Fretting about FRET: correlation between kappa and R

Abstract

Molecular dynamics simulations were used to examine the structural dynamics of two fluorescent probes attached to a typical protein, hen egg-white lysozyme (HEWL). The donor probe (D) was attached via a succinimide group, consistent with the commonly-used maleimide conjugation chemistry, and the acceptor probe (A) was bound into the protein as occurs naturally for HEWL and the dye Eosin Y. The <kappa(2)> is found to deviate significantly from the theoretical value and high correlation between the orientation factor kappa and the distance R is observed. The correlation is quantified using several possible fixed A orientations and correlation as high as 0.80 is found between kappa and R and as high as 0.68 between kappa(2) and R. The presence of this correlation highlights the fact that essentially all fluorescence-detected resonance energy transfer studies have assumed that kappa and R are independent--an assumption that is clearly not justified in the system studied here. The correlation results in the quantities <kappa(2)R(-)(6)> and <kappa(2)> < R(-)(6)> differing by a factor of 1.6. The observed correlation between kappa and R is caused by the succinimide linkage between the D and HEWL, which is found to be relatively inflexible.

FIGURE 1

Structures of the simulation system from three different snapshots. Each is labeled according to the probe orientation family it represents and its time. The HEWL protein is shown with gray bonds. The DACM donor and eosin acceptor dyes are shown with purple and orange Van der Waals spheres, respectively. In the family C structure, DACM interacts closely with Val-109, Arg-112, and Asn-113. Images generated using VMD (84).

FIGURE 2

Chemical structures showing the DACM dye, succinimide linkage, and cysteine side chain. The five dihedral angles discussed in the text are indicated with numbers near the central bond. Rotational freedom of these dihedral angles are shown in Fig. 8.

FIGURE 3

Two-dimensional RMSD plot for the HEWL protein (dyes were not included). Snapshots were taken every 200 ps and the RMSD was measured using all atoms. The vertical scale units are Å. Low RMSD values off-diagonal indicate structures that are similar to each other. Squares of low RMSD along the diagonal (e.g., 0–6 ns) indicate time periods when the protein structure fluctuated about some average position, but remained roughly the same. Junctions between these squares (e.g., ≈6 ns) indicate transitions from one structural family to another.

FIGURE 4

Trajectories of the distance between the D and A and of the κ factors for the three different A representations. Transitions between different probe orientation families were identified from these data. Transition times are given in Table 1.

FIGURE 5

Histograms showing the distributions of κ (upper panel) and R_DA (lower panel) values for four of the eight probe orientation families. The distributions of all eight families are given in Supplementary Material Figs. S3 and S4. These four families illustrate the familial parallels in κ and R_DA behavior.

FIGURE 6

Scatter plot of R_DA against κ showing the correlation between them. Each dot represents a single snapshot; snapshots were taken every 2 ps of the simulation.

FIGURE 7

Trajectories of κ (upper line) and R_DA (lower line), showing the high degree of correlation between them. The main plot shows correlation over the full length of the trajectory while the inset highlights the short timescale correlation.

FIGURE 8

Histograms of the five dihedral angles that are critical to motion of the DACM probe. The identities of the angles are shown in Fig. 2. Dihedrals 1, 2, and 4 predominantly occupy just one orientation, while dihedral-3 mainly occupies two orientations. Only dihedral-5 samples significantly from three orientations.