The aim was to evaluate the effects of backbone substituents on the stability of protein domains. The substituents used were a variety of peptidomimetics and uncommon amino acids. The highly constrained substituents were introduced by total chemical synthesis using solid phase peptide synthesis techniques. Protein G B2 domain analogues containing 6-amino hexanoic acid, bisthiazolidine dipeptide (BTD), aminoisobutyric acid, D-proline and N-methyl alanine were made using (O- (7-Azahydroxybenzotriaz-1-yl)-1,1,3,3,- tetramethyl uronium hexafluorophosphate (HATU) as a coupling agent for hindered residues. The stabilities, relative to wildtype, of nine synthetic analogues were analysed by denaturant unfolding, circular dichroism and differential scanning calorimetry. Binding to hA33 Fab was analysed using surface plasmon resonance methods. The wildtype sequence had an affinity for Fab of 35M. The mutant K15d-P T16N-MeA was 2kJmol-1 more stable than wildtype and had an affinity for Fab of 280M. The mutant Q37Aib was just as stable as wildtype and had an affinity for Fab of 250M. It is now possible to make analogues of protein domains with any amino acid residue, no matter how hindered, anywhere in the sequence. The introduction of restrained turn mimetics perturbs the folded state but does not destroy it. The introduction of uncommon side chains can make domains more stable than the wildtype structure and maintain the ability to bind antibodies to the wildtype. Structure/activity studies on small proteins can now be extended to use any uncommon amino acid in addition to the naturally occurring ones.