Significance of the C-Terminal Domain of Erwinia uredovora Ice Nucleation-Active Protein (Ina U)

Abstract

Ice nucleation-active (Ina) proteins of bacterial origin comprise three distinct domains, i.e., N-terminal (N-), central repeat (R-), and C-terminal (C-) domains, among which the R-domain is essential, and its length may be correlated with the ice nucleation activity. In addition, the short C-terminal domain of about 50 amino acid residues is indispensable for the activity. Using the Ina U protein of Erwinia uredovora, we carried out precise mutational analyses of its C-terminus. The ice nucleation activity (T50) assay showed that the C-terminal 12 amino acids were not necessary, and a deletion mutant (delta C29) with a new C-terminal, Met29 (numbered from the first amino acid residue of the C-domain and corresponding to Met1022), exhibited almost the same activity as the wild-type Ina U protein did. However, deletion of the C-terminal 13 residues including Met29 resulted in almost complete loss of the activity. In the deletion mutant (delta C29), amino acid replacement of the C-terminus, Met29, showed that the activity was retained when Met29 was replaced with a neutral, aromatic, or basic amino acid (Gly, Phe, or Lys), but was lost on the replacement with an acidic amino acid (Asp or Glu). In addition, two other residues in the C-terminal region commonly present in all Ina proteins were examined as to their importance, and it was shown that one of these residues, Tyr27, is important for the activity, although it is not exclusively required; the activity was lost to a great extent when this residue was replaced with Gly or Ala, but to a lesser extent when it was replaced with Leu. These results suggest that significance of the secondary and/or tertiary structure of the C-terminal region of the Ina U protein for the ice nucleation activity.

Enhanced production of extracellular ice nucleators from Erwinia herbicola.

“…Similar ECIN morphologies were also reported with thin sections (Phelps et al, 1986). A process such as minicell formation was suggested for the transport of the InaU protein from the inner membrane to the outer membrane, based on immunogold staining and electron microscopy (Michigami et al, 1995b). Vesicle formation may also result because the outer membrane grows more rapidly than the underlying peptidoglycan (Mayrand and Grenier, 1989).…”

“…This study demonstrates that maximal INA of cell-free filtrates was achieved at late log or early stationary phase. Comparable results were reported with E. uredovora (Michigami et al, 1995b). The timing of the expression of ice nucleation gene products and their correlation with the Ina ϩ phenotype were examined in P. syringae S203 and P. fluorescens MS1650 (Deininger et al, 1988).…”

Characterizations of Functions of Biological Materials Having Controlling-Ability Against Ice Crystal Growth

“…Also, the C-terminal domain is rich in basic amino acid residues and is very hydrophilic. Among C-terminal amino acid residues, Tyr27 in this domain is important for ice nucleation, although not exclusively required, since nucleation was lost to a great extent when this residue was replaced by Gly or Ala but to a much lesser extent when it was replaced by Leu [38]. These results point to the important of the secondary and/or tertiary structure of the C-domain region for the ice nucleation with the hydroxyl group in the surface of its protein, which may interact with water molecule.…”

Μοριακή Ανάλυση Και Εξέλιξη Γονιδίων Παγοπυρήνωσης

“…Συγκεκριμένα, ελλείψεις στην αμινοτελική περιοχή οδηγούν σε μείωση ή και εξάλειψη των παγοπυρήνων τύπου Ι και ΙΙ, ενώ δεν επηρεάζουν σημαντικά τους παγοπυρήνες τύπου ΙΙΙ. Αντίθετα, σταδιακή εξάλειψη της καρβοξυτελικής περιοχής της πρωτεΐνης οδήγησε σε πλήρη ανικανότητα σχηματισμού παγοπυρήνων όλων των τύπων (Green et al, 1988, Michigami et al, 1995. Πειράματα δημιουργίας ελλειμμάτων και αντικατάστασης αμινοξέων στην πρωτεΐνη ΙnaU έδειξαν ότι η ενεργότητα της πρωτεΐνης εξαρτάται από το μήκος του C-τελικού άκρου και από συγκεκριμένα κατάλοιπα αμινοξέων της περιοχής αυτής (Michigami et al, 1995).…”

“…Αντίθετα, σταδιακή εξάλειψη της καρβοξυτελικής περιοχής της πρωτεΐνης οδήγησε σε πλήρη ανικανότητα σχηματισμού παγοπυρήνων όλων των τύπων (Green et al, 1988, Michigami et al, 1995. Πειράματα δημιουργίας ελλειμμάτων και αντικατάστασης αμινοξέων στην πρωτεΐνη ΙnaU έδειξαν ότι η ενεργότητα της πρωτεΐνης εξαρτάται από το μήκος του C-τελικού άκρου και από συγκεκριμένα κατάλοιπα αμινοξέων της περιοχής αυτής (Michigami et al, 1995). Ελλείψεις στην επαναλαμβανόμενη περιοχή της πρωτεΐνης που διακόπτουν το μοτίβο των 48 -καταλοίπων επηρεάζουν τη συχνότητα σχηματισμού παγοπυρήνων τύπου Ι (Green et al, 1988).…”