Biomolecules interactions and competitions by non-immobilised ligand interaction assay by circular dichroism

Non-immobilised ligand interaction assay (NILIA) by CD spectroscopy provides an excellent technique to study molecular interactions in solution. Here are discussed molecular interactions of several systems that involve hosts and ligands with wide range of molecular sizes. Cytokine rhGM-CSF (14.6 kDa) bound to alpha-chain hGM-CSF receptor fragment (2 kDa, Kd = 35 microM), proline rich peptide (1.5 kDa) bound to fynSH3 domain (8 kDa, Kd = 28 microM), tumour imaging peptide (2 kDa) bound to mucin antigenic fragment (2 kDa, Kd = 20 microM), monoclonal antibody (150 kDa) bound to antigenic protein (120 kDa, Kd = 50 nM). Reconstitution of Cytochrome b5 (Cyt b5) from apo-Cyt b5 and hemin (Kd = 1.6 nM), correct protein folding of reconstituted porphobilinogen deaminase from apo-cofactorless form achieved using the product of the enzyme catalysis, preuroporphyrinogen, rather than porphobilinogen substrate. Competition studies of bound non-chiral drugs diclofenac and diazepam to carrier proteins such as HSA in the presence of fatty acids are few of the examples of the studies carried out by NILIA-CD spectroscopy. The CD changes in either backbone, aromatic side-chains and disulphide regions were used accordingly to screen qualitatively and quantitatively ligand binding in vitro. CD data were fitted by non-linear regression to the general equilibrium reaction of a single-binding site. NILIA-CD is fast compared to NMR, gives information on conformational changes due to interaction, avoids masking of the binding site due to immobilisation and requires no radiolabelling. NILIA-CD is thus an ideal technique for interaction, activity, screening studies.