3D Mapping of the SPRY2 Domain of Ryanodine Receptor 1 by Single-Particle Cryo-EM

The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca2+ release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform

New Insights into Alzheimer's Disease Progression: A Combined TMS and Structural MRI Study

BACKGROUND: Combination of structural and functional data of the human brain can provide detailed information of neurodegenerative diseases and the influence of the disease on various local cortical areas. METHODOLOGY AND PRINCIPAL FINDINGS: To examine the relationship between structure and function of the brain the cortical thickness based on structural magnetic resonance images and motor cortex excitability assessed with transcranial magnetic stimulation were correlated in Alzheimer's disease (AD) and mild cognitive impairment (MCI) patients as well as in age-matched healthy controls. Motor cortex excitability correlated negatively with cortical thickness on the sensorimotor cortex, the precuneus and the cuneus but the strength of the correlation varied between the study groups. On the sensorimotor cortex the correlation was significant only in MCI subjects. On the precuneus and cuneus the correlation was significant both in AD and MCI subjects. In healthy controls the motor cortex excitability did not correlate with the cortical thickness. CONCLUSIONS: In healthy subjects the motor cortex excitability is not dependent on the cortical thickness, whereas in neurodegenerative diseases the cortical thinning is related to weaker cortical excitability, especially on the precuneus and cuneus. However, in AD subjects there seems to be a protective mechanism of hyperexcitability on the sensorimotor cortex counteracting the prominent loss of cortical volume since the motor cortex excitability did not correlate with the cortical thickness. Such protective mechanism was not found on the precuneus or cuneus nor in the MCI subjects. Therefore, our results indicate that the progression of the disease proceeds with different dynamics in the structure and function of neuronal circuits from normal conditions via MCI to AD

C-13 and H-1-Nmr Studies of the Interaction of Tricyclic Antidepressant Drugs with Dodecyldimethylammonium Chloride Micelles

C-13 and H-1 NMR spectroscopy has been used to study the interaction of the tricyclic antidepressant (TCA) drugs imipramine and amitriptyline with dodecyldimethylammonium chloride (DDAC) micelles. C-13 NMR relaxation parameters, including T1 (the spin-lattice relaxation time) and NOE (the nuclear Overhauser effect), were measured at 75 and 100 MHz for mixtures of the TCA drugs and DDAC and were interpreted in terms of overall and internal motion of these micellar aggregates. NMR and fluorescence quenching experiments were used to determine the degree of partitioning of the drugs into the micelle in order to assist this interpretation. Under the conditions studied it was found that there is rapid exchange between free and micelle bound states and that the degree of partitioning of imipramine into the micelle (42%) is less than that for amitriptyline (76%). Both drugs were found to restrict the internal mobility of the micellar species, as evidenced by increased order parameters and correlation times for individual carbon sites in the DDAC alkyl chain on addition of drug. The drug molecules themselves were found to adopt an extended conformation in the micelle, with the dimethylammonium moiety residing close to the micelle surface and the tricyclic group penetrating the hydrophobic region of the micelle

C-13 and H-1-Nmr Studies of the Interaction of Tricyclic Antidepressant Drugs with Micellar Aggregates

C-13 NMR relaxation parameters including T1, the spin-lattice relaxation time, and NOE, the nuclear Overhauser effect, have been measured for mixtures of tricyclic antidepressants (TCAs) and sodium dodecyl sulfate (SDS) micelles. The relaxation parameters were measured at 75 and 100 MHz and were interpreted in terms of the overall and internal motion of these micellar aggregates. It is suggested that the drug molecules are partitioned almost totally within the SDS micelle, resulting in significant increases in micellar volume and a reduction in mobility of SDS alkyl chains. The mobility of the TCA molecules was also examined by using chemical shift data and 2D NMR techniques. It is found that the TCA molecules adopt an extended conformation, with the tricyclic moiety occupying the hydrophobic region of the micelle and the dimethylammonium moiety residing at the micelle/water interface

An Nmr and Theoretical-Study of the Conformation and Internal Flexibility of Butaclamol Hydrochloride

A theoretical (MM2) and experimental (H-1 and C-13 NMR) study of butaclamol hydrochloride in CDCl3 has been done in order to determine preferred conformations and internal molecular flexibility of this molecule. The theoretical calculations suggest the presence of four low-energy conformations, two of which involve a trans junction of the D and E rings, with the other two involving a cis I ring junction. An alternative cis junction (cis II) was excluded on energetic grounds. The H-1 NMR data strongly suggest the presence of a trans D-E ring junction and are consistent with a chair conformation of the E ring. C-13 spin-lattice relaxation time measurements show that most of the molecule is rigid, although there is some degree of mobility in the seven-membered B ring, associated with rapid flipping of the bridging C8 and C9 carbons between two skewed conformations, which have previously been referred to as conformer A and conformer B (Laus et al. Heterocycles 1984, 22, 311)

Direct measurement of the pK(a) of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: Implications for the catalytic mechanism

The ionization stale of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [C-13 gamma] aspartic acid and measuring the C-13 chemical shifts in the ape, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pK(a) greater than similar to 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pK(a) value of less than 4 in this complex. Such a low pK(a) value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pK(a) = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 C-13 gamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process

Effects of an alpha-helical ryanodine receptor C-terminal tail peptide on ryanodine receptor activity: Modulation by Homer.

We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 microM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 microM; but with 10 microM cytoplasmic Ca2+, skeletal ryanodine receptors were activated by < or = 1.0 microM peptide and inhibited by 10 microM peptide. Cardiac ryanodine receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruptio