Principles of Fluorescence Spectroscopy

494 TIME-RESOLVED ENERGY TRANSFER AND CONFORMATIONAL DISTRIBUTIONS OF BIOPOLYMERS
Figure 14.20. Intensity decays and distance distributions in a DNA
Holliday junction. Data revised and reprinted with permission from
[39]. Copyright © 1993, American Chemical Society.
Figure 14.21. Top: Structure of PGK showing the sites of cysteine
insertion. Bottom: Distance distributions recovered between various
sites on PGK. Revised and reprinted with permission from [43].
Copyright © 1997, American Chemical Society.
(Figure 14.20). This expectation is confirmed from the
recovered distance distributions. There is a narrow distance
distribution between one pair of ends and a wide distribution
between the other pair of ends (Figure 14.20). Results
from other laboratories have suggested somewhat different
structures. 40 Nonetheless, these data for a complex macromolecular
assembly illustrate how resolution of distance
distributions from the time-resolved energy transfer data
has become a widely used tool in structural biology. These
results on the ribozyme and Holliday junction represent just
two examples of a number of studies of distance distribution
in nucleic acids and DNA structure (see Representative
Publications on Measurement of Distance Distributions).
14.5.4. Distance Distributions and Unfolding of
Yeast Phosphoglycerate Kinase
Time-resolved RET was used to measure the distance distributions
between a number of sites on phosphoglycerate
kinase (PGK), which consists of a single peptide chain with
two domains (Figure 14.21). 42–43 A number of single- and
double-cysteine mutants were made to provide sites for
Figure 14.22. Top: Emission spectra of D–PGK–A, 135–290, in 0 and
2 M GuHCl. Bottom: Time-resolved decay of donor D-PGK and
donor–acceptor D–PGK–A, 135–290. Revised and reprinted with permission
from [43]. Copyright © 1997, American Chemical Society.