Supramolecular Systems as Protein Ligands
The standard substrate complexation mechanism engages natural binding sites. In contrast, supramolecular structures may form complexes with proteins by penetrating in regions which are either naturally unstable or become temporarily accessible due to structural rearrangements related to the protein’s function. This may result in enhancement of irreversible processes (e.g. immune complexation or complement activation) or inhibition of reversible processes (e.g. enzymatic catalysis). Only ribbon-like supramolecular structures may form complexes with proteins. Having anchored itself inside the protein, the supramolecular ligand is protected against environmental factors such as changes in pH. This type of interaction represents a unique, nonstandard phenomenon in the context of proteomics.
KeywordsProtein dynamics and Congo red binding Ribbon-like supramolecular micelles Congo red as supramolecular dye Self-assembled molecules form a unit protein ligand Unity of self-assembled molecules Congo red penetration to protein interior Congo red complexation properties Protection of bound Congo red by proteins
1.1 Mechanism of Complexation
Biological function is a critical aspect in proteomics, and is often defined as the capability to interact with specific ligands and form complexes. Protein ligands tend to be either small molecules or small fragments of larger systems. They bind to the target protein in a specific area called the active site (or active group). Typically, the active site is a pocket where the ligand may directly contact the nonpolar interior of the protein – an environment which excludes water. The result is a stable complex and the ability to carry out reactions which would not be possible in an aqueous solution.
Proteins are generally incapable of interaction in areas other than their active sites, since tight packing of polypeptide chains prevents penetration of random ligands. Nevertheless, the protein is not a monolith: its dynamic nature means that under certain conditions the packing of polypeptide chains may undergo relaxation, enabling small molecules to penetrate protein interior [1, 2, 3, 4, 5, 6]. Those ligands cannot form stable bonds due to low binding energy in an area otherwise unprepared for specific interaction with such compounds – to put it simply, a rigid molecule is not likely to exhibit good alignment with the conformation of a folded polypeptide chain. The high mobility of small ligands also discourages strong interactions.
In spite of the above, some supramolecular associations of organic compounds are able to penetrate and anchor themselves inside proteins. This unique property emerges as a result of association (or self-association) of individual molecules [7, 8, 9, 10, 11, 12], and is linked to the flexible structure and large interaction surfaces exposed by supramolecular ligands.
The presence of noncovalent bonds in supramolecular structures allows their components to shift with respect to one another, resulting in an adaptive ligand which has greater conformational alignment capabilities than polymers or small organic molecules.
Owing to its structural flexibility, the supramolecular ligand may interact with proteins as the specific component – although its presence may also alter the target protein due to the large interaction area and strength of binding. Both structures adapt finally to each other, producing a stable bond .
The large volume of supramolecular ligands undoubtedly hampers penetration. Consequently, supramolecular ligands prefer interaction with inherently unstable proteins – such as partly unfolded proteins and amyloids [23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34]. In some cases, however, even a tightly packed protein may – when binding its natural target ligand – undergo sufficient structural rearrangement to permit penetration of additional large supramolecular ligands penetrating outside of the primary binding site. This type of interaction, while temporary, often drastically modifies the function of the protein [35, 36].
It appears however that in order to form stable complexes with proteins without degrading their structure, supramolecular ligands must exhibit a ribbon-like conformation. It should also be noted that the distribution of polarity in a ribbon-like micelle approximates the properties of a beta fold, promoting formation of a stable complex.
Further adsorption of CR on Sephadex along the column eventually results in precipitation of insufficiently protected immunoglobulin molecules. To enhance the contrast of CR under EM we have added silver ions (AgNO3), which form weak complexes with the dye but remain in solution along the short Sephadex filtration path.
1.2 Structural Adaptability of Molecules Forming Supramolecular Structures
Taken together, the presented characteristics – flat ribbon-like structure , flexibility, large interaction surface and exposure of hydrophobicity – promote interaction and formation of stable complexes with proteins penetrating to areas which are not biologically configured for binding ligands. Another important factor which enhances the adaptability of the CR micelle is some kind of plasticity of individual molecules, permitting rotation about the central bond between aromatic rings, as well as about both lateral azo bonds.
The relation between degrees of rotational freedom, charge distribution and protein binding capabilities is also evident when comparing EB with Trypan blue (TB). Both dyes differ only with respect to the location of sulfonic groups. In Trypan blue this location is disadvantageous due to its proximity to both the azo bond and the central nonpolar region of the molecule (Fig. 1.12 – 4 and 5).
Regarding protein structures, supramolecular ligands tend to preferentially form complexes with beta-structure and random coils of polypeptide chains. Elongated non-helical polypeptides represent a good match for the supramolecular ribbon itself, providing the ligand with a convenient anchoring point (Fig. 1.4).
Susceptibility for supramolecular ligand penetration varies from protein to protein. In addition to the degree of packing and the protein’s intrinsic stability it also depends – as remarked above – upon function-related conformational rearrangement [35, 36, 38, 39].
The role of most proteins is to interact with specific targets. Such interaction affects the protein itself and often results in partial unfolding, which renders the protein susceptible to further penetration by a supramolecular ligand. This mechanism can be observed e.g. when analyzing the interaction of CR with serum proteins. Note that the bloodstream typically contains acute phase proteins, in complex with their respective ligands, and that such complexes are recognized and eliminated by macrophages and liver enzymes. We may therefore suspect that the capability for such selective elimination depends on function-related structural changes which occur in proteins introducing some local instability.
1.3 Specificity of Congo Red Complexation
The complexation capabilities of CR increase along with the dye’s concentration. This is related to increased probability of penetration into proteins as they undergo dynamic – and often temporary – structural changes. Furthermore, increased concentrations favor supramolecular association, resulting in longer micelles with more pronounced dipole characteristics. This effect can be revealed by measuring electrophoretic migration distance on the electrophoretic plate and present it as a function of dye concentration.
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. The observed shift towards greater wavelengths confirms the stated hypothesis (Fig. 1.16B).
In summary, complexes consisting of supramolecular dyes and proteins appear to result from penetration of associated dye molecules into the target protein. The capability for such penetration depends on the cohesiveness of the dye micelle, as well as its shape.
Another important property of supramolecular dyes is their capability to intercalate foreign bodies (other than the self-associating unit molecules), resulting in ligands which can introduce foreign substances into proteins even when the protein does not, by itself, react with such substances [17, 47]. Rhodamine B – a basic dye which exhibits strong fluorescence and is therefore useful in imaging studies – may be intercalated into CR micelles and bound to proteins. Other potential intercalants include heavy metal ions – such as in the case of TY, used as a carrier for silver ions to provide contrast for EM imaging of amyloid deposits . The same mechanism may be used to introduce some alterations to properties of proteins.
Work financially supported by Collegium Medicum – Jagiellonian University grant system – grant # K/ZDS/006363.
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