Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 585
Figure 17.13. Time-resolved fluorescence intensity and anisotropy
decay of RNase T 1 in buffered aqueous solution pH 5.5. Excitation at
295 nm. Data from [54].
17.3.2. Annexin V:A Calcium-Sensitive
Single-Tryptophan Protein
Ribonuclease T 1 provided an example of a single-tryptophan
protein that displayed single-exponential intensity and
anisotropy decays (Figure 17.13, bottom). For most proteins,
even with a single-tryptophan residue, the intensity
and anisotropy decay are more complex. One example is
annexin V, which possesses a single-tryptophan residue
(Figure 17.14). Annexins are a class of homologous proteins
that bind to cell membranes in a calcium-dependent
manner. The crystal structure is known to be different with
and without bound calcium. 58 The emission from the singletryptophan
residue is sensitive to calcium. 59 Addition of calcium
results in shift of the emission maximum from 324 to
348 nm (Figure 17.15).
In the absence of calcium the intensity decay is highly
non-exponential (Figure 17.16). Addition of calcium causes
the intensity decay to become more like a single expo-
Figure 17.14. Structure of Annexin V in the absence (top) and presence
(bottom) of Ca 2+ . The calcium atom is shown in red. Revised and
reprinted with permission from [60]. Copyright © 1994, American
Chemical Society.
nential. This dramatic change in calcium suggests the presence
of a nearby quenching group in the calcium-free form.
The crystal structures of annexin V are consistent with this
suggestion because the tryptophan residue moves away
from the protein in the presence of calcium (Figure 17.14).
As was described in Chapter 4 the multi-exponential
model can be used to fit almost any intensity decay, even if
the actual decay has some other functional form. When
examining tables of multi-exponential decay parameters it
is difficult to obtain an intuitive vision of the decays from
the numerical values. The forms of complex intensity
decays can be visualized from the lifetime distributions.