Introduction of potential helix‐capping residues into an engineered helical protein

Abstract

MB‐1 is an engineered protein that was designed to incorporate high percentages of four amino acid residues and to fold into a four‐ α‐helix bundle motif. Mutations were made in the putative loop I and III regions of this protein with the aim of increasing the stability of the helix ends. Four variants, MB‐3, MB‐5, MB‐11 and MB‐ 13, have replacements intended to promote formation of an ‘N‐ capping box’. The loop I and III sequences of MB‐3 (both GDLST) and MB‐11 (GGDST) were designed to cause αL C‐terminal ‘capping’ motifs to form in helices I and III. MB‐5 has a sequence, GPDST, that places proline in a favourable position for forming β‐turns, whereas MB‐13 (GLDST) has the potential to form Schellman C‐capping motifs. Size‐exclusion chromatography suggested that MB‐1, MB‐3, MB‐5, MB‐11 and MB‐13 all form dimers, or possibly trimers. Free energies for the unfolding of each of these variants were determined by urea denaturation, with the loss of secondary structure followed by CD spectroscopy. Assuming an equilibrium between folded dimer and unfolded monomer, MB‐13 had the highest apparent stability (40.5 kJ/mol, with ±2.5 kJ/mol 95% confidence limits), followed by MB‐11 (39.3±5.9 kJ/mol), MB‐ 3 (36.4±1.7 kJ/mol), MB‐5 (34.7±2.1 kJ/mol) and MB‐1 (29.3±1.3 kJ/mol); the same relative stabilities of the variants were found when a folded trimer to unfolded monomer model was used to calculate stabilities. All of the variants were relatively unstable for dimeric proteins, but were significantly more stable than MB‐1. These findings suggest that it might be possible to increase the stability of a protein for which the three‐dimensional structure is unknown by placing amino acid residues in positions that have the potential to form helix‐ and turn‐stabilizing motifs.