Ca(2+)-calmodulin can activate and inactivate cardiac ryanodine receptors.

BACKGROUND AND PURPOSE: Ca(2+)-calmodulin (Ca(2+)CaM) is widely accepted as an inhibitor of cardiac ryanodine receptors (RyR2); however, the effects of physiologically relevant CaM concentrations have not been fully investigated. EXPERIMENTAL APPROACH: We investigated the effects of low concentrations of Ca(2+)CaM (50-100 nmol.L(-1)) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol.L(-1) CaM on sarcoplasmic reticulum (SR) Ca(2+)-release. KEY RESULTS: Ca(2+)CaM activated or inhibited single RyR2, but activation was much more likely at low (50-100 nmol.L(-1)) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol x L(-1) CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca(2+)-release events. Application of caffeine demonstrated that 50 nmol x L(-1) CaM reduced SR Ca(2+) content. CONCLUSIONS AND IMPLICATIONS: We describe for the first time how Ca(2+)CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca(2+)-release and the use of caffeine suggests that this is a RyR2-mediated effect.

Studies of the aggregation of an amyloidogenic alpha-synuclein peptide fragment.

The deposition of alpha-syn (alpha-synuclein) fibrils in Lewy bodies is a characteristic feature of individuals with neurodegenerative disorders. A peptide comprising the central residues 71-82 of alpha-syn [alpha-syn(71-82)] is capable of forming beta-sheet-rich, amyloid-like fibrils with similar morphologies to fibrils of the full-length protein, providing a useful model of pathogenic alpha-syn fibrils that is suitable for detailed structural analysis. We have studied the morphology and gross structural features of alpha-syn(71-82) fibrils formed under different conditions in order to obtain reliable conditions for producing fibrils for further structural investigations. The results indicate that the rate of aggregation and the morphology of the fibrils formed are sensitive to pH and temperature.

The location of the mobile electron carrier ferredoxin in vascular plant photosystem I.

In this study, we present the location of the ferredoxin-binding site in photosystem I from spinach. Image analysis of negatively stained two-dimensional crystals indicates that the addition of ferredoxin and chemical cross-linkers do not significantly alter the unit cell parameters (for untreated photosystem I, a = 26.4 nm, b = 27.6 nm, and gamma = 90 degrees, space group p22(1)2(1) and for ferredoxin cross-linked photosystem I, a = 26.2 nm, b = 27.2 nm, and gamma = 90 degrees, space group p22(1)2(1)). Fourier difference analysis reveals that ferredoxin is bound on top of the stromal ridge principally interacting with the extrinsic subunits PsaC and PsaE. This location would be accessible to the stroma, thereby promoting efficient electron transfer away from photosystem I. This observation is significantly different from that of the ferredoxin binding site proposed for cyanobacteria. A model for the binding of ferredoxin in vascular plants is proposed and is discussed relative to observations in cyanobacteria.

A fragment of recombinant GABA(A) receptor alpha1 subunit forming rosette-like homo-oligomers.

The type A gamma-aminobutyric acid (GABA(A)) receptor plays a major role in inhibitory synaptic transmission in the central nervous system. A fragment consisting of residues Cys166 to Leu296 of the alpha1 subunit of the GABA(A) receptor was overexpressed in Escherichia coli and was found to have stable beta-rich structures. Here, results from laser scattering, gel electrophoresis and electron microscopy demonstrated that this recombinant protein formed rosette-like homo-oligomers, mainly pentamers in solution. Therefore, the fragment apparently provides a valuable model system for studying the pentameric holoreceptor assembly. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of the fragment showed that disulfide bonds formed between monomers contributed to the oligomerization of the fragment. The fact that this fragment alone could form pentamers in vitro strongly suggests that amino acid residues located within the Cys166-Leu296 region of the alpha1 subunit may contribute to the oligomerization of GABA(A) receptor in vivo.

Three-dimensional structure of higher plant photosystem I determined by electron crystallography.

We describe the three-dimensional structure of higher plant photosystem I (PSI) as obtained by electron microscopy of two-dimensional crystals formed at the grana margins of thylakoid membranes. The negatively stained crystalline areas displayed unit cell dimensions a = 26.6 nm, b = 27.7 nm, and gamma = 90(o), and p22121 plane group symmetry consisting of two monomers facing upward and two monomers facing downward with respect to the membrane plane. Higher plant PSI shows several structural similarities to the cyanobacterial PSI complex, with a prominent ridge on the stromal side of the complex. The stromal ridge is resolved into at least three separate domains that are interpreted as representing the three well characterized stromal subunits, the psa C, D, and E gene products. The lumenal surface is relatively flat but exhibits a distinct central depression that may be the binding site for plastocyanin. Higher plant PSI is of dimensions 15-16 x 11-12.5 nm, and thus leaves a larger footprint in the membrane than its cyanobacterial equivalent (13 x 10.5 nm). It is expected that additional membrane-bound polypeptides will be present in the higher plant PSI. Both higher plant and cyanobacterial complexes span about 8-9 nm in the direction orthogonal to the membrane. This report represents the first three-dimensional structure for the higher plant PSI complex.

Two-dimensional crystals of photosystem I in higher plant grana margins.

In this report, we present new structural data on the size, shape, and oligomeric form of higher plant photosystem I (PSI) formed within the thylakoid grana margins. We show that PSI complexes can be assembled into ordered molecular monolayers (two-dimensional crystals) using thylakoid membranes from a variety of higher plant sources. Digital image analysis of negatively stained two-dimensional crystals (a = 26.9 nm, b = 28.0 nm, gamma = 90 degrees, p22121 plane group) resulted in a projection map consisting of 4 monomers/unit cell. Higher plant PSI is slightly larger than its cyanobacterial equivalent but shows many similar features. Structural changes after urea and salt washing of the crystals supported the biochemical characterization and were mainly assigned to the stromal side of the complex where the psaC, psaD, and psaE gene products are known to be bound. Labeling with ferredoxin-colloidal gold complexes provided direct evidence for a segregated PSI population, with 5 nm diameter ferredoxin-gold particles enriched in the thylakoid grana margins and the two-dimensional crystals. This lateral segregation of photosynthetic complexes is important for the understanding of the kinetics of electron transfer between photosystem II and PSI in higher plants.

Arthrobacter D-xylose isomerase: partial proteolysis with thermolysin.

The pattern and kinetics of partial proteolysis of Arthrobacter D-xylose isomerase tetramer was studied in order to determine the flexibility of surface loops that may control its stability. It was completely resistant to trypsin, chymotrypsin and elastase at 37 degrees C, but thermolysin cleaved specifically and quantitatively at Thr-347-Leu-348 between helices 10 and 11 to remove 47 residues from the C-terminus of each 43.3 kDa subunit. At high temperatures, helices 9 and 10 were removed from each 38 kDa subunit to give a 36 kDa tetramer. The kinetics of nicking by thermolysin indicated that the Thr-347-Leu-348 loop is locked at low temperatures, but 'melts' at 25 degrees C and is fully flexible above 34 degrees C. The flexibility appears to be associated with binding of Ca2+ ions at the active site, since Co2+, Mg2+ and xylitol protect in proportion to their ability to displace Ca2+. The missing C-terminal helices make many intersubunit contacts that appear in the structure to stabilize the tetramer, but the properties of the purified nicked proteins are almost indistinguishable from the native enzyme. Both the 38 kDa tetramer and the 36 kDa tetramer are identically active and dissociate similarly in urea or SDS to fully active dimers, but the nicked dimers are slightly less stable to urea at 62 degrees C. In the Mg2+ form the thermostability of the 38 kDa tetramer is identical with that of the native enzyme, but the 36 kDa tetramer has a slightly lower 'melting point' (70 degrees C versus 80 degrees C), which may be due to unravelling from the end of helix 8. Since elimination of all the C-terminal helices and many intersubunit contacts has so little effect, one can conclude that the 'weak point' that controls the protein's thermostability lies within the N-terminal beta-barrel domain.