Folding without charges

Abstract

Surface charges of proteins have in several cases been found to function as "structural gatekeepers," which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the charge-depleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.

Fig. 1.

Charge removal of S6. A. Wild-type S6^+17-17 comprises 16 negative (red) and 16 positive (blue) charges on the protein surface. B. Supercharged S6^+1-17 was produced by mutation of all K and R to S. C. Charge-depleted S6⁺¹ was finally obtained by transferring S6^+1-17 to pH 2.3 where all negative side-chain charges as well as the C-terminal becomes neutralized by protonation. D. Positions of positively- (blue) and negatively (red) charged side chains in the S6 sequence.

Fig. 2.

NMR HSQC spectra of the different charge variants of S6. All spectra show wild-type like dispersion, suggesting that the supercharged S6^+1-17 and the charge-depleted S6⁺¹ maintain fixed, three-dimensional structures. Additional NMR evidence for conserved structure of S6^+1-17 is presented in Fig. S2, whereas detailed structural analysis of S6⁺¹ has so far been precluded by aggregation on longer timescales (cf. Fig. 5).

Fig. 5.

Charge depletion leads to S6 aggregation. A. The three charge variants of S6 were incubated at 11 μM protein concentration for 48 h and then centrifuged at 17,000  × g for 2 h. SDS-PAGE gels show that wild-type S6^+17-17 and supercharged S6^+1-17 remain soluble in the supernatant, whereas the charge-depleted S6⁺¹ aggregates go into the pellet. The different densities of the bands are due to weakened interaction between the SYPRO Orange stain and the supercharged S6^+1-17. During the electrophoresis at pH 6.8, charge-depleted S6 deprotonates and becomes supercharged S6^+1-17. B. Photograph of S6 samples showing aggregation as increased scatter upon flash illumination. Charge-depleted S6⁺¹ aggregates both in its folded and unfolded states at high concentration of denaturant.

Fig. 3.

The folding kinetics of the different charge variants of S6 analyzed by stopped-flow mixing. A. The chevron plots of wild-type S6^+17-17, supercharged S6^+1-17 and charge-depleted S6⁺¹ are all classically v-shaped, showing that the folding transition remains cooperative and does not rely on side-chain charges. Of particular interest is that complete charge removal even speeds-up folding: at the transition midpoint S6⁺¹ folds > 300 times faster than wild-type S6^+17-17. B. Na₂SO₄ titration of the refolding reaction at 0.4 M GdmCl shows that the propensity of the coil to undergo premature collapse in the mixing dead-time slightly increases upon removal of all positively charged side chains. C. At 1.6 M GdmCl, it is seen that charge-depleted S6⁺¹ has the highest collapse propensity of the three proteins and also displays a change of rate-limiting step at high [Na₂SO₄] (). The origin of this change could be the population of an alternative, parallel, folding route to the native state according to Scheme 2.

Scheme 2.

Fig. 4.

Folding free-energy profiles of wild-type S6^+17-17 (blue), supercharged S6^+1-17 (red), and charge-depleted S6⁺¹ (black). Barrier heights were calculated from k_f and a prefactor of 10⁶s^-1 (28). Charge removal leads to faster folding kinetics and an apparent stabilization of the transition-state ensemble (‡), whereas the native state is destabilized. The positions of D, ‡ and N along the progress coordinate have been scaled according to the m-values in Table 1 and normalized to N.

Scheme 1.