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2. nanoDSF Technology
nanoDSF is an advanced Differential Scanning Fluorimetry technology. It detects smallest
changes in the fluorescence of tryptophan and tyrosine residues present in virtually all proteins.
The fluorescence of tryptophans and tyrosines in a protein is strongly dependent on their close
surroundings. When located in the hydrophobic core of proteins, tryptophan is shielded from
the surrounding aqueous solvent. Upon unfolding however, tryptophan is exposed, which
alters its photo-physical properties. By following changes in fluorescence, the unfolding of
proteins can be monitored in real time in a truly label-free fashion (
).
Thermal and chemical unfolding experiments are highly appreciated methods to quantify
protein stability. While thermal unfolding experiments use a temperature ramp to monitor
protein conformational changes, chemical unfolding experiments use chaotropes such as urea
to unfold proteins. Both types of experiments are easily done with the Prometheus Series
instruments.
Figu
The unique dual-UV technology of the Prometheus Series monitors intrinsic tryptophan and
tyrosine fluorescence at the emission wavelengths of 330 nm and 350 nm. To generate an
unfolding curve, either the fluorescence change in one of the two channels, or, alternatively,
the ratio of the fluorescence intensities (F350 nm/F330 nm), is plotted against temperature or
concentration of denaturant (see
). The fluorescence ratio monitors changes in
fluorescence intensity as well as the tryptophan-specific shift of the fluorescence emission
maximum. It is therefore extremely robust and allows the investigation of virtually all buffer
conditions and even fluorescent additives.
Figure 2:
Schematic representation of protein unfolding.
Charged, solvent exposed regions of the protein are colored in
red and blue, the hydrophobic core in gray. Tryptophan residues
are represented as green sticks. The illustration shows their
increasing exposure to the solution upon unfolding. Below, the
respective curve shows the transition from folded to unfolded
protein as a function of temperature or the concentration of a
chemical denaturant.
