Ice nucleation proteins (INPs) form oligomeric structures by self-assembly and aggregation.
We looked for the presence of potential aggregating sequences inside the INP from Pseudomonas syringae
by a computational approach with the AGGRESCAN, FOMDAMYLOID and TANGO softwares.
A total of 38 hot spots of aggregation were predicted in the INP sequence: 7 localized in the Nterminal
domain, 2 in the C-terminal region, 28 in the highly repetitive central (HRC) region and 1
shared between the HRC and the Carboxyl-terminus regions of the protein. All the hot spots of aggregation
identified in the HRC domain overlapped a 8-residue low fidelity repeat including a LIAGYrelated
sequence. We confirmed the predictions by an experimental approach using synthetic peptides corresponding to
different parts of the INP central sequence, absorbance spectroscopy and fluorescence spectroscopy in the presence of
Congo red (CR) or Thioflavin T (ThT), respectively. Peptide 620-SFIIAGYG-627 predicted to aggregate by the three
softwares induced an increase in fluorescence of ThT. Peptide 729-GFKSILTAGY-738 predicted to aggregate by AGGRESCAN
and FOLDAMYLOID induced a shift in the maximum of absorbance of CR. Peptide 1124-SVLTAGA-1130
predicted to aggregate only by TANGO did not interfere with CR absorbance or ThT fluorescence. In conclusion, the use
of three aggregation prediction algorithms and two biochemical assays showed that the hexapeptide repeated segment
LIAGY, previously shown to form a hairpin loop may be involved in the aggregation of the P. syringae INP.
Keywords: Aggregation, ice nucleation, INP, Pseudomonas syringae.
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