaMKII-mediated phosphorylation moderately suppresses SynGAP condensation, and also increases condensate liquidity. While PSD95 fails to form assemblies under these conditions, it is recruited to SynGAP condensates by specifically binding to the PDZ-binding motif of SynGAP. SynGAP[PSD95] condensates selectively immobilize postsynaptic transmembrane proteins, Neuroligin1 and AMPAR-TARP2 complexes, in a manner dependent on their oligomerization state, indicating cooperative recruitment dynamics among SynGAP, PSD95, and transmembrane components, which might mimic initial PSD assembly. These findings suggest that SynGAP may act as a primary nucleator of postsynaptic density assembly, challenging the PSD95-centered models. ### Competing Interest Statement The authors have declared no competing interest.

The alpha-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable alpha-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of alpha-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered "undruggable", such as protein-protein interactions. To date, no general method for discovering alpha-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized alpha-helix recognition sites, and restricting the starting chemical matter to those known alpha-helical binders. Here, we report a general and rapid screening method to empirically map the alpha-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated alpha-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct alpha-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein-protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here, we show that during the conjugation of ATG8 to single membranes (CASM), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal capacity. GABARAP directly binds to a previously unidentified LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex and mediates sequestration to GABARAP-conjugated membrane compartments. This disrupts FLCN/FNIP GAP function toward RagC/D, resulting in impaired substrate-specific mTOR-dependent phosphorylation of TFEB. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here we show that during single membrane ATG8 conjugation (SMAC), Parkin-dependent mitophagy, and Salmonella -induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal homeostasis and capacity. GABARAP directly binds to a novel LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex with picomolar affinity and regulates its relocalization to these GABARAP-conjugated membrane compartments. This disrupts the regulation of RagC/D by the FLCN/FNIP GAP complex, resulting in impaired mTOR-dependent phosphorylation of TFEB without changing mTOR activity towards other substrates. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a universal molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

SynGAP is a Ras and Rap GTPase-activating protein (GAP) found in high concentration in the postsynaptic density (PSD) fraction from mammalian forebrain where it binds to PDZ domains of PSD-95. Phosphorylation of pure recombinant synGAP by Ca2+/calmodulin-dependent protein kinase II (CaMKII) shifts the balance of synGAP's GAP activity toward inactivation of Rap1; whereas phosphorylation by cyclin-dependent kinase 5 (CDK5) has the opposite effect, shifting the balance toward inactivation of HRas. These shifts in balance contribute to regulation of the numbers of surface AMPA receptors, which rise during synaptic potentiation (CaMKII) and fall during synaptic scaling (CDK5). Polo-like kinase 2 (Plk2/SNK), like CDK5, contributes to synaptic scaling. These two kinases act in concert to reduce the number of surface AMPA receptors following elevated neuronal activity by tagging spine-associated RapGAP protein (SPAR) for degradation, thus raising the level of activated Rap. Here we show that Plk2 also phosphorylates and regulates synGAP. Phosphorylation of synGAP by Plk2 stimulates its GAP activity toward HRas by 65%, and toward Rap1 by 16%. Simultaneous phosphorylation of synGAP by Plk2 and CDK5 at distinct sites produces an additive increase in GAP activity toward HRas (∼230%) and a smaller, non-additive increase in activity toward Rap1 (∼15%). Dual phosphorylation also produces an increase in GAP activity toward Rap2 (∼40-50%), an effect not produced by either kinase alone. As we previously observed for CDK5, addition of Ca2+/CaM causes a substrate-directed doubling of the rate and stoichiometry of phosphorylation of synGAP by Plk2, targeting residues also phosphorylated by CaMKII. In summary, phosphorylation by Plk2, like CDK5, shifts the ratio of GAP activity of synGAP to produce a greater decrease in active Ras than in active Rap, which would produce a shift toward a decrease in the number of surface AMPA receptors in neuronal dendrites.

SynGAP is a Ras/Rap GTPase-activating protein (GAP) that is a major constituent of postsynaptic densities (PSDs) from mammalian forebrain. Its α1 isoform binds to all three PDZ (PSD-95, Discs-large, ZO-1) domains of PSD-95, the principal PSD scaffold, and can occupy as many as 15% of these PDZ domains. We present evidence that synGAP-α1 regulates the composition of the PSD by restricting binding to the PDZ domains of PSD-95. We show that phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Polo-like kinase-2 (PLK2) decreases its affinity for the PDZ domains by several fold, which would free PDZ domains for occupancy by other proteins. Finally, we show that three critical postsynaptic signaling proteins that bind to the PDZ domains of PSD-95 are present in higher concentration in PSDs isolated from mice with a heterozygous deletion of synGAP.

​We report an improved methodology for the production of cephalopod proteins known as reflectins. Our findings may afford new opportunities for the study of these proteins’ multifaceted materials properties.

SynGAP is a Ras/Rap GTPase-activating protein (GAP) present in high concentration in postsynaptic densities (PSDs) from mammalian forebrain where it binds to all three PDZ ( P SD-95, D iscs-large, Z O-1) domains of PSD-95. We show that phosphorylation of synGAP by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) decreases its affinity for the PDZ domains as much as 10-fold, measured by surface plasmon resonance. SynGAP is abundant enough in postsynaptic densities (PSDs) to occupy about one third of the PDZ domains of PSD-95. Therefore, we hypothesize that phosphorylation by CaMKII reduces synGAP’s ability to restrict binding of other proteins to the PDZ domains of PSD-95. We support this hypothesis by showing that three critical postsynaptic signaling proteins that bind to the PDZ domains of PSD-95 are present at a higher ratio to PSD-95 in PSDs isolated from synGAP heterozygous mice.

The skin morphology of cephalopods endows them with remarkable camouflage capabilities. Herein, we report infrared invisibility stickers inspired by the structures and proteins found in cephalopod skin. These stickers enable arbitrary objects to acquire reconfigurable infrared camouflage patterning. Our work represents an initial step towards wearable biomimetic infrared stealth technologies.

PDZ domains function in nature as protein‐binding domains within scaffold and membrane‐associated proteins. They comprise approximately 90 residues and undergo specific, high‐affinity interactions with complementary C‐terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain‐binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. © 2015 by John Wiley & Sons, Inc.

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.

In nature, cephalopods employ unique dynamic camouflage mechanisms. Herein, we draw inspiration from self-assembled structures found in cephalopods to fabricate tunable biomimetic camouflage coatings. The reflectance of these coatings is dynamically modulated between the visible and infrared regions of the electromagnetic spectrum in situ. Our studies represent a crucial step towards reconfigurable and disposable infrared camouflage for stealth applications.

Cephalopods are known as the chameleons of the sea - they can alter their skin's coloration, pattern, texture, and reflectivity to blend into the surrounding environment. Despite much research effort, there are few known strategies (natural or artificial) for emulating the unique dynamic reflectivity and coloration of cephalopods. We have developed an approach to the fabrication of stimuli-responsive cephalopod-inspired thin films with emergent optical properties. We have furthermore enhanced the properties of such films through a synergistic combination of bioconjugate chem., bottom up self-assembly, and top down nanofabrication. Our findings hold significant implications for the development of reconfigurable biomimetic camouflage coatings.

Graphene-templated, camouflage coatings that possess several important advantages are demonstrated by Alon A. Gorodetsky and co-workers on page 5621. These coatings are inspired by the unique dynamic camouflage mechanisms or cephalopods. A squid's ability to control transmission and reflection of light can be applied to a substrate and made controllable. Such substrates are shown to be a home window that reflects long wave light (IR) and transmits short wave light. The thin film fabrication strategy is inexpensive, robust, and compatible with a wide range of substrates of arbitrary form factor.

Criteria for sub-typing of microbial organisms by DNA sequencing proposed by Olive and Bean were applied to several genes in Escherichia coli to identify targets for the development of microbial source tracking assays. Based on the aforementioned criteria, the icd (isocitrate dehydrogenase), and putP (proline permease) genes were excluded as potential targets due to their high rates of horizontal gene transfer; the rrs (16S rRNA) gene was excluded as a target due to the presence of multiple gene copies, with different sequences in a single genome. Based on the above criteria, the mdh (malate dehydrogenase) gene was selected as a target for development of a microbial source tracking assay. The mdh assay was optimized to analyze a 150 bp fragment corresponding to residues G191 to R240 (helices H10 and H11) of the Mdh catalytic domain. 295 fecal isolates (52 horse, 50 deer, 72 dog, 52 seagull and 69 human isolates) were sequenced and analyzed. Target DNA sequences for isolates from horse, dog plus deer, and seagull formed identifiable groupings. Sequences from human isolates, aside from a low level (ca. 15%) human specific sequence, did not group; nevertheless, other hosts could be distinguished from human. Positive and negative predictive values for two- and three-way host comparisons ranged from 60% to 90% depending on the focus host. False positive rates were below 10%. Multiple E. coli isolates from individual fecal samples exhibited high levels of sequence homogeneity, i.e. typically only one to two mdh sequences were observed per up to five E. coli isolates from a single fecal sample. Among all isolates sequenced from fecal samples from each host, sequence homogeneity decreased in the following order: horse > dog > deer > human and gull. For in-library isolates, blind analysis of fecal isolates (n = 12) from four hosts known to contain host specific target sequences was 100% accurate and 100% reproducible for both DNA sequence and host identification. For blind analysis of non-library isolates, 18/19 isolates (94.7%) matched one or more library sequences for the corresponding host. Ten of eleven geographical outlier fecal isolates from Florida had mdh sequences that were identical to in-library sequences for the corresponding host from California. The mdh assay was successfully applied to environmental isolates from an underground telephone vault in California, with 4 of 5 isolates matching sequences in the mdh library. 146 sequences of the 645 bp mdh fragment from five host sources were translated into protein sequence and aligned. Seven unique Mdh protein sequences, which contained eight polymorphic sites, were identified. Six of the polymorphic sites were in the NAD+ binding domain and two were in the catalytic domain. All of the polymorphic sites were located in surface exposed regions of the protein. None of the non-silent mutations of the Mdh protein were in the 150 bp mdh target. The advantages and disadvantages of the assay compared to established source tracking methods are discussed.