Principles of Fluorescence Spectroscopy

490 TIME-RESOLVED ENERGY TRANSFER AND CONFORMATIONAL DISTRIBUTIONS OF BIOPOLYMERS
14.5. BIOCHEMICAL APPLICATIONS OF
DISTANCE DISTRIBUTIONS
14.5.1. Calcium-Induced Changes in the
Conformation of Troponin C
Troponin C (TnC) is one of several proteins involved in
muscle contraction. The structure of troponin C in solution
is known to be sensitive to calcium. The structure of troponin
C consists of two domains linked by a helical peptide
(Figure 14.15).
Troponin C typically lacks tryptophan residues. This is
fortunate because it allows insertion of tryptophan donors at
any desired location by site-directed mutagenesis. In the
mutant shown in Figure 14.15 a single-tryptophan residue
was placed at position 22 to serve as the donor. As is typical
in the design of mutant proteins, the tryptophan (W) was
a conservative replacement for phenylalanine (F). A uniquely
reactive site for the acceptor was provided by replacing
asparagine 52 with a cysteine residue (N52C). This site was
labeled with IAEDANS.
The upper panel in Figure 14.16 shows the time-dependent
donor decays of the donor-alone and the donor-acceptor
protein in the absence of bound calcium. 8 Without acceptor
the intensity decay of trp-22 is close to a single expo-
Figure 14.15. The crystal structure of turkey skeletal
2Ca–Troponin C.
Figure 14.16. Tryptophan-22 intensity decays of Troponin C without
(D) and with (DA) an IAEDANS on cysteine 52. Top, without Ca 2+ .
Bottom, with Ca 2+ . From [8].
nential. The intensity decay of trp-22 becomes strongly heterogeneous
in the presence of IAEDANS acceptor at position
52. This change in the donor decay was used to recover
the trp-to-IAEDANS distance distribution, which is seen
to be centered near 11 Å and to be about 10 Å wide (Figure
14.17). The close D–A distance and relatively wide distribution
comprise one reason for the strongly heterogeneous
intensity decay. Close inspection of Figure 14.16 shows that
at longer times the intensity decay of the D–A pair is the
same as the donor-only protein. This is due to less than
100% labeling by the acceptor. The extent of acceptor labeling
is claimed to be about 95%. Because the donor is
strongly quenched by the nearby acceptor, even 5% donoralone
protein contributes significantly to the measured
intensity decay. The distance distribution shown in Figure
14.17 was corrected for acceptor underlabeling. The data in
Figure 14.16 illustrates how only a small amount of donoralone
molecules can distort the data. Complete labeling by