Munich, Jan 20, 2023
Scientists study such dynamic processes and conformational changes with a highly sensitive spectroscopic ruler called Förster resonance energy transfer (FRET). In this process, they read the distance-dependent energy transfer between two organic dyes. The method is so sensitive that scientists can detect distance changes of a single FRET pair on a single biomolecular machine. Often, the fluctuations in the fluorescence signal are so rapid that scientists must rely on statistical analysis of the intensity signal from a single biomolecular machine. The analysis of the fluctuations is known as fluorescence correlation spectroscopy (FCS), but this method only provide the time scale of the conformational changes, not the forward and reverse transition rates. In addition, the interpretation of intensity correlation can be ambiguous because the photophysics of fluorescent dyes can lead to artifacts. To rule these out, additional experiments are often required, involving a large amount of work.
LMU scientists Dr. Tim Schröder, Prof. Don C. Lamb, and Prof. Philip Tinnefeld and their team now report an elegant approach to analyzing intensity fluctuations by exploiting the excited-state lifetime of organic dyes. The change in fluorescence lifetime proved to fingerprint the conformational changes of biomolecules in a FRET experiment. In contrast, perturbing photophysical processes do not change the fluorescence lifetime of organic dyes. Here, the scientists filter the intensity with respect to the fluorescence lifetime and use only subsets of the intensity signal for intensity correlation. In this way, photophysical artifacts of the organic dyes can be easily distinguished and the sought-after signal of the biomolecule can be isolated. "The new method, which we call shrinking gate (sg)-FCS, greatly speeds up the analysis of intensity fluctuations and provides information that was previously difficult to extract. We don't need any prior assumptions or additional experiments and can focus on interpreting the data," says Dr. Tim Schröder, who led the development of the new technique.
The scientists demonstrated the potential of their technique on well-defined model structures before applying it to other projects at the lab. The first application was to isolate the Brownian motion of a biosensor on graphene. Binding of a protein significantly slows this motion, and a binding event is detected. In the second application, they deciphered the mechanism of a new FRET-based membrane charge sensor they developed to study the membrane charge of cells. The charge sensor is anchored to the membrane, and the scientists were able to show that the sensor works because of an equilibrium of temporary attachment and detachment from the membrane. This equilibrium is determined by the surface charge of the membrane. "For more than 20 years, FCS has been analyzed in concert with fluorescence lifetime, but we can improve this interaction today. This is very exciting!" says Prof. Philip Tinnefeld. "Our new general approach offers a wide range of applications and we will work to study fast conformational changes in complex environments like living cells."
Prof. Dr. Philip Tinnefeld
Physikalische Chemie / Nanobiochemie
Butenandtstr. 5 - 13