Ribosome-SRP-FtsY cotranslational targeting complex in the closed state.

The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome-nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP-RNC complex results in GTP-independent binding of the SRP-FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP-FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP-FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.

A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end.

Microtubules are cytoskeleton filaments consisting of αß-tubulin heterodimers. They switch between phases of growth and shrinkage. The underlying mechanism of this property, called dynamic instability, is not fully understood. Here, we identified a designed ankyrin repeat protein (DARPin) that interferes with microtubule assembly in a unique manner. The X-ray structure of its complex with GTP-tubulin shows that it binds to the ß-tubulin surface exposed at microtubule (+) ends. The details of the structure provide insight into the role of GTP in microtubule polymerization and the conformational state of tubulin at the very microtubule end. They show in particular that GTP facilitates the tubulin structural switch that accompanies microtubule assembly but does not trigger it in unpolymerized tubulin. Total internal reflection fluorescence microscopy revealed that the DARPin specifically blocks growth at the microtubule (+) end by a selective end-capping mechanism, ultimately favoring microtubule disassembly from that end. DARPins promise to become designable tools for the dissection of microtubule dynamic properties selective for either of their two different ends.

Kif2C minimal functional domain has unusual nucleotide binding properties that are adapted to microtubule depolymerization.

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

Molecular stiffness cues of an interpenetrating network hydrogel for cell adhesion.

Understanding cells' response to the macroscopic and nanoscale properties of biomaterials requires studies in model systems with the possibility to tailor their mechanical properties and different length scales. Here, we describe an interpenetrating network (IPN) design based on a stiff PEGDA host network interlaced within a soft 4-arm PEG-Maleimide/thiol (guest) network. We quantify the nano- and bulk mechanical behavior of the IPN and the single network hydrogels by single-molecule force spectroscopy and rheological measurements. The IPN presents different mechanical cues at the molecular scale, depending on which network is linked to the probe, but the same mechanical properties at the macroscopic length scale as the individual host network. Cells attached to the interpenetrating (guest) network of the IPN or to the single network (SN) PEGDA hydrogel modified with RGD adhesive ligands showed comparable attachment and spreading areas, but cells attached to the guest network of the IPN, with lower molecular stiffness, showed a larger number and size of focal adhesion complexes and a higher concentration of the Hippo pathway effector Yes-associated protein (YAP) than cells linked to the PEGDA single network. The observations indicate that cell adhesion to the IPN hydrogel through the network with lower molecular stiffness proceeds effectively as if a higher ligand density is offered. We claim that IPNs can be used to decipher how changes in ECM design and connectivity at the local scale affect the fate of cells cultured on biomaterials.

Production of Multi-subunit Membrane Protein Complexes.

Membrane proteins constitute an important class of proteins for medical, pharmaceutical, and biotechnological reasons. Understanding the structure and function of membrane proteins and their complexes is of key importance, but the progress in this area is slow because of the difficulties to produce them in sufficient quality and quantity. Overexpression of membrane proteins is often restricted by the limited capability of translocation systems to integrate proteins into the membrane and to fold them properly. Purification of membrane proteins requires their isolation from the membrane, which is a further challenge. The choice of expression system, detergents, and purification tags is therefore an important decision. Here, we present a protocol for expression in bacteria and isolation of a seven-subunit membrane protein complex, the bacterial holo-translocon, which can serve as a starting point for the production of other membrane protein complexes for structural and functional studies.

A Non-immunogenic Bivalent d-Protein Potently Inhibits Retinal Vascularization and Tumor Growth.

We report a general approach to engineering multivalent d-proteins with antibody-like activities in vivo. Mirror-image phage display and structure-guided design were utilized to create a d-protein that uses receptor mimicry to antagonize vascular endothelial growth factor A (VEGF-A). Selections against the d-protein form of VEGF-A using phage-displayed libraries of two different domain scaffolds yielded two proteins that bound distinct receptor interaction sites on VEGF-A. X-ray crystal structures of the d-protein/VEGF-A complexes were used to guide affinity maturation and to construct a heterodimeric d-protein VEGF-A antagonist with picomolar activity. The d-protein VEGF-A antagonist prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration and slowed tumor growth in the MC38 syngeneic mouse tumor model with efficacies comparable to those of approved antibody drugs, and in contrast with antibodies, the d-protein was non-immunogenic during treatment and following subcutaneous immunizations.

Printed Soft Optical Waveguides of PLA Copolymers for Guiding Light into Tissue.

The application of optical technologies in treating pathologies and monitoring disease states requires the development of soft, minimal invasive and implantable devices to deliver light to tissues inside the body. Here, we present soft and degradable optical waveguides from poly(d,l-lactide) and derived copolymers fabricated by extrusion printing in the desired dimensions and shapes. The obtained optical waveguides propagate VIS to NIR light in air and in tissue at penetration depths of tens of centimeters. Besides, the printed waveguides have elastomeric properties at body temperature and show softness and flexibility in the range relevant for implantable devices in soft organs. Printed waveguides were able to guide light across 8 cm tissue and activate photocleavage chemical reactions in a photoresponsive hydrogel (in vitro). The simplicity and flexibility of the fiber processing method and the optical and mechanical performance of the obtained waveguides exemplify how rational study of medically approved biomaterials can lead to useful inks for printing cost-effective and flexible optical components for potential use in medical contexts.

Near-infrared-light regulated angiogenesis in a 4D hydrogel.

Light-responsive hydrogels are useful platforms to study cellular responses. Current photosensitive motifs need UV light to be activated, which is intrinsically cytotoxic and has a low penetration depth in tissues. Herein we describe a strategy for near-infrared (NIR) controlled activation of cellular processes (3D cell spreading and angiogenesis) by embedding upconverting nanoparticles (UCNPs) in a hydrogel modified with light-activatable cell adhesive motifs. The UCNPs can convert NIR light (974 nm) into local UV emission and activate photochemical reactions on-demand. Such optoregulation is spatially controllable, dose-dependent and can be performed at different timepoints of the cell culture without appreciable photodamage of the cells. HUVEC cells embedded in this hydrogel can form vascular networks at predefined geometries determined by the irradiation pattern. The penetration depth of NIR light enabled activation of the angiogenesis response through skin tissue with a thickness of 2.5 mm. Our strategy opens a new avenue for 4D cell cultures, with the potential to be extended to dynamically manipulate cell-matrix interactions and derived cellular processes in vivo.

Influence of furfural concentration on growth and ethanol yield of Saccharomyces kluyveri.

Furfural is an important inhibitor in ethanol fermentation process using lignocellulosic hydrolysates as raw materials. In order to find out the furfural concentration range in which furfural inhibits the fermentation process, we used one strain Saccharomyces kluyveri selected from soil and cultured in several different furfural content media under low glucose concentration condition. Experiment results showed that microorganism growth was stimulated and dry cell weight decreased when furfural concentration in the medium was 0.25 mg/ml. Furfural had negative effect on cell growth when its concentration was above 1.00 mg/ml. At the same time, the strain growed better and had a higher glucose consumption rate in 5% original glucose concentration condition than in 3% original glucose concentration condition. The results showed that appropriate exaltation of original glucose concentration in stalk hydrolysates will increase the strain resistance to furfural.

A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion.

The conserved SecYEG protein-conducting channel and the accessory proteins SecDF-YajC and YidC constitute the bacterial holo-translocon (HTL), capable of protein-secretion and membrane-protein insertion. By employing an integrative approach combining small-angle neutron scattering (SANS), low-resolution electron microscopy and biophysical analyses we determined the arrangement of the proteins and lipids within the super-complex. The results guided the placement of X-ray structures of individual HTL components and allowed the proposal of a model of the functional translocon. Their arrangement around a central lipid-containing pool conveys an unexpected, but compelling mechanism for membrane-protein insertion. The periplasmic domains of YidC and SecD are poised at the protein-channel exit-site of SecY, presumably to aid the emergence of translocating polypeptides. The SecY lateral gate for membrane-insertion is adjacent to the membrane 'insertase' YidC. Absolute-scale SANS employing a novel contrast-match-point analysis revealed a dynamic complex adopting open and compact configurations around an adaptable central lipid-filled chamber, wherein polytopic membrane-proteins could fold, sheltered from aggregation and proteolysis.

Advances and challenges of membrane-protein complex production.

Most membrane-proteins exist in complexes rather than isolated entities. To fully understand their biological function it is essential to study the intact membrane-protein assemblies. The overexpression and purification of many essential membrane-protein complexes is still a considerable and often unsurmountable challenge. In these cases, extraction from source is the only option for many large multi-subunit cellular machines. Here, we describe recent advances in overexpression of multi-subunit membrane-protein complexes, the strategies to stabilize these complexes and highlight major achievements in membrane-protein structural research that were facilitated by the prospect of achieving subnanometer to near-atomic resolution by electron cryo-microscopy.

Targeted mutagenesis in soybean using the CRISPR-Cas9 system.

Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2-9.7% using the pCas9-AtU6-sgRNA vector and 14.7-20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules.

The structure of apo-kinesin bound to tubulin links the nucleotide cycle to movement.

Kinesin-1 is a dimeric ATP-dependent motor protein that moves towards microtubules (+) ends. This movement is driven by two conformations (docked and undocked) of the two motor domains carboxy-terminal peptides (named neck linkers), in correlation with the nucleotide bound to each motor domain. Despite extensive data on kinesin-1, the structural connection between its nucleotide cycle and movement has remained elusive, mostly because the structure of the critical tubulin-bound apo-kinesin state was unknown. Here we report the 2.2 Å structure of this complex. From its comparison with detached kinesin-ADP and tubulin-bound kinesin-ATP, we identify three kinesin motor subdomains that move rigidly along the nucleotide cycle. Our data reveal how these subdomains reorient on binding to tubulin and when ATP binds, leading respectively to ADP release and to neck linker docking. These results establish a framework for understanding the transformation of chemical energy into mechanical work by (+) end-directed kinesins.

Structure of a kinesin-tubulin complex and implications for kinesin motility.

The typical function of kinesins is to transport cargo along microtubules. Binding of ATP to microtubule-attached motile kinesins leads to cargo displacement. To better understand the nature of the conformational changes that lead to the power stroke that moves a kinesin's load along a microtubule, we determined the X-ray structure of human kinesin-1 bound to αß-tubulin. The structure defines the mechanism of microtubule-stimulated ATP hydrolysis, which releases the kinesin motor domain from microtubules. It also reveals the structural linkages that connect the ATP nucleotide to the kinesin neck linker, a 15-amino acid segment C terminal to the catalytic core of the motor domain, to result in the power stroke. ATP binding to the microtubule-bound kinesin favors neck-linker docking. This biases the attachment of kinesin's second head in the direction of the movement, thus initiating each of the steps taken.