Ta-Hsien Lin

The correlation between neurotoxicity, aggregative ability and secondary structure studied by sequence truncated Aβ peptides

Aggregated β‐amyloid (Aβ) peptides are neurotoxic and cause neuronal death both in vitro and in vivo. Although the formation of a β‐sheet structure is usual required to form aggregates, the relationship between neurotoxicity and the Aβ sequence remains unclear. To explore the correlation between Aβ sequence, secondary structure, aggregative ability, and neurotoxicity, we utilized both full‐length and fragment‐truncated Aβ peptides. Using a combination of spectroscopic and cellular techniques, we demonstrated that neurotoxicity and aggregative ability are correlated while the relationship between these characteristics and secondary structure is not significant. The hydrophobic C‐terminus, particularly the amino acids of 17–21, 25–35, and 41–42, is the main region responsible for neurotoxicity and aggregation. Deleting residues 17–21, 25–35 or 41–42 significantly reduced the toxicity. On the other hand, truncation of the peptides at either residues 22–24 or residues 36–40 had little effect on toxicity and aggregative ability. While the N‐terminal residues 1–16 may not play a major role in neurotoxicity and aggregation, a lack of N‐terminal fragment Aβ peptide, (e.g. Aβ17–35), does not display the neurotoxicity of either full‐length or 17–21, 25–35 truncated Aβ peptides.

Structural analysis of the extracellular domain of vaccinia virus envelope protein, A27L, by NMR and CD spectroscopy

This study presents the molecular structure of the extracellular domain of vaccinia virus envelope protein, A27L, determined by NMR and CD spectroscopy. A recombinant protein, eA27L-aa, containing this domain in which cysteines 71 and 72 were replaced with alanine, was constructed to prevent self-assembly due to intermolecular disulfide bonds between these two cysteines. The soluble eA27L-aa protein forms an oligomer resembling that of A27L on vaccinia virions. Heteronuclear correlation NMR spectroscopy was carried out on eA27L-aa in the presence or absence of urea to determine backbone resonance assignments. Chemical shift index (CSI) propensity analysis showed that eA27L-aa has two distinct structural domains, a relatively flexible 22-amino acid random coil in the N-terminal region and a fairly rigid α-helix structure in the remainder of the structure. Binding interaction studies using isothermal titration calorimetry suggest that a 12-amino acid lysine/arginine-rich segment in the N-terminal region is responsible for glycosaminoglycan binding. The rigid α-helix portion of eA27L-aa is probably involved in the intrinsic self-assembly, and CSI propensity analysis suggests that region N37-E49, with a residual α-helix tendency, is probably the self-assembly core. Self-assembly was ascribed to three hydrophobic leucine residues (Leu41, Leu45, and Leu48) in this segment. The folding mechanism of eA27L-aa was analyzed by CD spectroscopy, which revealed a two-step transition with a Gibbs free energy of 2.5 kcal/mol in the absence of urea. Based on these NMR and CD studies, a residue-specific molecular model of the extracellular domain of A27L is proposed. These studies on the molecular structure of eA27L-aa will help in understanding how vaccinia virus enters cells.

Phosphorylation by glycogen synthase kinase of inhibitor-2 does not change its structure in free state

Inhibitor‐2 (I2) is a thermostable protein that specifically binds to the catalytic subunit of protein phosphatase‐1 (PP1), resulting in the formation of the inactive holoenzyme, ATP‐Mg‐dependent phosphatase. Phosphorylation of I2 at Thr‐72 by glycogen synthase kinase‐3 (GSK‐3) results in activation of the phosphatase, suggesting that kinase action triggers conformational change in the complex. In this paper, we characterize the effect of GSK‐3 phosphorylation on the structure of free state I2[1–172] by nuclear magnetic resonance and circular dichroism spectroscopy, and show that phosphorylation has no significant effect on its conformation. We conclude that the conformational changes of ATP‐Mg‐dependent phosphatase induced by GSK‐3 phosphorylation must depend on the interactions between PP1 and I2.

Purification, crystallization and preliminary crystallographic studies of a calmodulin-OLFp hybrid molecule

A hybrid molecule consisting of calmodulin (CaM) and the CaM-binding domain of olfactory nucleotide-gated ion-channel peptide (CaM-OLFp) was purified and crystallized by the hanging-drop vapour-diffusion method at 298 K. The crystals diffracted to a maximum resolution of 1.85 A at cryogenic temperature (100 K) using X-rays from a rotating anode (Cu, wavelength 1.54 A). The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 64.76, b = 36.23, c = 70.96 A, alpha = gamma = 90, beta = 109.4 degrees. Analysis of the packing density shows that the asymmetric unit contains one CaM-OLFp hybrid molecule with a solvent content of 36.42%.