The more we learn about complex processes in cells and organisms the more we are astonished that they can exist quite stable over long periods of time. This is partly due to the fact that evolution has developed in cells alternative ways in case of emergency. However, for many diseases it was discovered that dysfunction of a single protein is lethal. The dysfunction of a protein is caused by a change in its conformation or its dynamics. It should be noticed that the amino acid sequence not only determines the conformation of proteins but also its dynamics. The notion dynamics contains two contributions. In most cases the working cycle of a protein starts by an external stimulus like the binding of a ligand or the absorption of a photon. Evolution has created one or several ways within the protein how it is passing from this excited state via several intermediates back to its resting state. These processes are one type of the protein dynamics, which is imprinted in the system by to the amino acid sequence together with the cellular environment. Intermediate states are separated by energy barriers which normally can be overcome by thermal equilibrium fluctuations. These fluctuations are the second type of dynamics also imprinted in the protein by the amino acid sequence and the environment. It was clearly shown that different domains of a protein have different amplitudes and frequencies of these fluctuations. The decay times of intermediate states in the functional cycle of a protein are often slowed down with decreasing temperatures caused by decreasing amplitudes of the equilibrium fluctuations. In order to understand a protein in depth, the research goal of the Dept. of Molecular Biophysics I (work group Büldt) is to understand and investigate these aspects of protein mechanisms by applying different biophysical techniques:

• Protein folding by fluorescence spectroscopy
• Structures of ground and intermediate states by protein crystallography
• The kinetics of the working cycle of a protein by time-resolved absorption spectroscopy in the visible and infrared and fluorescence spectroscopy
• Thermal equilibrium fluctuations by quasi-elastic neutron scattering and molecular dynamics simulations

Besides many different projects the main interest of the work group Büldt is focussed on proteins in signalling pathways and energy conversion systems. From early days until now the group is investigating the mechanism of the light-driven proton pump bacteriorhodopsin by the above mentioned methods. They obtained high-resolution structures of the K, L and late M state of wild-type bacteriorhodopsin (Sass et al. Nature 406, 649-653, 2000). By improving the resolution to 1.3 A more and more details of the structure of bacteriorhodopsin became visible.
Two signalling pathways are of main interest, the amplification cascade of the eye and the two-component system in archaebacteria. The engagement of INB-2/I started with a collaboration of Dr. Granzin in Büldt's groupe with Dr. Wilden in Kaupp's group on the crystallisation of arrestin. Arrestins are proteins, which stop signalling pathways in all G-protein coupled receptor systems. They were able to solve the structure of the first arrestin (Fig.1) to 3.3 A resolution (Granzin et al., Nature 391, 918-921, 1998). Büldt's group is still working on the interaction of arrestin with MetaII-rhodopsin by fluorescence spectroscopy. In addition, together with Kaupp's group they are working on the crystallization of a cGMP sensitive ion channel which transforms the chemical signal of the visual system into an electrical potential.

Fig.1: Ribben diagram of the structure of arrestin: ß-strands in red, α-helix in blue and loops in yellow. Right side: N-terminal domain, left side: C-terminal domain.

The second system, under investigation, can be entitled "The little brain of an archaebacterium". So far, 16 different receptors for photo- and chemo-taxis are discovered in the archaebacterial membrane which control the swimming behaviour of the bacterium via the so-called two-component system. Here the signal is developed in the receptor after excitation and transferred to a second membrane protein a transducer which activates a kinase.

Dr. Gordeliy in Büldt's group crystallized the functional 2:2 complex of two receptor molecules of sensory rhodopsin II with two C-terminal truncated transducer molecules and solved the structure to 1.9 A resolution (Fig. 2) in collaboration with Prof. Engelhard's group, MPI Dortmund. In addition, they followed-up the signal development in the receptor and its transfer to the transducer by solving the crystal structures of an early intermediate and of the signalling state. Research is going on to solve the structure of the full-length transducer in order to investigate the molecular details of the kinase activation.

Fig.2: Complex of two molecules of sensory rhodopsin II (red) and two transducer molecules (green), ES: extracellular side, CS: cytoplasmic side.