The Trp-cage: optimizing the stability of a globular miniprotein

Abstract

The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).

Fig. 1

The Trp-cage fold showing the secondary structure features and the buried Trp side chain as well as the residues that shield the Trp indole ring from solvent exposure.

Fig. 2

Representative plots of CSDs versus temperature for a stable Trp-cage (TC10b, dark lines) and its destabilized S14A-mutant (grey lines and symbols). a illustrates the L7α shift melts, the melting of the downfield shifts at P12Hβ3 and R16α are illustrated in b, the melting of the larger upfield ring-current shifts due to the indole ring appear in (c) and (d). The lines through the data are polynomial fits with no theoretical significance. Similar effects are observed for acidification-induced decreases in extent of folding (see Fig. S1, Supplementary data available at PEDS online).

Fig. 3

‘Cage’ (best fit lines through solid symbols) and ‘helix’ (open symbols) CSD melts, χ_U versus T (°C), for representative mutants from Table I, with the exception of Q5A-TC9b (at pH 2.5), all melts were measured at pH 7. The (L7A)-mutant of TC10b illustrates the large deviations between helix and cage melts that can be observed for mutants with significantly destabilized cage structures. In more typical Trp-cage sequences, the helix measure yields χ_U values that are only slightly smaller than the values based on the shifts that measure cage formation.

Fig. 4

Trp-cage NMR structure ensembles: (a) The TC10b structure ensemble (28 structures): heavy atoms are displayed for all residue side chains and the backbone, the hydrophobic cluster residues are shown in color in the version of this figure in the Supplementary data available at PEDS online. (b) An overlay of one member each of the TC10b and TC5b structure ensembles. All atoms are displayed for Tyr³, Trp⁶, Leu⁷, Pro¹², Pro¹⁸ and Pro¹⁹ with only N, CA, C′ shown for the remaining residues.

Fig. 5

(a) CD-monitored pH titration of fold stability for TC5a at 25°C; comparable effects were observed at both the maximum (190 nm) and the classical helix minimum (observed at 223.4 nm in this case). (b) Structure melting monitored at the 223 nm CD minimum; the unfolded baseline assumed for calculating fraction folded values (see Methods) is also given. The systems illustrated, in order of increasing stability, are: L7A-TC10b, TC5a at pH 2.5, TC5a at pH 7 and TC10b.

Fig. 6

Differential melting effects upon destabilization of the Trp-cage: fractional CSDs versus T (°C) for TC10b (squares) and its S14A-(circles) and P19A-mutants (triangles)–(a) the ‘cage’ (dashed lines) and ‘helix’ (solid line, filled symbols) measures of folding, (b) the G11α2 (dashed lines) and G11α3 (solid line, filled symbols) CSDs, (c) the P12δ3 CSDs, TC10b at 7°C is the calibration standard for CSD=1.0 in all panels. The P12δ3 CSDs (upfield) for the less stable systems are larger than those observed for TC10b. In the case of the P19A mutant, the only cage measure available for (a) are the P18α and β3 CSDs.

Fig. 7

Unfolding (melting) scenarios for Trp-cages with different degrees of intrinsic helix stability: (a) Trp-cage formation as Pro^17,18,19 docking onto a stable helix, (b) Trp-cage melting producing an unfolded ensemble retaining a measurable population of a residual hydrophobic cluster, a ‘half-cage’ structure.

Fig. 8

(a) The temperature dependence of the CD spectrum of TC10b at pH 7, traces are shown for every 10° increment from 0°C to 90°C. (b) Melting curves (χ_U versus T) for TC10b and some of its single site-mutants, in order of increasing stability: Y3A-TC10b, TC11b, TC9a and unaltered TC10b. NMR χ_U values from ‘cage CSDs’ as open symbols are superimposed on the CD melts. When the cage is substantially destabilized, illustrated here by Y3A-TC10b, some residual helicity remains after the cage structure melts.