Crystal structure of a ten-amino acid protein.

What is the smallest protein? This is actually not such a simple question to answer, because there is no established consensus among scientists as to the definition of a protein. We describe here a designed molecule consisting of only 10 amino acids. Despite its small size, its essential characteristics, revealed by its crystal structure, solution structure, thermal stability, free energy surface, and folding pathway network, are consistent with the properties of natural proteins. The existence of this kind of molecule deepens our understanding of proteins and impels us to define an "ideal protein" without inquiring whether the molecule actually occurs in nature.

Peripheral region for core cross-beta plays important role in amyloidogenicity.

The role of the peripheral sequence neighboring the core cross-beta region was investigated using a peptide library constructed with all possible combinations of Lys, Glu, Ser, and Leu at three residue positions (X1-X3) forming the N-terminal region linked to the amyloid core sequence of the barnase-derived segment (A4-K22). By means of CD spectra and thioflavin T binding assay for 64 peptides, not only the composition but also the sequence in the peripheral region were found to be responsible for amyloid formation. The preferences of amino acid residues in the peripheral region of the amyloid-forming peptides were in the order of Leu approximately SerGlu>>Lys. A balance of positive and negative charges was found to be essential for amyloid formation, suggesting that the electrostatic interaction at the surface of the amyloid fibrils is relevant to their stability. On the basis of the maximum fluorescence wavelength of fibril-bound thioflavin T, the highly amyloidogenic peptides were classified into two classes, which exhibited the sequence preferences of (Leu, Ser/Glu, and Leu) and (Glu, Leu, and Ser) for the peripheral sequence (X1, X2, and X3). The former class can be rationally assigned to the structural model with deep grooves along the fibril axis. Thus, the peripheral sequence regulates the manner of molecular packing in the fibrils as well as the amyloidogenicity. In addition, the chains of the peripheral sequence are most likely to form thioflavin T binding sites.

Thermodynamic and kinetic aspects of antibody evolution during the immune response to hapten.

We determined thermodynamic and kinetic parameters for the antigen-antibody interaction using a group of anti-(4-hydroxy-3-nitrophenyl)acetyl monoclonal antibodies whose differences in amino acid sequences had arisen only from somatic hypermutation. These monoclonal antibodies were considered to have originated from a common ancestor clone and to represent progression along the affinity maturation pathway. The kinetic measurements showed that both association and dissociation rate constants of the antigen-antibody interaction decreased during maturation. Thermodynamic measurements revealed that an increase in affinity was obtained by an increase in entropy without any significant change in enthalpy. These results suggested that the mechanism for the antigen-antibody interaction shifted from a "zipper" type to a "lock-and-key" type during antibody evolution.

Interactions of wheat-germ agglutinin with GlcNAc beta 1,6Gal sequence.

The interactions of wheat-germ agglutinin (WGA) with the GlcNAc beta 1,6Gal sequence, a characteristic component of branched poly-N-acetyllactosaminoglycans, were investigated using isothermal titration calorimetry and X-ray crystallography. GlcNAc beta 1,6Gal exhibited an affinity greater than GlcNAc beta 1,4GlcNAc to all WGA isolectins, whereas Gal beta 1,6GlcNAc showed much less affinity than GlcNAc beta 1,4GlcNAc. X-ray structural analyses of the glutaraldehyde-crosslinked WGA isolectin 3 crystals in complex with GlcNAc beta 1,6Gal, GlcNAc beta 1,4GlcNAc and GlcNAc beta 1,6Gal beta 1,4Glc were performed at 2.4, 2.2 and 2.2 A resolution, respectively. In spite of different glycosidic linkages, GlcNAc beta 1,6Gal and GlcNAc beta 1,4GlcNAc exhibited basically similar binding modes to each other, in contact with side chains of two aromatic residues, Tyr64 and His66. However, the conformations of the ligands in the two primary binding sites were not always identical. GlcNAc beta 1,6Gal showed more extensive variation in the parameters defining the glycosidic linkage structure compared to GlcNAc beta 1,4GlcNAc, demonstrating large conformational flexibility of the former ligand in the interaction with WGA. The difference in the ligand binding conformation was accompanied by alterations of the side chain conformation of the amino acid residues involved in the interactions. The hydrogen bond between Ser62 and the non-reducing end GlcNAc was always observed regardless of the ligand type, indicating the key role of this interaction. In addition to the hydrogen bonding and van der Waals interactions, CH--pi interactions involving Tyr64, His66 and Tyr73 are suggested to play an essential role in determining the ligand binding conformation in all complexes. One of the GlcNAc beta 1,6Gal ligands had no crystal packing contact with another WGA molecule, therefore the conformation might be more relevant to the interaction mode in solution.