Formation of compact structure in Cytochrome c in 40 ms.
- Jones et al., Proc. Natl. Acad. Sci. USA 90 (1993) 11860
- Pascher et al., Science 271 (1996) 1558.

For some heme proteins, under carefully chosen solvent conditions, the folded structure only is stable if there is no ligand attached to the heme. A ligand (CO), therefore, leads to the protein being present in the unfolded structure. After photolysis of the ligand, the protein will begin to fold into the native structure, thus the folding process can be triggered on a very short time scale in these proteins. In a similar way, again under carefully chosen solvent conditions, the folded structure is stable only for one redox state of the heme, so that electron transfer from an external donor can induce the folding.

Although these methods are almost ideal for studying fast processes of protein folding, they are limited to only very few proteins and to very special solvent conditions.

Formation of compact structure in apomyoglobin in 3.5 ms.
- Ballew et al., Nat. Struct. Biol. 3 (1996) 9123.

Loss of tertiary contacts in apomyoglobin in 130 ms.
- Gilmanshin et al., Proc. Natl. Acad. Sci. USA 94 (1997) 3709.

For more details on the temperature-jump method, see below.

Formation of partially unfolded state in a few ms.
- Abbruzzetti et al., Biophys. J. 78 (2000) 405.

Helix-coil-transition in homopolymers in 10 ns - 10 ms.
- Schwarz et al., Biopolymers 6 (1968) 1263
- Hammes et al., J. Am. Chem. Soc. 91 (1969), 1812

Various relaxation methods (dielectric relaxation, ultrasonic attenuation) were employed for measuring helix-coil transition times of homopolymers. Unfortunately, these methods do not allow to directly follow the transitions and the time scales which are accessible are rather limited.

Folding of a-helices in cold-denatured apomyoglobin in 250 ns.
- Ballew et al., Nat. Struct. Biol. 3 (1996) 923.

Unfolding of a-helices in apomyoglobin in 50 ns.
- Gilmanshin et al., Proc. Natl. Acad. Sci. USA 94 (1997) 3709.

Unfolding of a-helix in short peptide (suc-F_S 21) in 160 ns.
- Williams et al, Biochemistry 35 (1996) 691.

Unfolding of the end of a-helix in suc-F_S-Peptid in 20 ns.
- Thompson et al., Biochemistry 36 (1997) 9200.

Unfolding of b-sheets in Rnase A in a few ns.
- Phillips et al., Proc. Natl. Acad. Sci. USA 92 (1995) 7292.

Unfolding of b-hairpin in 3 ms.
- Munoz et al., Nature 390 (1997) 196.

Transition from a_II- to a_I-helix in bacteriorhodopsin in 65 ns.
- Wang et al., Biophys. J. 76 (1999) 2777.

Raising the temperature of the solvent above a certain value (the melting temperature) leads to the unfolding of a protein. In a temperature-jump experiment, the solution is kept at a temperature just below the melting temperature. With a short laser flash the temperature of the solution then can be raised to a value above the melting point, thus inducing the unfolding of the protein. From the unfolding dynamics, a lot can be learned about the folding dynamics, which we actually are more interested in. In a few proteins, again under carefully chosen solvent conditions, cold denaturation occurs, i.e. the protein unfolds upon cooling. Here, a temperature jump even can be used to observe folding directly.

The temperature-jump method is a universal method which can be employed for most proteins or peptides and thus is the most commonly used method. Its main disadvantage is the fact that in general it only allows the observation of unfolding.

Folding of a-helices in short peptides slower than a few ns.
- Lu et al., J. Am. Chem. Soc. 119 (1997) 7173
- Volk et al., J. Phys. Chem. B 101 (1997) 8607

In these studies, a synthetic peptide, which in its native state adopts an a-helical conformation, initially is constrained to a less helical cyclic conformation by an aromatic disulfide bond between the peptide ends (click here to see the peptides investigated so far). This bond can be photolyzed by a short UV-laser flash, after which the peptide will begin to relax to its native helical conformation. Thus, helix folding can be followed directly. The use of synthetic peptides also makes it possible to alter the primary sequence and thus investigate helix formation in a detailed way, similar to the planned approach for the Ph.D.-project described here.

In a first investigation, we found that using an aromatic disulfide bond as photochemical trigger suffers from the fast recombination of the disulfide bond which occurs on a similar time scale as helix formation and thus constitutes a competitive process. I have some ideas how to improve on that, but their realization still lies somewhere in the future.