Defective chicken skin collagen molecules, hydrolyzed by actinidain protease, assemble to form loosely packed fibrils that promote cell spheroid formation.

Cells recognize collagen fibrils as the first step in the process of adherence. Fibrils of chicken skin actinidain-hydrolyzed collagen (low adhesive scaffold collagen, LASCol), in which the telopeptide domains are almost completely removed, cause adhering cells to form spheroids instead of adopting a monolayer morphology. Our goal was to elucidate the ultrastructure of the LASCol fibrils compared with pepsin-hydrolyzed collagen (PepCol) fibrils.

Actinidain-hydrolyzed type I collagen reveals a crucial amino acid sequence in fibril formation.

We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of alpha1 and alpha2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized by poor fibrillogenesis.

Characterization of type I collagen fibril formation using thioflavin T fluorescent dye.

Collagen is composed of fibrils that are formed by self-assembly of smaller units, monomers which are triple-helical polypeptide. However, the mechanism of fibril formation at the level of individual molecules has remained to be clarified. We found that the fluorescence of thioflavin T, which has been widely used as a specific dye for amyloid fibrils, also increased by binding with fibrils of atelocollagen prepared from yellowfin tuna skin.

Preparation and structural analysis of actinidain-processed atelocollagen of yellowfin tuna (Thunnus albacares).

Pepsin-hydrolyzed collagen (atelocollagen) is a trimer, consisting of alpha 1 and alpha 2 monomers, and shows molecular species corresponding to a monomer, dimer (beta chain), and trimer (gamma chain) by SDS-polyacrylamide gel electrophoresis. Atelocollagen was purified from yellowfin tuna (Thunnus albacares) by salt precipitation and cation-exchange chromatography. Enzymatic hydrolysis of the atelocollagen by actinidain, a cysteine protease purified from kiwifruit, was analyzed by SDS-polyacrylamide gel electrophoresis.

Presence of protein complex is prerequisite for aragonite crystallization in the nacreous layer.

We have isolated a protein complex from the nacreous layer of pearl beads and oyster shells. This complex was mainly composed of pearlin and pearl keratin. Addition of a minute amount of the complex to a calcium-carbonate-saturated solution containing Mg2+ induced aragonite crystallization.

Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction.

Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of L-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess L-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20A away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the L-lysine activation reaction.

Functional tolerance of Streptomyces subtilisin inhibitor toward conformational and stability changes caused by single-point mutations in the hydrophobic core.

Single amino acid mutations of Met103 in the hydrophobic core of a serine protease inhibitor, Streptomyces subtilisin inhibitor, caused little change in the inhibitory activity, as measured by the inhibitor constant, although some altered the thermodynamic stability of the protein considerably. (1)H NMR investigations showed that the conformational stress caused by the replacement of Met103 with Gly, Ala, Val, and Ile, namely, the effects of the cavities generated by replacements with smaller side-chains and of the steric distortions generated by beta-branched side-chains, caused considerable changes in the structural arrangement of the side-chains within the core. However, these structural changes were absorbed within the hydrophobic core, without distorting the structure of the reactive site essential for the protein function.