Heat-induced formation of a specific binding site for self-assembled congo red in the V domain of immunoglobulin L chain ?

Abstract

Moderate heating (40–50°C) of immunoglobulins makes them accessible for binding with Congo Red and some related highly associated dyes. The binding is specific and involves supramolecular dye ligands presenting ribbon‐like micellar bodies. The L chain λ dimer, which upon heating disclosed the same binding requirement with respect to supramolecular dye ligands, was used in this work to identify the site of their attachment. Two clearly defined dye–protein (L λ chain) complexes arise upon heating, here called complex I and complex II. The first is formed at low temperatures (up to 40–45°C) and hence by a still native protein, while the formation of the second one is associated with domain melting above 55°C. They contain 4 and 8 dye molecules bound per L chain monomer, respectively. Complex I also forms efficiently at high dye concentration even at ambient temperature. Complex I and its formation was the object of the present studies. Three structural events that could make the protein accessible to penetration by the large dye ligand were considered to occur in L chains upon heating: local polypeptide chain destabilization, VL‐VL domain incoherence, and protein melting. Of these three possibilities, local low‐energy structural alteration was found to correlate best with the formation of complex I. It was identified as decreased packing stability of the N‐terminal polypeptide chain fragment, which as a result made the V domain accessible for dye penetration. The 19‐amino acid N‐terminal fragment becomes susceptible to proteolytic cleavage after being replaced by the dye at its packing locus. Its splitting from the dye–protein complex was proved by amino acid sequence analysis. The emptied packing locus, which becomes the site that holds the dye, is bordered by strands of amino acids numbered 74–80 and 105–110, as shown by model analysis. The character of the temperature‐induced local polypeptide chain destabilization and its possible role in intramolecular antibody signaling is discussed. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 446–456, 2001

Metal Ions Introduced to Proteins by Supramolecular Ligands

“…Prior to aggregation, V domains undergo partial unfolding initiated by displacement of N-terminal chain. This uncovers the interior of domains [21] making available a pocket in which the CR aggregate may anchor itself. It appears that unfolding affects the so-called upper core [36,37] and occurs symmetrically in the dimeric structure of the protein.…”

“…Such ligands can form stable complexes with proteins which have become susceptible to penetration as a result of function-related structural changes, or which possess such properties natively [21][22][23]. Certain proteins can be induced to undergo complexation with supramolecular ligands e.g.…”

Supramolecular Systems as Protein Ligands

“…This effect results from transitioning between water and an environment characterized by lower values of the dielectric constant ( Fig. 1.16A) [45,46]. To further illustrate this effect, the spectrum of CR has been analyzed in the presence of alcohols containing increasingly larger nonpolar components: methanol, ethanol and propanol.…”

Protein Conditioning for Binding Congo Red and Other Supramolecular Ligands

“…Such instabilities can, however, be artificially exacerbated, e.g. through heating [3]. Under natural conditions Congo red (CR) is spontaneously bound by partly unfolded proteins capable of forming aggregationssuch as amyloids and some abnormal [4][5][6][7][8][9].…”