Characterization of the internal motions of a chimeric protein by 13C NMR highlights the important dynamic consequences of the engineering on a millisecond time scale

Abstract

By transferring the central curaremimetic β hairpin of the snake toxin α into the scaffold of the scorpion charybdotoxin, a chimeric protein was constructed that reproduced the three-dimensional structure and partially reproduced the function of the parent β hairpin, without perturbing the three-dimensional structure of the scaffold [1]. Picosecond to hour time scale motions of charybdotoxin and the engineered protein were observed, in order to evaluate the dynamic consequences of the six deletions and eight mutations differentiating the two molecules. The chimeric protein dynamics were also compared to that of toxin α, in order to examine the β hairpin motions in both structural contexts. Thus, 13C R1, R(1ρ) and 1H→13C nOe were measured for all the C(α)H(α) and threonine C(β)H(β) vectors. As the proteins were not labeled, accordion techniques combined to coherence selection by pulsed field gradients and preservation of magnetization following equivalent pathways were used to considerably reduce the spectrometer time needed. On one hand, we observed that the chimeric protein and charybdotoxin are subjected to similar picosecond to nanosecond time scale motions except around the modified β sheet region. The chimeric protein also exhibits an additional millisecond time scale motion on its whole sequence, and its β structure is less stable on a minute to hour time scale. On the other hand, when the β hairpin dynamics is compared in two different structural contexts, i.e. in the chimeric protein and the curaremimetic toxin α, the picosecond to nanosecond time scale motions are fairly conserved. However, the microsecond to millisecond time scale motions are different on most of the β hairpin sequence, and the β sheet seems more stable in toxin α than in the chimera. The slower microsecond to hour time scale motions seem to be extremely sensitive to the structural context, and thus poorly transferred from one protein to another.