Measurement of Covalent Bond Formation in Light-Curing Hydrogels Predicts Physical Stability under Flow.

Photo-crosslinking hydrogels are promising for tissue engineering and regenerative medicine, but challenges in reaction monitoring often leave their optimization subject to trial and error. The stability of crosslinked gels under fluid flow, as in the case of a microfluidic device, is particularly challenging to predict, both because of obstacles inherent to solid-state macromolecular analysis that prevent accurate chemical monitoring and because stability is dependent on size of the patterned features. To solve both problems, we obtained H NMR spectra of cured hydrogels which were enzymatically degraded.

Multi-site esterification: a tunable, reversible strategy to tailor therapeutic peptides for delivery.

Peptides are naturally potent and selective therapeutics with massive potential; however, low cell membrane permeability limits their clinical implementation, particularly for hydrophilic, anionic peptides with intracellular targets. To overcome this limitation, esterification of anionic carboxylic acids on therapeutic peptides can simultaneously increase hydrophobicity and net charge to facilitate cell internalization, whereafter installed esters can be cleaved hydrolytically to restore activity. To date, however, most esterified therapeutics contain either a single esterification site or multiple esters randomly incorporated on multiple sites.

Measurement of covalent bond formation in light-curing hydrogels predicts physical stability under flow.

Photocrosslinking hydrogels are promising for tissue engineering and regenerative medicine, but challenges in reaction monitoring often leave their optimization subject to trial and error. The stability of crosslinked gels under fluid flow, as in the case of a microfluidic device, is particularly challenging to predict, both because of obstacles inherent to solid-state macromolecular analysis that prevent accurate chemical monitoring, and because stability is dependent on size of the patterned features. To solve both problems, we obtained H NMR spectra of cured hydrogels which were enzymatically degraded.

Backbone H, N, and C resonance assignments of the Phafin2 pleckstrin homology domain.

Phafin2 is a peripheral protein that triggers cellular signaling from endosomal and lysosomal compartments. The specific subcellular localization of Phafin2 is mediated by the presence of a tandem of phosphatidylinositol 3-phosphate (PtdIns3P)-binding domains, the pleckstrin homology (PH) and the Fab-1, YOTB, Vac1, and EEA1 (FYVE) domains. The requirement for both domains for binding to PtdIns3P still remains unclear.

Structural, in silico, and functional analysis of a Disabled-2-derived peptide for recognition of sulfatides.

Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin αβ receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with the pro-aggregatory mediators, the integrin αβ receptor and sulfatides, blocking their association to fibrinogen and P-selectin, respectively.

Membrane targeting of TIRAP is negatively regulated by phosphorylation in its phosphoinositide-binding motif.

Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM).

Structural, thermodynamic, and phosphatidylinositol 3-phosphate binding properties of Phafin2.

Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns(3)P) binding protein involved in the regulation of endosomal cargo trafficking and lysosomal induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both its PH and FYVE domains. However, there are no studies on the structural basis, conformational stability, and lipid interactions of Phafin2 to better understand how this protein participates in signaling at the surface of endomembrane compartments.

Backbone H, N, and C resonance assignments of the Tom1 VHS domain.

Efficient trafficking of ubiquitinated receptors (cargo) to endosomes requires the recruitment of adaptor proteins that exhibit ubiquitin-binding domains for recognition and transport. Tom1 is an adaptor protein that not only associates with ubiquitinated cargo but also represents a phosphoinositide effector during specific bacterial infections. This phosphoinositide-binding property is associated with its N-terminal Vps27, Hrs, STAM (VHS) domain.

Structure of the GAT domain of the endosomal adapter protein Tom1.

Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain's association to Tollip's Tom1-binding domain (TBD).

Molecular architecture of the nucleoprotein C-terminal domain from the Ebola and Marburg viruses.

The Filoviridae family of negative-sense, single-stranded RNA (ssRNA) viruses is comprised of two species of Marburgvirus (MARV and RAVV) and five species of Ebolavirus, i.e. Zaire (EBOV), Reston (RESTV), Sudan (SUDV), Taï Forest (TAFV) and Bundibugyo (BDBV).

Tom1 Modulates Binding of Tollip to Phosphatidylinositol 3-Phosphate via a Coupled Folding and Binding Mechanism.

Early endosomes represent the first sorting station for vesicular ubiquitylated cargo. Tollip, through its C2 domain, associates with endosomal phosphatidylinositol 3-phosphate (PtdIns(3)P) and binds ubiquitylated cargo in these compartments via its C2 and CUE domains. Tom1, through its GAT domain, is recruited to endosomes by binding to the Tollip Tom1-binding domain (TBD) through an unknown mechanism.

The structure of the C-terminal domain of the Zaire ebolavirus nucleoprotein.

Ebolavirus (EBOV) causes severe hemorrhagic fever with a mortality rate of up to 90%. EBOV is a member of the order Mononegavirales and, like other viruses in this taxonomic group, contains a negative-sense single-stranded (ss) RNA. The EBOV ssRNA encodes seven distinct proteins.

The SNARE motif of synaptobrevin exhibits an aqueous-interfacial partitioning that is modulated by membrane curvature.

The structure and interfacial association of the full-length vesicle SNARE, synaptobrevin, were compared in four different lipid environments using nuclear magnetic resonance and electron paramagnetic resonance spectroscopy. In micelles, segments of the SNARE motif are helical and associated with the interface. However, the fraction of helix and interfacial association decreases as synaptobrevin is moved from micelle to bicelle to bilayer environments, indicating that the tendency toward interfacial association is sensitive to membrane curvature.

Synaptotagmin 1 modulates lipid acyl chain order in lipid bilayers by demixing phosphatidylserine.

Synaptotagmin 1 (syt1) functions as the Ca(2+) sensor in neuronal exocytosis, and it has been proposed to act by modulating lipid bilayer curvature. Here we examine the effect of the two C2 domains (C2A and C2B) of syt1 on membrane lipid order and lateral organization. In mixtures of phosphatidylcholine and phosphatidylserine (PS), attenuated total internal reflection Fourier transform infrared spectroscopy indicates that a fragment containing both domains (C2AB) or C2B alone disorders the lipid acyl chains, whereas the C2A domain has little effect upon chain order.

Membrane thickness varies around the circumference of the transmembrane protein BtuB.

BtuB is a large outer-membrane β-barrel protein that belongs to a class of active transport proteins that are TonB-dependent. These TonB-dependent transporters are based upon a 22-stranded antiparallel β-barrel, which is notably asymmetric in its length. Here, site-directed spin labeling and simulated annealing were used to locate the membrane lipid interface surrounding BtuB when reconstituted into phosphatidylcholine bilayers.

Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation.

The synaptic vesicle protein synaptobrevin engages with syntaxin and SNAP-25 to form the SNARE complex, which drives membrane fusion in neuronal exocytosis. In the SNARE complex, the SNARE motif of synaptobrevin forms a 55-residue helix, but it has been assumed to be mostly unstructured in its prefusion form. NMR data for full-length synaptobrevin in dodecylphosphocholine micelles reveals two transient helical segments flanked by natively disordered regions and a third more stable helix.

Solution and membrane-bound conformations of the tandem C2A and C2B domains of synaptotagmin 1: Evidence for bilayer bridging.

Synaptotagmin 1 (syt1) is a synaptic vesicle membrane protein that functions as the Ca(2)(+) sensor in neuronal exocytosis. Here, site-directed spin labeling was used to generate models for the solution and membrane-bound structures of a soluble fragment of syt1 containing its two C2 domains, C2A and C2B. In solution, distance restraints between the two C2 domains of syt1 were measured using double electron-electron resonance and used in a simulated annealing routine to generate models for the structure of the tandem C2A-C2B fragment.

The calcium-dependent and calcium-independent membrane binding of synaptotagmin 1: two modes of C2B binding.

The Ca2+-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca2+-independent manner with a lipid partition coefficient of approximately 3.0 x 10(2) M(-1).

Spin-diffusion couples proton relaxation rates for proteins in exchange with a membrane interface.

Changes in nuclear spin-lattice relaxation rates that are induced by a freely diffusing paramagnetic relaxation agent are examined for a protein in solution and compared to the case where the protein binds to a membrane. In the solution case, the intramolecular cross-relaxation rates are modest and large differences are observed in the oxygen induced protein-proton relaxation rates. In the case where a dynamic equilibrium between solution and membrane-bound environments is established, the intramolecular (1)H cross-relaxation rates for the protein protons increase dramatically because of the slow reorientational motion in the membrane-bound environment.

Substrate-dependent unfolding of the energy coupling motif of a membrane transport protein determined by double electron-electron resonance.

BtuB is a TonB-dependent transport protein that binds and carries vitamin B(12) across the outer membrane of Gram negative bacteria such as Escherichia coli. Previous work has demonstrated that the Ton box, a highly conserved segment near the N-terminus of the protein, undergoes an order-to-disorder transition upon the binding of substrate. Here, we incorporate pairs of nitroxide spin labels into membrane reconstituted BtuB and utilize a four-pulse double electron-electron resonance (DEER) experiment to measure distances between the Ton box and the periplasmic surface of the transporter with and without substrate.

Membrane position of a basic aromatic peptide that sequesters phosphatidylinositol 4,5 bisphosphate determined by site-directed spin labeling and high-resolution NMR.

The membrane interactions and position of a positively charged and highly aromatic peptide derived from a secretory carrier membrane protein (SCAMP) are examined using magnetic resonance spectroscopy and several biochemical methods. This peptide (SCAMP-E) is shown to bind to membranes containing phosphatidylinositol 4,5-bisphosphate, PI(4,5)P2, and sequester PI(4,5)P2 within the plane of the membrane. Site-directed spin labeling of the SCAMP-E peptide indicates that the position and structure of membrane bound SCAMP-E are not altered by the presence of PI(4,5)P2, and that the peptide backbone is positioned within the lipid interface below the level of the lipid phosphates.

Location of the myristoylated alanine-rich C-kinase substrate (MARCKS) effector domain in negatively charged phospholipid bicelles.

The effector domain of the myristoylated alanine-rich C-kinase substrate (MARCKS-ED) is a highly basic, unstructured protein segment that is responsible for attaching MARCKS reversibly to the membrane interface. When attached to the interface, it also has the capacity to sequester phosphoinosities, such as PI(4,5)P(2), within the plane of the bilayer. Here, the position of the MARCKS-ED was determined when bound to phospholipid bicelles using high-resolution NMR methods.

Perfluorooctyl bromide has limited membrane solubility and is located at the bilayer center. Locating small molecules in lipid bilayers through paramagnetic enhancements of NMR relaxation.

There is considerable interest in the use of perfluorocarbons as oxygen carriers in clinical settings; however, little is known regarding the molecular interactions made by these apolar compounds with biological membranes or their effect on membrane structure. NMR spectroscopy was used to investigate the interaction of perfluorooctyl bromide (PFOB) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. (19)F NMR spectra demonstrate that PFOB partitions into POPC bilayers but that it saturates at a remarkably low membrane concentration of approximately 2 mol %.