Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 457
Figure 13.19. Emission spectra of the MLCK peptides when free in
solution and when bound to AEDANS-calmodulin. The upper and
lower spectra correspond to the peptide emission spectra in the
absence and presence of AEDANS-calmodulin, respectively. The peptide
sequences are shown in the top panel, and the calculated distance
shown with the spectra. Excitation at 295 nm. Revised and reprinted
with permission from [43]. Copyright © 1992, American Chemical
Society.
Emission spectra of the tryptophan-containing peptides
are shown in Figure 13.19. The excitation wavelength was
295 nm to avoid excitation of the tyrosine residues in
calmodulin. Upon binding of AEDANS–calmodulin the
tryptophan emission of each peptide is quenched. One of
the peptides showed a transfer efficiency of 54%, and the
remaining three peptides showed efficiencies ranging from
5 to 16%. These results demonstrated that the C-terminal
region of the peptides bound closely to the N-terminal
domain of calmodulin, and illustrate how structural information
can be obtained by comparative studies of analogous
structures.
13.4.5. Protein Binding to Semiconductor
Nanoparticles
Semiconductor nanoparticles, or Quantum Dots (QDs),
have become widely used as fluorescent probes. They can
have high quantum yields, narrow emission spectra, and
good photostability (Chapter 3). Understanding the interactions
of Quantum Dots with proteins is important for their
use as intracellular probes and for making the surfaces
functional. RET has been used to determine the interaction
of the E. coli maltose-binding protein (MBP) when bound
to a QD. 44
The orientation of MBP when bound to a QD was
determined using six single-cysteine mutants of MBP. The
individual proteins were labeled with Rhodamine Red (RR)
at the sites shown in the top panel of Figure 13.20. The QDs
were then titrated with the RR-labeled MBP. The donor was
the QDs with a diameter near 60 Å and emitting at 555 nm.
Binding of RR–MBP resulted in quenched QD emission
and increased RR emission (middle panels). For some of
the MBP mutants strong donor quenching was observed, for
Figure 13.20. Top: E. coli malliose-binding protein shows six sites
where Rhodamine Red was bound. Middle: Emission spectra of the
QDs titrated with two RR-MBP. Bottom: Orientation of bound MBP
relative to the center of the QD (pink dot). Revised from [44].