Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 559
Figure 16.54. Emission spectra of the trp-92 mutant of RNase A in the
native and denatured state at pH 5. Excitation at 280 nm. Revised from
[158].
residue inserted at position 92 would be quenched by the
nearby carboxyl group in the folded state.
Emission spectra of RNase A with the W92 insertion
are shown in Figure 16.54. The excitation wavelength was
280 nm, so that both tyrosine and tryptophan are excited.
The surprising feature of these spectra is that the relative
tyrosine contribution is highest in the native protein. This is
Figure 16.55. Positions of tryptophan probes in the lactate dehydrogenase
subunit from B. stearothermophilus. The backbone of the protein
is shown as a ribbon, and the positions of each single change of
tyrosine to tryptophan are indicated by the residue number. Reprinted
with permission from [149]. Copyright © 1991, American Chemical
Society.
the opposite of what is observed for most proteins. This
unusual result occurs because W92 is quenched in the folded
state by the nearby aspartate residue. The large amount
of quenching by D38 suggests the carboxyl group is in the
protonated form, which is known to quench tryptophan.
16.9.2. Folding of Lactate Dehydrogenase
In order to determine the folding pathway it is important to
examine multiple positions in a protein. 149,159 This is possible
using mutant proteins. One example is the folding–
unfolding transition of lactate dehydrogenase from Bacillus
stearothermophilus. This protein typically contains three
tryptophans that were replaced by tyrosines. Nine singletryptophan
mutants were produced with the residues dispersed
throughout the protein matrix (Figure 16.55). These
mutant proteins were studied in increasing concentrations
of guanidium hydrochloride. Three of the nine unfolding
curves are shown (Figure 16.56). The unfolding transitions
occur at different denaturant concentrations for each of the
Figure 16.56. Equilibrium unfolding of a single-tryptophan mutants
of lactate dehydrogenase monitored by tryptophan fluorescence intensity.
Tryptophan fluorescence was excited at 295 nm and measured at
345 nm. Revised and reprinted with permission from [149]. Copyright
© 1991, American Chemical Society.