Circular Permutation of the Trp-cage: Fold Rescue upon Addition of a Hydrophobic Staple

Abstract

The Trp-cage, at 20 residues in length, is generally acknowledged as the smallest fully protein-like folding motif. Linking the termini by a two-residue unit and excising one residue affords circularly permuted sequences that adopt the same structure. This represents the first successful circular permutation of any fold of less than 50-residue length. As was observed for the original topology, a hydrophobic staple near the chain termini is required for enhanced fold stability.

Fig. 1

Comparison of a circularly permuted (left panel) and typical non-cyclic Trp-cage (right panel) illustrating the permutation strategy adopted. The conformational features in the left panel are from the X-ray structure of cyclo-TC1, those in the right panel are from an NMR structure: the full backbone and the heavy atoms of Y3, W6, L7, P18 and P19 (the buried Trp and the residues that form the hydrophobic core) are displayed. The added Gly-Gly loop of the cyclic species is shown in blue, the residue excised in producing circular permutants appears in red (right panel).

Fig. 2

A comparison of structuring shifts (as CSDs at 280 K) along the sequence of the cyclic ‘starting point’ and a circularly permuted Trp-cage (cp-TC1). The data for cp-TC1 are shown for aqueous medium (pH 7) and upon addition of 30 vol-% trifluoroethanol. Except as noted, the CSDs are for the single Hα of each residue.

Fig. 3

Local χ_F measures (CSDobs/CSDref) for Trp-cage circular permutants and a C-terminal truncated model (Ac-DAAAAAYA-QWLADGGPASG) of a half-cage structure. The folding measures are shown below each set of histograms. For each site, the analogs are shown in the following order, from left to right: – cp-TC1, cp-TC2 (RPPPSGGDAYAQWLADaGPSS, a = D-Ala), cp-TC2 with the following GGDA loop replacements – cp-TC2b, TADA and cp-TC2c, DUAA (U = Aib), cp-T²C3, and the half-cage model. The CSDref values are taken from χ_F ≥ 0.99 examples with each mutation. Additional comparisons of fold stabilities for the circular permutants appear in the Supporting Information (Table S1) together with CD melt comparisons for cp-TC2 and cp-T²C3 and their closest hyperstable, non-permuted analogs (Figure S1).

Fig. 4

A comparison of representative structures from the NMR ensembles of [P12W]-T²C16b (green) and cp-T²C3b (gray). In the permutant, ring current shifts at W6, P17 and P18 due to the added Trp were in the same ratio as those observed in [P12W]-T²C16b indicating the same orientation. See the Supporting Information for complete ensembles (Figure S3), statistics and dihedral angles (Tables 2S,3S,4S)chemical shifts (Table 5S), NOE constraints and CSD comparisons (Tables 5S & 6S).