concomitant to the water molecules’ structuration around hydrophobic moieties with
the result of reducing the entropy.
A few years ago, Tolstorukov et al. [87] tried to demonstrate the existence
of hydrophobic association between proteins and nucleic acids. Deformations of
nucleic acids generate the rearrangement of sugars which directly influence the
width of the grooves. This deformation frees up enough space for protein associations by means of their nonpolar sites, where hydrophobic amino acids dominate. It
has been clearly demonstrated that the hydrophobic interaction and consequent
protein-DNA recognition occur in the minor groove. These interaction areas are
impermeable to water, strengthening thus the hydrophobic association. While the
presence of salts does not influence the hydrophobic associations, changes in the pH
environment may modulate the strength of this interaction. These peculiar properties
are probably due to the presence of a large number of negatively charged amino
acids on the protein moiety contributing to increase the global hydrophilicity of the
entire construct. In fact, at low pH, the change of protein conformation generates an
intensification of hydrophobic associations due to the rearrangements of loop-helix
structures [88]. These considerations are of importance and will probably open the
way to further developments (see Sect. 7 below).
3.3.4 Thermodynamics Bases of the Molecular Interaction
Electrostatic interactions, hydrogen bonding, and hydrophobic association taken
together mainly generate the molecular recognition, characterized by an affinity
constant of an aptamer for a given protein, like any other complex in affinity
chromatography [89]. Thus the density of the aptamer-grafted ligand on solid
support is critical for the binding capacity properties of the affinity sorbent. Moreover the degree of freedom of the grafted aptamer must be relatively large to reach
the protein aptatope to dock there intimately. To this end it appears useful to have a
flexible spacer between the oligonucleotide ligand and the solid matrix. On the other
side, proteins are large constructs that surpass the mass of the oligonucleotide by
factors ranging from 1 to about 3. Proteins are folded on themselves and expose
generally a limited surface area, compared to the length of the amino acid sequence.
This situation largely enhances the probability to have a highly specific molecular
interaction happening.
Figure 5 depicts the interaction between a grafted aptamer and a protein in
solution schematically. Therefore the formation of this intimate couple is only
possible if complementary/synergistic association forces are present and located in
the right position.
However, all these considerations are static and may not be sufficient to describe
the dynamics of the interaction phenomena that comprise an intense competition
among interaction forces present.
Thermodynamic equilibria are also very sensitive to several environmental
parameters such as pH, ionic strength, temperature, and the presence of additives
(e.g., mineral ions). This situation is fundamental in affinity chromatography, since it
is at the basis of the capture and the harvesting of the protein to purify.
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
105
the result of reducing the entropy.
A few years ago, Tolstorukov et al. [87] tried to demonstrate the existence
of hydrophobic association between proteins and nucleic acids. Deformations of
nucleic acids generate the rearrangement of sugars which directly influence the
width of the grooves. This deformation frees up enough space for protein associations by means of their nonpolar sites, where hydrophobic amino acids dominate. It
has been clearly demonstrated that the hydrophobic interaction and consequent
protein-DNA recognition occur in the minor groove. These interaction areas are
impermeable to water, strengthening thus the hydrophobic association. While the
presence of salts does not influence the hydrophobic associations, changes in the pH
environment may modulate the strength of this interaction. These peculiar properties
are probably due to the presence of a large number of negatively charged amino
acids on the protein moiety contributing to increase the global hydrophilicity of the
entire construct. In fact, at low pH, the change of protein conformation generates an
intensification of hydrophobic associations due to the rearrangements of loop-helix
structures [88]. These considerations are of importance and will probably open the
way to further developments (see Sect. 7 below).
3.3.4 Thermodynamics Bases of the Molecular Interaction
Electrostatic interactions, hydrogen bonding, and hydrophobic association taken
together mainly generate the molecular recognition, characterized by an affinity
constant of an aptamer for a given protein, like any other complex in affinity
chromatography [89]. Thus the density of the aptamer-grafted ligand on solid
support is critical for the binding capacity properties of the affinity sorbent. Moreover the degree of freedom of the grafted aptamer must be relatively large to reach
the protein aptatope to dock there intimately. To this end it appears useful to have a
flexible spacer between the oligonucleotide ligand and the solid matrix. On the other
side, proteins are large constructs that surpass the mass of the oligonucleotide by
factors ranging from 1 to about 3. Proteins are folded on themselves and expose
generally a limited surface area, compared to the length of the amino acid sequence.
This situation largely enhances the probability to have a highly specific molecular
interaction happening.
Figure 5 depicts the interaction between a grafted aptamer and a protein in
solution schematically. Therefore the formation of this intimate couple is only
possible if complementary/synergistic association forces are present and located in
the right position.
However, all these considerations are static and may not be sufficient to describe
the dynamics of the interaction phenomena that comprise an intense competition
among interaction forces present.
Thermodynamic equilibria are also very sensitive to several environmental
parameters such as pH, ionic strength, temperature, and the presence of additives
(e.g., mineral ions). This situation is fundamental in affinity chromatography, since it
is at the basis of the capture and the harvesting of the protein to purify.
Aptamer-Based Affinity Chromatography for Protein Extraction and Purification
105