The presented thesis examines two cases of protein binding from a structural and a thermodynamic point of view in which protein recognition occurs through the structural adaptation of one of the binding partners.
First, we focused on the interactions of intrinsically disordered proteins (IDPs) which consist of sequences that fold into α-helices upon binding to their globular protein targets. Using data from literature and from our own measurements, we compiled a representative dataset of α-helical IDPs. We found that IDPs in general possess significant residual helicity in their unbound state (pre-folded structure) and gain helicity upon binding their targets. The presence of such a transient helical structure in the unbound state is advantageous from a thermodynamic point of view, as it reduces the entropic penalty of folding and thereby increases interaction affinity. It turns out that the reason for the observed ordered structure in the unbound state is, among other things, the presence of leucine – a hydrophobic amino acid residue – and some typical binding motifs that are an integral part of intrinsically disordered transactivation domains (the LXXLL leucine motif is especially common). Due to its properties, leucine significantly affects the stability of the pre-folded structure and forms important stabilizing interactions between an IDP and its target.
In the second part of this thesis, the interactions of camelid antibody fragments (nanobodies) and their targets (HigB2 and MazF antigens) were analyzed using isothermal titration calorimetry (ITC). The goal was to explain the thermodynamic contributions of binding using the properties of the amino acid sequences of the hypervariable regions (CDR loops) of nanobodies. We observed that some conformational plasticity of the hypervariable loops is present but found that it doesn’t represent a major contribution to the high binding affinity of nanobodies. A relatively high content of hydrophobic amino acid residues in the CDR3 regions and its apparent connection with binding affinity both suggest a pronounced hydrophobic character of nanobody-antigen interactions.
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