Werner Jahn

Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide

Muscle contraction involves the cyclic interaction of the myosin cross-bridges with the actin filament, which is coupled to steps in the hydrolysis of ATP. While bound to actin each cross-bridge undergoes a conformational change, often referred to as the “power stroke”, which moves the actin filament past the myosin filaments; this is associated with the release of the products of ATP hydrolysis and a stronger binding of myosin to actin. The association of a new ATP molecule weakens the binding again, and the attached cross-bridge rapidly dissociates from actin. The nucleotide is then hydrolysed, the conformational change reverses, and the myosin cross-bridge reattaches to actin. X-ray crystallography has determined the structural basis of the power stroke, but it is still not clear why the binding of actin weakens that of the nucleotide and vice versa. Here we describe, by fitting atomic models of actin and the myosin cross-bridge into high-resolution electron cryo-microscopy three-dimensional reconstructions, the molecular basis of this linkage. The closing of the actin-binding cleft when actin binds is structurally coupled to the opening of the nucleotide-binding pocket.

Argiotoxin detects molecular differences in AMPA receptor channels

Argiotoxin, a component of the spider venom from Argiope lobata, blocks AMPA receptor channels expressed in homomeric and heteromeric configuration in Xenopus oocytes. Argiotoxin acts as an open channel blocker in a voltage-dependent manner and discriminates between the functionally diverse AMPA receptors. Importantly, a transmembrane region 2 determinant for divalent cation permeability also determines argiotoxin sensitivity. Subunit-specific differences in the time courses of block and recovery demonstrate that heteromeric AMPA receptors can assemble in variable ratios. Thus, argiotoxin can be used as a tool in analyzing the subunit composition of AMPA receptors in native membranes.

Subunit-specific block of cloned NMDA receptors by argiotoxin636

Cloned NMDA receptor channels of the NR1‐NR2A, NR1‐NR2B and NR1‐NR2C type show differences in argiotoxin636 block. Mutations of an asparagine residue located at a homologous position in the TM2 region of all NMDA receptor subunits, which corresponds to the Q/R site of the AMPA receptors, alters the argiotoxin636‐induced block. The results suggest that the toxin interacts at this amino acid position with the putative pore forming TM2 region of the NMDA receptor subunits. Sequence differences in the TM2 segment of NR2A and NR2C subunits are not responsible for the subtype‐specific sensitivity to argiotoxin636 as revealed by site‐directed mutagenesis.

Methods of preparing well-orientated sols of f-actin containing filaments suitable for X-ray diffraction

In this paper we describe methods of preparing orientated f-actin and reconstituting thin filaments that are suitable for X-ray diffraction that allow us to analyse the structure of f-actin to at least 15 A resolution (1 A = 0.1 nm). We described problems that occur during the process of orientation and ways of solving them.

Crystallography of ribosomal particles

Abstract Several forms of three-dimensional crystals and two-dimensional sheets of intact ribosomes and their subunits have been obtained as a result of: (a) an extensive systematic investigation of the parameters involved in crystallization, (b) a development of an experimental procedure for controlling the volumes of the crystallization droplets, (c) a study of the nucleation process, and (d) introducing a delicate seeding procedure coupled with variations in the ratios of mono- and divalent ions in the crystallization medium. In all cases only biologically active particles could be crystallized, and the crystalline material retains its integrity and activity. Crystallographic data have been collected from crystals of 50S ribosomal subunits, using synchrotron radiation at temperatures between + 19 and - 180°C. Although at 4°C the higher resolution reflections decay within minutes in the synchrotron beam, at cryo-temperature there was hardly any radiation damage, and a complete set of data to about 6Aresolution could be collected from a single crystal. Heavy-atom clusters were used for soaking as well as for specific binding to the surface of the ribosomal subunits prior to crystallization. The 50S ribosomal subunits from a mutant of B. stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with in the native ones. Models, aimed to be used for low resolution phasing, have been reconstructed from two-dimensional sheets of 70S ribosomes and 50S subunits at 47 and 30A, respectively. These models show the overall structure of these particles, the contact areas between the large and small subunits, the space where protein synthesis might take place and a tunnel which may provide the path for the nascent protein chain.

Argiotoxin636 inhibits NMDA−activated ion channels expressed in Xenopus oocytes

Argiotoxin636, a component of the spider venom of argiope species, was chemically synthesized together with a number of derivatives in order to analyse their blocking activity on mammalian glutamate receptors. Xenopus laevis oocytes injected with rat brain mRNA served as assay system. The results showed that argiotoxin636 had a higher affinity for N-methyl-D-aspartate (NMDA) than for kainate receptors, blocking the corresponding ion channels in a voltage-dependent manner. Modifications of the polyamine tail or the terminal arginine residue strongly reduced the blocking potency. The iodinated monohydroxyl phenylderivatives, however, retained their NMDA-selective binding and could serve as non-competitive antagonists for radioligand binding assays aiding in the biochemical isolation of glutamate receptors.

Towards Atomic Resolution of Prokaryotic Ribosomes: Crystallographic, Genetic and Biochemical Studies

The studies reported here were initiated and inspired by the late Prof. H.G. Wittmann. From the early stages of this project, when it was widely believed that even the initial steps in determining the molecular structure of ribosomes are impossible, until his last days, Prof. Wittmann was actively involved in the experimental design and in the actual studies. We have no doubt that without his motivation, optimism, guidance and support, this project would not have reached its current stage.

Phalloidinwirkung an der erythrocytenfrei perfundierten Rattenleber

Summary1.The first symptom of phalloidin poisoning in the isolated rat liver is an increase in the light scattering of the liver tissue, which begins 2–3 min after exposing the tissue to the toxin. Simultanously the oxygen consumption increases, reaching a new plateau 15±11% above the starting level after 10 min. 2.In a low-Ca medium (2·10−4M Ca++) phalloidin produces a Ca++ release of 0,22 ± 0,08 ΜM Ca/g liver which starts after 2–4 min and finishes after 15 to 20 min. In the presence of 1.4·10−3M Ca++ there is a transient Ca++ release of about 0.1 ΜM Ca/g liver (only detectable by indirect methods) followed probably by a slow Ca++ uptake. 3.At 27‡ C a potassium efflux starts 11.5 ± 3.5 min after phalloidin, preceeded by an increase in the weight of the liver starting after 6 ± 1,5 min. 4.EDTA (10−3 M, in a medium containing 2·10−4M Ca++ and 5·10−4 M Mg++) produces a swelling of the liver, a Ca++ release (0.3 ΜM/g liver after 30 min) and a limited K+ release (11±4 ΜM/g liver after 30 min).Phalloidin has no further effect on the Ca or K content of the liver. The only effect of phalloidin in the presence of EDTA is a small increase of the light scattering of the liver tissue. 5.Addition of EDTA (0.4–1.4 mM free EDTA) to a phalloidin poisoned liver which has lost most of its potassium, leads to a re-uptake of the released K+ of up to 75% in 120 min. The K+ uptake is not correlated with a decrease in liver weight. 6.A decrease of temperature from 27‡ to 19‡ C diminishes the rate of K++ release caused by phalloidin to 33±15% of the rate at 27‡. The analogous changes of the rate of the K+ re-uptake are in the same range.

Derivatization of ribosomes and of tRNA with an undecagold cluster: crystallographic and functional studies

An undecagold cluster was covalently attached to whole ribosomes and to their small and large subunits prior to their crystallization. X-ray crystallographic data were collected from crystals of the first two. The same cluster was bound to tRNAphe fromE. coli at base 47. It was found that the modified tRNA molecule binds to the ribosome and can be aminoacylated by its cognate synthetase. The gold cluster modified tRNAphe may be used for phasing diffraction data of crystals of complexes containing it, mimicking defined states in the process of protein biosynthesis.

Aufnahme von Wasser und hochmolekularen Substanzen durch die isoliert perfundierte Leber nach Phalloidinvergiftung und nach Erhöhung des posthepatischen Druckes

Summary1.Livers of rats were perfused with solutions containing dextran or albumin.2.Changes in the concentration of dextran or albumin in the perfusion medium due to shifts of water between the intra- and extracellular space of the liver were measured by continous recording the optical rotation of the perfusion medium.3.The volume of the perfusion medium (i.e. the dextran space of the perfusion apparatus and of the liver) was estimated by measuring the changes in optical rotation of the perfusion medium due to a known dilution.4.The intracellular space of the liver was calculated from measurements of the dextran or albumin space, the wet weight and the dry weight of the liver.5.The swelling and the potassium release of the liver poisoned by phalloidin depends strongly on the perfusion pressure.6.At high perfusion pressures, phalloidin poisoning is followed by an increase of the intracellular space of the liver by about 100%. Accordingly the volume of the perfusion medium diminishes during the poisoning, while the change in the concentration of dextran or albumin in the perfusion medium is very small. Thus the poisoned liver takes up water together with colloids.7.When the posthepatic pressure is increased, potassium is released and water with dextran (or albumin) is accumulated. The potassium release and the water uptake are reversible. The potassium release is inhibited by 4,7-phenanthroline.8.The histological changes, due to increased posthepatic pressure, are similar to those in livers poisoned with phalloidin.9.The results are discussed in respect to the similarity between the effects observed in the phalloidin poisoned livers as well as in the livers perfused with high posthepatic pressure.