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 b hairpin of the snake toxin a 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 b 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 a, in order to examine the b hairpin motions in both structural contexts. Thus, 13 C R 1 , R 1r and 1 H3 13 C nOe were measured for all the C a H a and threonine C b H b 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 b sheet region. The chimeric protein also exhibits an additional millisecond time scale motion on its whole sequence, and its b structure is less stable on a minute to hour time scale. On the other hand, when the b hairpin dynamics is compared in two different structural contexts, i.e. in the chimeric protein and the curaremimetic toxin a, the picosecond to nanosecond time scale motions are fairly conserved. However, the microsecond to millisecond time scale motions are different on most of the b hairpin sequence, and the b sheet seems more stable in toxin a 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.

Motions and structural variability within toxins: Implication for their use as scaffolds for protein engineering

“…Order parameters reflecting the amplitudes of the ps to ns time‐scale motions were calculated from the 10‐ns trajectory (see Materials and Methods), and were compared to experimental values determined from NMR relaxation data (Wolff et al 2000). The experimental and simulated values are in agreement (data not shown).…”

“…By now, engineering of the scorpion toxin scaffold was based on the introduction of ponctual mutations (Vita et al 1995, 1999; Mer et al 1998). Suppression of the first six amino acids of charybdotoxin, in conjunction with mutations of 8 residues in the β‐sheet, was shown to create important msec time‐scale motions on the whole protein, thus deeply modifying the energy landscape of the toxin (Wolff et al 2000).…”

“…5B (2) and 5B (3) ). From the NMR data, very few residues exhibit μsec‐msec time‐scale motions in charybdotoxin: Only three residues—Ser10 (N‐terminus of the helix), Cys17, and Gln 18 (C‐terminus of the helix)— present fast exchange motions (10–100 μsec), and four residues dispersed in the sequence show slower time scale motions (>250 μsec) (Wolff et al 2000). The small number of residues exhibiting μsec to msec time‐scale motions in charybdotoxin might be correlated with the low structural variability of the short α/β scorpion.…”

Low-Frequency Cooperative Dynamics in l-, d-, and dl-Alanine Crystals: A 13C and 15N Cross-Polarization Magic-Angle-Spinning NMR Study

“…Proteins display a hierarchy of dynamic processes that are characterized by a wide range of time scales, typically ranging from seconds to picoseconds. Slow time scale motions in the nano- to millisecond range are of particular interest because biologically important processes such as enzyme catalysis, signal transduction, and ligand binding are expected to occur on this time scale. , Low-frequency processes are also known to exist in structurally simpler amino acid crystals and are expected to be sensitive to the long-range intermolecular interaction. However, a systematic comparative study of these processes in d -, l -, and dl -amino acid crystals, including their dependence on temperature and molecular packing density, has not been attempted to date.…”