Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 553
Figure 16.41. Relative changes in the fluorescence intensity of Trp 99
(!), Trp 135 (Q), Trp 26 (∆), and Trp 62 (O) in single-tryptophan
mutants of calmodulin with changes in the amount of bound calcium.
The arrows show the location of the tryptophan residues and the
orange dots are calcium atoms. Reprinted with permission from [121].
Copyright © 1992, American Chemical Society.
DNA is tightly packed in chromatin, but DNA replication
occurs rapidly nonetheless. Hence it is of interest to
determine the rates of DNA folding around the SSB. This
can be accomplished using rapid mixing stopped-flow
measurements, while monitoring the intrinsic tryptophan
fluorescence of SSB (Figure 16.43). Upon mixing with
(dT) 70 there is a rapid decrease in the tryptophan emission.
128 By studies with various concentrations of DNA and
different lengths of DNA it was possible to determine the
rate constants for initial DNA binding and subsequent folding
of DNA around the protein. The rates of DNA folding
Figure 16.42. Ribbon structure for the E. coli single-stranded binding
protein (SSB) with a bound 70-mer. Figure from [128] and provided
by Dr. Alexander Kozlov and Dr. T. Lohman, Washington University,
MO.
around the protein were also measured using RET between
DNA oligomers labeled with Cy5 and Cy3 at the 5' and 3'
ends, respectively. These probes were brought into closer
contact when DNA wrapped around SSB (not shown). The
changes in RET and intrinsic protein fluorescence occurred
on the same timescale, indicating that wrapping of DNA
around the protein proceeds very rapidly after binding
occurs.
The intensity changes can be used to determine the
fraction of the total emission that is quenched by DNA. The
Myb oncoprotein is associated with chromatin and appears
to function as a regulator of gene expression. The N-terminal
region of this protein, which contains the DNA binding
region, consists of three domains: R 1 , R 2 , and R 3 . The subscripts
refer to the domain number, not the number of
domains. Each domain contains three highly conserved
tryptophan residues, which suggests these residues are
involved in binding to DNA.
The intrinsic protein fluorescence of the R 2 R 3 fragment
of the Myb oncoprotein is partially quenched by DNA. 129