Protein Conformation

The fact that proteins with a single tryptophan generally exhibit multi-exponential decay is well known. To what degree the components from a
multi-exponential fit actually represent distinct structures the protein can adapt is still an open question. In a collaborative project with the
University of Minnesota - Duluth, we have generated data over a range of temperatures for the C2A domain of the protein synaptotagmin. The
fluorescence lifetime of this molecule is one of the highest known, approaching 10 ns. The protein is also very resistant to aggregation upon
melting.

Figure 1 shows a two-component analysis of the fluorescence decay curve at 20 C. Fluorescence Innovations tunable UV source was used to
excite at 295 nm. The acquisition time per waveform was 4 seconds and the data were collected at magic angle. The long component accounts
for approximately 93% of the total fluorescence intensity.

Figure 2 illustrates the outstanding quality of the two-component fit. The residuals indicate less than a 0.2% difference between the
observed and fitted decay curves at peak intensity.

The fluorescence decay of synaptotagmin C2A was measured at 5-degree intervals from 5 C to 90 C. The lifetime shows a sharp
decrease at around 60 C as the protein melts, i.e., goes from a folded to a denatured form. The mole fraction averaged lifetime as a
function of temperature is shown in Figure 3. Up to 50 C, the lifetime of the long component drops linearly with temperature, whereas

the short component lifetime changes very little. The mole
fractions (amplitudes in the fit) of the two components are
nearly unchanged in the 5 to 50 C range. As the melting
begins, the mole fraction of the long component drops
relative to the mole fraction of the shorter component and
additional components are needed to get a good fit. Fully
understanding the molecular implications of these
phenomena is a significant research endeavor, but it is
clear the speed and precision of the Fluorescence
Innovations's UV lifetime technology makes these kinds of
experiments easy to conduct.

Using the NovaFluor PR Fluorescence Lifetime Plate Reader
to automate the process described here, one can measure
protein conformation on as many as 5 samples per second
using microgram quantities of each protein. Contact us to
learn more.