The information obtained by studying fluorescence decay of labeled biopolymers is a major resource for understanding the dynamics of their conformations and interactions. The lifetime of the excited states of probes attached to macromolecules is in the nanosecond time regime, and hence, a series of snapshot decay curves of such probes might - in principle - yield details of fast changes of ensembles of labeled molecules down to sub-microsecond time resolution. Hence, a major current challenge is the development of instruments for the low noise detection of fluorescence decay curves within the shortest possible time intervals. Here, we report the development of an instrument, picosecond double kinetics apparatus, that enables recording of multiple fluorescence decay curves with picosecond excitation pulses over wide spectral range during microsecond data collection for each curve. The design is based on recording and averaging multiphoton pulses of fluorescence decay using a fast 13 GHz oscilloscope during microsecond time intervals at selected time points over the course of a chemical reaction or conformational transition. We tested this instrument in a double kinetics experiment using reference probes (N-acetyl-tryptophanamide). Very low stochastic noise level was attained, and reliable multi-parameter analysis such as derivation of distance distributions from time resolved FRET (fluorescence resonance excitation energy transfer) measurements was achieved. The advantage of the pulse recording and averaging approach used here relative to double kinetics methods based on the established time correlated single photon counting method, is that in the pulse recording approach, averaging of substantially fewer kinetic experiments is sufficient for obtaining the data. This results in a major reduction in the consumption of labeled samples, which in many cases, enables the performance of important experiments that were not previously feasible.
For the processing of fluorescence decay data it is often necessary to obtain the time profile of the flash lamp which excites the fluorescence as well as the variation with time of the fluorescence intensity. If these intensity profiles are collected in consecutive experiments, instrumental drift may introduce serious errors. This problem is shown to be minimized by alternate collection of the data for the fluorescence and for the excitation lamp. An automatic setup for performing such experiments is outlined.
The extremely fast and efficient folding transition (in seconds) of globular proteins led to the search for some unifying principles embedded in the physics of the folding polypeptides. Most of the proposed mechanisms highlight the role of local interactions that stabilize secondary structure elements or a folding nucleus as the starting point of the folding pathways, i.e., a "bottom-up" mechanism. Nonlocal interactions were assumed either to stabilize the nucleus or lead to the later steps of coalescence of the secondary structure elements. An alternative mechanism was proposed, an "up-down" mechanism in which it was assumed that folding starts with the formation of very few non-local interactions which form closed long loops at the initiation of folding. The possible biological advantage of this mechanism, the "loop hypothesis", is that the hydrophobic collapse is associated with ordered compactization which reduces the chance for degradation and misfolding. In the present review the experiments, simulations and theoretical consideration that either directly or indirectly support this mechanism are summarized. It is argued that experiments monitoring the time-dependent development of the formation of specifically targeted early-formed sub-domain structural elements, either long loops or secondary structure elements, are necessary. This can be achieved by the timeresolved FRET-based "double kinetics" method in combination with mutational studies. Yet, attempts to improve the time resolution of the folding initiation should be extended down to the sub-microsecond time regime in order to design experiments that would resolve the classes of proteins which first fold by local or non-local interactions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.