Self-assembly of coherently dynamic, auxetic, two-dimensional protein crystals.

Two-dimensional (2D) crystalline materials possess unique structural, mechanical and electronic properties that make them highly attractive in many applications. Although there have been advances in preparing 2D materials that consist of one or a few atomic or molecular layers, bottom-up assembly of 2D crystalline materials remains a challenge and an active area of development. More challenging is the design of dynamic 2D lattices that can undergo large-scale motions without loss of crystallinity. Dynamic behaviour in porous three-dimensional (3D) crystalline solids has been exploited for stimuli-responsive functions and adaptive behaviour. As in such 3D materials, integrating flexibility and adaptiveness into crystalline 2D lattices would greatly broaden the functional scope of 2D materials. Here we report the self-assembly of unsupported, 2D protein lattices with precise spatial arrangements and patterns using a readily accessible design strategy. Three single- or double-point mutants of the C4-symmetric protein RhuA were designed to assemble via different modes of intermolecular interactions (single-disulfide, double-disulfide and metal-coordination) into crystalline 2D arrays. Owing to the flexibility of the single-disulfide interactions, the lattices of one of the variants ((C98)RhuA) are essentially defect-free and undergo substantial, but fully correlated, changes in molecular arrangement, yielding coherently dynamic 2D molecular lattices. (C98)RhuA lattices display a Poisson's ratio of -1-the lowest thermodynamically possible value for an isotropic material-making them auxetic.

Precise Fabrication of De Novo Nanoparticle Lattices on Dynamic 2D Protein Crystalline Lattices.

The science of protein self-assembly has experienced significant development, from discrete building blocks of self-assembled nanoarchitectures to advanced nanostructures with adaptive functionalities. Despite the prominent achievements in the field, the desire of designing de novo protein-nanoparticle (NP) complexes and constructing dynamic NP systems remains highly challenging. In previous works, l-rhamnulose-1-phosphate aldolase (C98RhuA) tetramers were self-assembled into two-dimensional (2D) lattices via disulfide bond interactions. These interactions provided 2D lattices with high structural quality and a sophisticated assembly mode. In this study, we devised a rational design for RhuA building blocks to fabricate 2D functionalized protein lattices. More importantly, the lattices were used to direct the precise assembly of NPs into highly ordered and diverse nanoarchitectures. These structures can be employed as an excellent tool to adequately verify the self-assembly mode and structural quality of the designed RhuA crystals. The subsequent redesign of RhuA building blocks enabled us to predictably produce a novel protein lattice whose conformational dynamics can be controllably regulated. Thus, a dynamic system of AuNP lattices was achieved. Transmission electron microscopy and small-angle X-ray scattering indicated the presence of these diverse NP lattices. This contribution enables the fabrication of future NP structures in a more programmable manner with more expected properties for potential applications in nanoelectronics and other fields.

Heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of KDO8P synthase from Arabidopsis thaliana.

3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) [EC 4.1.2.16] is the first and rate-limiting enzyme in the 3-deoxy-d-manno-octulosonate (KDO) biosynthetic pathway. The enzyme is widely expressed in bacteria and plants. Their well conserved protein sequences imply a similar oligomeric arrangement. However, the reported size exclusion chromatrographic analysis suggested a species-dependent self-assembling. To clarify the discrepancy and explore the self-assembling property of KDO8PS, we expressed and purified the Arabidopsis enzyme in Escherichia coli system. The enzyme was highly purified using a two-step purification strategy including nickel affinity and size exclusion chromatography with an expected pH activity profile. The identity of the purified enzyme was confirmed by Western-blot and mass fingerprints. Further analysis by analytical ultracentrifugation indicated that both bacteria and Arabidopsis enzymes are homotetramer. Furthermore, the purified enzyme from the plant has been crystallized and a complete set of X-ray data to 2.1Å resolution has been collected.

Segmented flow generator for serial crystallography at the European X-ray free electron laser.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.

Structural insights into catalytic mechanism and product delivery of cyanobacterial acyl-acyl carrier protein reductase.

Long-chain alk(a/e)nes represent the major constituents of conventional transportation fuels. Biosynthesis of alkanes is ubiquitous in many kinds of organisms. Cyanobacteria possess two enzymes, acyl-acyl carrier protein (acyl-ACP) reductase (AAR) and aldehyde-deformylating oxygenase (ADO), which function in a two-step alkane biosynthesis pathway. These two enzymes act in series and possibly form a complex that efficiently converts long chain fatty acyl-ACP/fatty acyl-CoA into hydrocarbon. While the structure of ADO has been previously described, structures of both AAR and AAR-ADO complex have not been solved, preventing deeper understanding of this pathway. Here, we report a ligand-free AAR structure, and three AAR-ADO complex structures in which AARs bind various ligands. Our results reveal the binding pattern of AAR with its substrate/cofactor, and suggest a potential aldehyde-transferring channel from AAR to ADO. Based on our structural and biochemical data, we proposed a model for the complete catalytic cycle of AAR.

Structures of almond hydroxynitrile lyase isoenzyme 5 provide a rationale for the lack of oxidoreductase activity in flavin dependent HNLs.

Hydroxynitrile lyases (HNLs) catalyze the asymmetric addition of HCN to aldehydes producing enantiomerically pure cyanohydrins. These enzymes can be heterologously expressed in large quantities making them interesting candidates for industrial applications. The HNLs from Rosaceae evolved from flavin dependent dehydrogenase/oxidase structures. Here we report the high resolution X-ray structure of the highly glycosylated Prunus amygdalus HNL isoenzyme5 (PaHNL5 V317A) expressed in Aspergillus niger and its complex with benzyl alcohol. A comparison with the structure of isoenzyme PaHNL1 indicates a higher accessibility to the active site and a larger cavity for PaHNL5. Additionally, the PaHNL5 complex structure with benzyl alcohol was compared with the structurally related aryl-alcohol oxidase (AAO). Even though both enzymes contain an FAD-cofactor and histidine residues at crucial positions in the active site, PaHNL5 lacks the oxidoreductase activity. The structures indicate that in PaHNLs benzyl alcohol is bound too far away from the FAD cofactor in order to be oxidized.