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Research examines the role of hydrophobic core substitutions, contributed by the EF subdomain fragments, on the reconstitution and stabilization of a protein as exemplified by the calcium-binding protein calbindin D. Data show a strong correlation between the affinity and stability indicating to the importance of the hydrophobic core residues.

A study on the effect of mutations of hydrophobic amino acids on the structure and folding of calbindin D(sub 9k) is presented. Calbindin D(sub 9k) is a four-helix calcium-binding protein that does not easily undergo conformational change. Eleven mutations have been made at eight sites in the hydrophobic core of calbindin D(sub 9k). Results show that calbindin D(sub 9k) can absorb large changes in stability without any changes in its fold and calcium-binding properties.

Proteoglycan modification is essential for development and early cell division in Caenorhabditis elegans. The specification of proteoglycan attachment sites is defined by the Golgi enzyme polypeptide xylosyltransferase. Here we evaluate the substrate specificity of this xylosyltransferase for its downstream targets by using reporter proteins containing proteoglycan modification sites from C. elegans syndecan/SDN-1. The N terminus of the SDN-1 contains a Ser-Gly proteoglycan site at Ser71, flanked by potential mucin and N-glycosylation sites. However, Ser71 was exclusively used as a proteoglycan site in vivo, based on mapping studies with a Ser71 reporter protein, glycosyltransferase RNA interference, and co-expression of worm polypeptide xylosyltransferase. To elucidate the substrate requirements of this enzyme, a library of 42 point mutants of the Ser71 reporter was expressed in tissue culture. The nematode proteoglycan modification site in SDN-1 required serine (not threonine), two flanking glycine residues (positions -1 and +1), and either one proximal acidic N-terminal amino acid (positions -4, -3, and -2) or a pair of distal N-terminal acidic amino acids (positions -6 and -5). C-terminal acidic amino acids, although present in many proteoglycan modification sites, had minimal impact on xylosylation at Ser71. Proline inhibited glycosylation when present at -1, +1, or +2. The position of glycine, proline, and acidic amino acids allows the glycosylation machinery to discriminate between mucin and proteoglycan modification sites. The key residues that define proteoglycan modification sites also function with the Drosophila polypeptide xylosyltransferase, indicating that the specificity in the glycosylation process is evolutionarily conserved. Using a neural network method, a preliminary proteoglycan predictor has been developed. [ABSTRACT FROM AUTHOR]