Id3, inhibitor of DNA binding and cell differentiation: synthesis and conformational analysis of the full-length protein and its truncated analogues

The Dissertation deals with the synthesis and characterization of Id3 which is the smallest member of the Id protein family. It is a negative regulator of the bHLH transcription factors; by the formation of heterodimers with the bHLH proteins, Id3 prevents their binding to DNA and hence the transcriptional activation. This eventually leads to increased cell proliferation and reduced cell differentiation. In general, the Id proteins are overexpressed in proliferating, undifferentiated normal and cancer cells, while their expression is low or they are absent in non-proliferating, terminally differentiated cells. Id3 is a valuable target for the automated solid phase peptide synthesis. The full-length protein and its truncated and modified analogues were assembled on Wang or Rinkamide resin using Fmoc protocol. Native chemical ligation was considered as an alternative approach, and various peptide thioesters were therefore prepared. The best method to prepare the thioesters was the one performed on solid support with side-chain-anchored peptides. Obtained polypeptides were purified by preparative RP-HPLC and characterized by CD and fluorescence spectroscopy and mass spectrometry. The conformational properties of all synthesized Id3 polypeptides were studied by circular dichroism spectroscopy and revealed a complex interplay between the Id3 subdomains. The 41-residue long helix-loop-helix motif (spanning the Id3 residues 41-81) which is responsible for the interaction with the bHLH proteins was found to be sensitive to amino acid replacement with the two helices. In particular, mutation of the two tyrosine residues located in helix-1 and helix-2 which are conserved in the Id family, but not in the HLH family, has shown that the hydroxyl group of the tyrosine residue in helix-1 is not important for the conformation, whereas the tyrosine residue in helix-2 is critical for the stabilization of the Id3 HLH motif within the dimer. Also the cysteine residue in helix-1, conserved within the Id family, seems to be important for the helix stability. Whereas the Id3 HLH motif is highly helical, the regions which precede and follow it do not adopt any preferred conformation as isolated fragments. Nevertheless, they are critical for correct folding of the central HLH motif. Presence of N-terminal part favors the formation of beta-sheet strands, while the C-terminal part stabilizes the helix component. Phosphorylation of the N-terminus at position 5 (CDK2 phosphorylation site) also induces conformational change, as shown by increased beta-strand content after the modification. Flexibility of the N- and C-tails is probably a structural prerequisite for the modulation of the protein fold during dimerization processes with different bHLH transcription factors. The FRET properties of some fluorescence-labeled Id3 segments suggest that the N-terminus folds back to the compact HLH motif. Tryptophan/dansyl pair was introduced into various positions along the sequence, and spatial arrangement of the segments was deduced from the FRET efficiency and hence distance between the two chromophores. Recruitment and/or expression of matrix metalloproteinase MMP-2 required for tumor growth and metastasis may be controlled by the Id proteins. Fluorescent tools for the detection of MMP�s activity are therefore of interest. Fluorogenic MMP-2 substrate which contains FAM/TAMRA FRET pair and a C-terminal oligoarginine segment was prepared. Additionally, two peptides with a fibrillogenic sequence were prepared to probe the possibility to use the enzymatic process not only for spectroscopic properties modulation, but also to trigger other events, such as conformational transitions and aggregation. In accordance with the expectations, the oligoarginine-containing peptide was cleaved by MMP-2, as proved by a significant increase of FAM emission. In contrast, the two fibrillogenic-domain-containing peptides exhibited slight decrease of the FAM emission which likely reflects the triggering of the aggregation process with consequent self-quenching of FAM. Obtained results contribute to the synthetic and conformational characterization of this biologically highly relevant biomolecule and build a solid ground for future investigations.