Proline 235 plays a key role in the regulation of the oligomeric states of Thermotoga maritima Arginine Binding Protein

• The replacement of Pro235 leads to the formation of distinct TmArgBP oligomers.
• TmArgBP swapping is driven by the conformational restraints produced by Pro235.
• Trimers are endowed with distinctive properties including the ability to bind ThT.
• Sequence control of hinge regions is essential to avoid the formation of undesired species.

The Arginine Binding Protein isolated from Thermotoga maritima (TmArgBP) is a protein endowed with several peculiar properties. We have previously shown that TmArgBP dimerization is a consequence of the swapping of the C-terminal helix. Here we explored the structural determinants of TmArgBP domain swapping and oligomerization. In particular, we report a mutational analysis of the residue Pro235, which is located in the hinge region of the swapping dimer. This residue was either replaced with a Gly-Lys dipeptide (TmArgBPP235GK) or a Gly residue (TmArgBPP235G).Different forms of these mutants were generated and extensively characterized using biophysical techniques.For both TmArgBPP235GK and TmArgBPP235G mutants, the occurrence of multiple oligomerization states (monomers, dimers and trimers) was detected. The formation of well-folded monomeric forms for these mutants indicates that the dimerization through C-terminal domain swapping observed in wild-type TmArgBP is driven by conformational restraints imposed by the presence of Pro235 in the hinge region. Molecular dynamics studies corroborate this observation by showing that Gly235 assumes conformational states forbidden for Pro residues in the TmArgBPP235G monomer. Unexpectedly, the trimeric forms present: (a) peculiar circular dichroism spectra, (b) a great susceptibility to heating, and (c) the ability to bind the Thioflavin T dye.The present findings clearly demonstrate that single-point mutations have an important impact on the TmArgBP oligomerization process. In a wider context, they also indicate that proteins endowed with an intrinsic propensity to swap have an easy access to states with altered structural and, possibly, functional properties.

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