Principles of Fluorescence Spectroscopy

456 ENERGY TRANSFER
Figure 13.16. Emission spectra of IEDANS-labeled GroEL and fluorescein-labeled
GroES without (top) and with (bottom) ATP. Revised
from [42].
Figure 13.17. Dissociation of doubly labeled GroEL–GroES. Revised
from [42].
unlabeled GroES associates with labeled GroEL the donor
intensity increases and the acceptor intensity decreases.
This result shows that information about binding can be
obtained using either the donor or acceptor emission.
13.4.4. Orientation of a Protein-Bound Peptide
In the presence of calcium, calmodulin is known to interact
with a number of proteins and peptides. For example, a peptide
from myosin light-chain kinase (MLCK) binds to
calmodulin. 43 Such peptides are known to bind in the cleft
between the two domains of calmodulin (Figure 13.18).
When bound to calmodulin the MLCK peptide was known
to adopt a α-helical conformation. However, the direction
of peptide binding to calmodulin was not known.
Information on the direction of binding was obtained
by studying of four similar peptides, each containing a single-tryptophan
residue that served as the donor (Figure
13.19). Calmodulin typically contains only tyrosine
residues, so an intrinsic acceptor was not available. This
problem was solved by using calmodulin from spinach,
which contains a single-cysteine residue at position 26. This
residue was labeled with 1,5-IAEDANS, which contains a
thiol-reactive iodoacetyl group. The Förster distances for
trp-to-AEDANS energy transfer ranged from 21 to 24 Å.
Figure 13.18. Structure of spinach calmodulin. The acceptor is
AEDANS on cysteine-26 of calmodulin. The tryptophan donor is on
the myosin light chain kinase peptides shown in Figure 13.19. Revised
and reprinted with permission from [43]. Copyright © 1992,
American Chemical Society.