Protein L is a multidomain cell wall protein from Peptostreptococcus magnus that is one of a group of proteins capable of binding to antibodies without producing an immune response. In contrast to other immunoglobulin (Ig) binding proteins, protein L binds exclusively to the V_L domain of κ-chains. It has previously been shown that a single Ig binding domain of protein L (PpL) has two sites of interaction with the V_L domain, with the affinity of the second site up to 50 fold less than that of the first, depending on the nature of the κ-chain. The binding interaction between recombinant PpL and a recombinant humanised mouse 12.6 kDa variable light chain domain antibody (dAb) has been examined using a range of techniques, including stopped-flow fluorimetry, circular dichroism, isothermal titration calorimetry and NMR. The wild-type dAb is bound with the same affinity as seen previously with κ1 chain, with Kd values of 51.5 ± 2.6 nM for site 1 and 1.5 ± 0.2 μM for site 2 of PpL. This confirmed that the loss of the C_L domain does not affect binding. Thermodynamic studies of the binding interaction have shown that unusually, the thermodynamic constants obtained for the binding interactions between PpL and dAb either directly or indirectly are in very good agreement. In addition, these constants are significantly different to those seen with κ-chain in previous studies, indicating altered entropic and enthalpic contributions to the free energy of these interactions. To further understand the interaction between PpL and dAb, in particular at the second binding site, mutagenesis of both species has been undertaken. These changes show a range of effects on the affinity of the PpL-dAb interaction, and indicate a large degree of plasticity in the interfaces between the proteins.