Alternate Soaking Technique for Micropatterning Alginate Hydrogels on Wettability-patterned Substrates.

Techniques for patterning hydrogels are important for fabrication of cell culture, analytical, and actuator devices at the micro- and nanometer length scales. In this study, we fabricated alginate hydrogels cross-linked by divalent cations on wettability-patterned substrates by alternate soaking of precursor solutions of sodium alginate and divalent cations. The wettability-patterned substrates were fabricated on hydrophilic glass plates modified with hydrophobic self-assembled monolayers of hexamethyldisilazane followed by exposure to an ultraviolet/ozone atmosphere through a metal mask. The film thickness of alginate gels with a width and length of 0.1 and 4 mm were tuned stepwise from 30 nm to 200 nm by adjusting the precursor conditions, including the pH, type of divalent metal ions, and sodium alginate concentration, and the alternate soaking conditions, including the dipping/withdrawal speed and number of alternate soaking cycles. This technique can be applied to other functional gels and will contribute to fabrication of hydrogel devices at the micro- and nanometer scales in the future.

Crystal structures of the carbamoylated and cyanated forms of HypE for [NiFe] hydrogenase maturation.

Hydrogenase pleiotropically acting protein (Hyp)E plays a role in biosynthesis of the cyano groups for the NiFe(CN)2CO center of [NiFe] hydrogenases by catalyzing the ATP-dependent dehydration of the carbamoylated C-terminal cysteine of HypE to thiocyanate. Although structures of HypE proteins have been determined, until now there has been no structural evidence to explain how HypE dehydrates thiocarboxamide into thiocyanate. Here, we report the crystal structures of the carbamoylated and cyanated forms of HypE from Thermococcus kodakarensis in complex with nucleotides at 1.53- and 1.64-Å resolution, respectively. Carbamoylation of the C-terminal cysteine (Cys338) of HypE by chemical modification is clearly observed in the present structures. In the presence of ATP, the thiocarboxamide of Cys338 is successfully dehydrated into the thiocyanate. In the carbamoylated state, the thiocarboxamide nitrogen atom of Cys338 is close to a conserved glutamate residue (Glu272), but the spatial position of Glu272 is less favorable for proton abstraction. On the other hand, the thiocarboxamide oxygen atom of Cys338 interacts with a conserved lysine residue (Lys134) through a water molecule. The close contact of Lys134 with an arginine residue lowers the pKa of Lys134, suggesting that Lys134 functions as a proton acceptor. These observations suggest that the dehydration of thiocarboxamide into thiocyanate is catalyzed by a two-step deprotonation process, in which Lys134 and Glu272 function as the first and second bases, respectively.

Structural basis of [NiFe] hydrogenase maturation by Hyp proteins.

[NiFe] hydrogenases catalyze reversible hydrogen production/consumption. The active site of [NiFe] hydrogenases contains a complex NiFe(CN)2CO center, and the biosynthesis/maturation of these enzymes is a complex and dynamic process, primarily involving six Hyp proteins (HypABCDEF). HypA and HypB are involved in the Ni insertion, whereas the other four Hyp proteins (HypCDEF) are required for the biosynthesis, assembly and insertion of the Fe(CN)2CO group. Over the last decades, a large number of functional and structural studies on maturation proteins have been performed, revealing detailed functions of each Hyp protein and the framework of the maturation pathway. This article will focus on recent advances in structural studies of the Hyp proteins and on mechanistic insights into the [NiFe] hydrogenase maturation.

Structure of the [NiFe]-hydrogenase maturation protein HypF from Thermococcus kodakarensis KOD1.

HypF is involved in the biosynthesis of the CN ligand of the NiFe(CN)(2)CO centre of [NiFe]-hydrogenases. Here, the full-length structure of HypF from Thermococcus kodakarenesis is reported at 4.5 Šresolution. The N-terminal acylphosphatase-like (ACP) domain interacts with the zinc-finger domain with some flexibility in its relative position. Molecular-surface analysis shows that a deep pocket formed between the ACP and zinc-finger domains is highly conserved and has positive potential. These results suggest that the positively charged pocket identified is involved in the hydrolysis of carbamoyl phosphate and the formation of a carbamoyl intermediate.