Tryptophan rotamers as evidenced by X-ray, fluorescence lifetimes, and molecular dynamics modeling

Abstract

We investigated the native-state dynamics of the Bacillus caldolyticus cold-shock protein mutant Bc-Csp L66E, using fluorescence and appropriate molecular dynamics methods. Two fluorescence lifetimes were found, the amplitudes of which agree very well with tryptophan rotamer populations, obtained from parallel tempering calculations. Rotamer lifetimes were predicted by transition-state theory from high-temperature simulations. Transition pathways were extracted from the transition rates between individual rotameric states. The molecular dynamics also reveal the loop fluctuations in the native state.

FIGURE 1

Energy contour maps in units of kJ mol⁻¹. (a) Free energy as a function of Q and bRMSD, from PT. The highest bRMSD well corresponds with a loss of the 3₁₀-helix structure. (b) Potential energy in function of Trp⁸ χ1 and χ2, from DEE. The energies are calculated as the total nonbonded energies E_totnb for all structures with a negative E_totnb. Free energy in function of Trp⁸ χ1 and χ2 from (c) PT and (d) 500 K MD. The relative free energy ΔG between two states i and j with relative populations P_i and P_j is calculated as , where R denotes the universal gas constant.

FIGURE 2

Superposition of 15 native-state structures of 1HZB from PT. The structures were carefully chosen to represent the occupied conformational space. The color scheme is as follows: 3₁₀-helix (mauve), extended β-strand (yellow), isolated β-bridge (orange), hydrogen-bonded turn (cyan), and coil (white). To these ribbon models were added in ball-and-stick models the side chains of (a) Trp⁸ in the p−90° state, and (b) 118 Arg⁵⁶ conformations.

FIGURE 3

(a) bRMS fluctuation (bars) and total RMS fluctuation (dotted line) as a function of the residue number, from PT. The black bars indicate β-sheet residues. (b and c) Secondary-structure content in function of residue number for states belonging to (b) the low-bRMSD free-energy well and (c) the high-bRMSD free-energy well (see Fig. 1 a). Curves represent the 3₁₀-helix (solid lines), isolated β-bridge (dashed lines), and extended β-strand (dotted lines).

FIGURE 4

Eyring plots from the high-temperature dynamics for the three most stable rotamers, based on the total transition rate constants k_tot,i (T). p−90° (○), t80° (⋄), and p80° (□).

FIGURE 5

χ1 × χ2 map of the six stable rotamers and a schematic representation of the observed transitions at 500 K, with transition rates given in ns⁻¹. The dashed arrows indicate the reaction path between p−90° and t80°. The contour lines enclose 95% of the population of each rotamer.

FIGURE 6

Tryptophan rotamers in Bc-Csp 1HZB. Superposition of the six Trp⁸ rotamers in their ideal positions and the surface-exposed residues within a 5-Å radius of Trp⁸. (a) m−90°, p−90°, and t−90°. (b) m90°, p90°, and t90°.