The experimental approach which is used in this project for measuring fast dynamics of protein folding essentially consists of two parts:

• Laser-induced temperature jump as the fast trigger event and
• time-resolved IR-spectroscopy for detecting changes in the peptides' secondary structure.

I will give a brief description of the relevant components below. Obviously, these time-resolved measurements will be supported by the usual range of analytic methods, in particular absorbance spectroscopy and temperature-dependent FTIR-measurements.

For raising the temperature of water in the reaction volume (typically 0.5 x 0.5 x 0.1 mm³) by 5-10^oC, it is sufficient to absorb less than 1 mJ of light energy into the reaction volume. Ideally, one may use IR-light at wavelengths of >1.6 mm, which is directly absorbed by weak overtone vibrational bands of water itself. However, it is a bit complicated to make short laser pulses at these wavelengths, whereas it is much easier to make laser pulses in the UV or visible. To induce a temperature jump with UV or visible laser pulses, one adds a dye to the peptide solution which undergoes fast internal conversion, i.e. relaxes from its photo-excited state to the ground state without the emission of a photon, but converts all of the photon energy to vibrational energy. Initially, this energy is localised in the dye molecule, but vibrational relaxation and heat diffusion are sufficiently fast to reach a uniform temperature distribution within 100 ps, given typical parameters for a temperature jump experiment (heating a volume of 0.5 x 0.5 x 0.1 mm³ by 5 - 10^oC). This is much faster than the dynamics of secondary structure formation, which occurs on the time scale of 10s of nanoseconds or slower (see previous experiments). As a matter of fact, it also is much faster than the pulse widths of the lasers used in our lab, so there certainly is nothing to worry about.

In our lab, we make use of an excimer laser, which yields UV laser flashes of sufficient energy with pulse widths of approx. 10 ns. This can be used to directly excite the dye, alternatively we may have to use it to pump a dye laser running at 600 nm, where the main absorption band of the dye is located. [There are some advantages and disadvantages for either method and I haven't decided yet which method to use finally - maybe we'll just have to try both of them and see which one works better.] The light then is focused into the sample, absorbed by the dye, converted to vibrational energy and thus induces the temperature jump. Once the temperature is raised, it remains at the higher value until the excess energy diffuses away, which occurs typically within 100 ms.

For measuring the IR-absorption of the peptides (in the amide I band), which changes upon the formation or melting of secondary structure, we will use a cw IR-laser diode, i.e. a laser diode which continuously emits IR-light at a certain wavelength. This light is focused into the reaction volume (where the excimer/dye laser light induces the temperature jump) and measured behind the sample using a fast IR-sensitive detector. The signal of this detector then is amplified and displayed and digitized with a so-called digitizing oscilloscope. [Digitizing the signal allows one to read the data into a computer and average over many single measurements.]

The time-resolution of these measurements is determined by the speed of IR-detector, amplifier and oscilloscope. Typically a resolution of 20 ns can be achieved nowadays. Again, this is sufficient for the observation of secondary structure dynamics.

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