Susanne Liemann

Extremely rapid folding of the C-terminal domain of the prion protein without kinetic intermediates.

The kinetics of folding of mPrP(121–231), the structured 111-residue domain of the murine cellular prion protein PrPC, were investigated by stopped-flow fluorescence using the variant F175W, which has the same overall structure and stability as wild-type mPrP(121–231) but shows a strong fluorescence change upon unfolding. At 22 °C and pH 7.0, folding of mPrP(121–231)–F175W is too fast to be observable by stopped-flow techniques. Folding at 4 °C occurs with a deduced half-life of ~170 μs without detectable intermediates, possibly the fastest protein-folding reaction known so far. Thus, propagation of the abnormal, oligomeric prion protein PrPSc, which is supposed to be the causative agent of transmissible spongiform encephalopathies, is unlikely to follow a mechanism where kinetic folding intermediates of PrPC are a source of PrPSc subunits.

Annexin V: the key to understanding ion selectivity and voltage regulation?

Annexin V is a Ca(2+)-dependent membrane-binding protein that forms voltage-dependent Ca2+ channels in phospholipid bilayers and is the first ion channel to be structurally and functionally characterized. Data outlined here indicate that key amino acid residues act as selectivity filters and voltage sensors, thereby regulating the permeability of the channel pore to ions.

THREE-DIMENSIONAL STRUCTURE OF ANNEXINS

Abstract. Annexins constitute a family of structurally related calcium- and phospholipid-binding proteins whose molecular structure has been investigated in detail in the crystalline and membrane-bound form. Their polypeptide chain is folded into four or eight α-helical domains of similar structure with a central hydrophilic pore. Bound to phospholipid membranes, the four-domain arrangement of the annexin molecule is conserved. A peripheral binding mode has been well documented by electron microscopy and a variety of other techniques.

Annexins: a novel family of calcium- and membrane-binding proteins in search of a function

Although the annexins have been extensively studied and much detailed structural information is available, their in vivo function has yet to be established.

Voltage dependent binding of annexin V, annexin VI and annexin VII-core to acidic phospholipid membranes

Annexin V, VI and VII-core (delta1-107) are members of the annexin protein family and bind to acidic phospholipid membranes in a calcium dependent manner. They also show ion channel activity under certain conditions. As annexins bind peripherally to lipid membranes, ion channel formation must consist of at least two steps: An adsorption reaction regulating the binding of annexin to the membrane surface and the opening and closing of the active species controlling the channel activity. By using the baseline current through the patch clamp seal as a probe for unoccupied binding sites at the membrane, we show that the adsorption of annexins to membranes is not only calcium dependent but also strongly voltage dependent. Whereas the free transfer energies at low calcium concentrations are similar for all three annexins, the binding of annexin V becomes much tighter with higher calcium levels, compared to annexin VI and VII-core. This correlates with the finding that annexin VI and VII-core display channel activity much more often than annexin V if one assumes that a high coverage of the membrane surface with annexins stabilizes the bilayer. At higher protein concentrations weaker binding is observed in agreement with the previously reported anti-cooperativity of membrane binding.

Annexin V: Structure-function analysis of a voltage-gated, calcium-selective ion channel

The structural basis of ion channel function remained mostly a mystery for more than 140 years since the existence of water-filled pores through membranes was first postulated (Brucke, 1843). Despite intense efforts in the determination of the electrophysiological properties of ion channels (for a review see Hille, 1992), starting with the seminal work of Hodgkin and Huxley, the field still suffers from the lack of high-resolution structures of ion channel proteins. The first determination of the structure of a membrane protein, the photosynthetic reaction center (Deisenhofer et al., 1985), was a flash of hope for the crystallization of membrane-integrated ion channel proteins in order to understand their selectivity, conductance regulation, and voltage-gating. But still, no crystals of ion channel proteins diffracting x-rays to high resolution could be obtained. Therefore, channel model systems with known crystal structures, such as the porins (e.g., Weiss et al., 1991) or colicins (e.g., Parker et al., 1989), were valuable. Unfortunately, these proteins do not combine the main features of typical ion channels mentioned above. To compensate for this shortcoming extensive investigations by mutational analysis (see Catterall, 1988 or Miller, 1991 for reviews) and molecular modeling (see Guy and Seetharamulu, 1992; Durell and Guy, 1992) of ion channels and of pore-forming peptides (see Sansom, 1991 for a review) were carried out. In 1990, annexin V was structurally characterized as the first ion channel protein, thus paving the way, in combination with electrophysiological analysis of annexin V mutants, for an understanding of ion channel function in molecular terms.