Ultra-fast and extra-long: a novel laser T-jump apparatus for time-resolved spectroscopy of conformational dynamics of proteins.
I.Chizhov and D.J. Manstein
Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany
Development of the Laser T-jump experimental method with two infra-red lasers. One laser generates in the aqueous solution the rise of temperature within 10 nanoseconds and the second laser sustains the high temperature up to 10 seconds or longer. This innovation expands the range of the applicability of method. Relaxation techniques such as a pressure-jump, temperature-jump and flash photolysis are powerful tools for the kinetic study of ligand binding and conformational changes of proteins and nucleic acids. We developed a nanosecond T-jump technique that uses two infra-red (IR) lasers. The first laser pulse of 5 ns duration induces a temperature rise in an aqueous solution of 2 μl volume. A so-called Raman laser with a wavelength of 1.9 μm and ca. 100 mJ energy is used for this initial pulse to achieve a 10°C rise in temperature. The heat from this initial temperature jump starts to dissipate significantly after approximately 10 ms due to the heat transfer to the quartz cover glasses that form most of the reaction chamber surface. To avoid this, we use a second IR laser to keep the temperature constant. A continuous wave (cw) Thulium Fiber laser (TLR-15-1940; IPG Laser GmbH) that generates up to 15 W of power at 1.94 μm wavelength is used for this purpose. This laser alone can heat the sample to 100oC within less than 100 ms. Combining both laser systems and using specially developed computer algorithms for modulation of the Thulium laser’s power, we have achieved the flat temporal profile of following the initial T-jump over nine and more time decades, i.e. from 10 ns to 10 seconds or longer. T-jump profiles were calibrated using a temperature sensitive dye Phenol Red. In Figure 1 the transient absorption of Phenol Red at 560 nm is shown at different T-jump experiments. Note that the vertical axis is scaled to the temperature changes. The observed rise of the Phenol Red signal with a characteristic time of 100 ns matches the rate-limiting step of the temperature-induced deprotonation of the dye. The experimentally obtained temporal profile of the Raman T-jump is depicted as a red line. The blue line shows the T-jump profile when only the Thulium laser is used. In the case of the profile shown, the Thulium laser was switched on for 20 ms with the power set to 15 W followed by a gradual reduction to a final power of about 0.1 W. Power was completely switched off after approximately 9 s. As in the case of the Raman laser the initial rise of temperature can be easily modulated to achieve a higher or lower plateau temperature. Once the plateau temperature has been reached, the time over which the temperature is kept constant can be extended by modifying the computer algorithm that controls the power modulation of the Thulium laser. An example of temperature dependent conformational equilibrium obtained in the Bacteriorhodopsin photocycle illustrates the method in figure 2. The T-jump system described here will be of great use in the fields of Protein Science, Biotechnology, Material Science, Nanotechnology and Chemistry. The temporal overlap between the time domain that can be experimentally observed using our T-jump system and advanced computational approaches that are used to simulate macromolecule dynamics is of particular importance.
Figure 1. T-jump profiles induced either by Raman (red), Thulium (blue) or both lasers (magenta). Note the logarithmic scale of time. Dots: Temperature values are derived from the transient absorption of Phenol Red at 560 nm, lines: data fit.
Figure 2. T-jump experiments provide access to the full kinetic and thermodynamic information of the reaction. In this example, the B state of the molecule has a 80 kJ mol-1 higher enthalpy and 260 J mol-1 K-1 higher entropy than the A state. The rise of temperature shifts the equilibrium in favor of B. The steady state temperature titration (red and blue lines) shows the thermodynamic potentials. The T-jump experiment provides information about the re-equilibration kinetics, i.e. the rate constants of forward and back reactions and the enthalpy and entropy values of the activation barrier.