Molecular dissection of ALS-linked TDP-43 - involvement of the Gly-rich domain in interaction with G-quadruplex mRNA.

TDP-43 is the major pathogenic protein of amyotrophic lateral sclerosis (ALS). Previously, we identified that TDP-43 interacts with G-quadruplex (G4)-containing RNA and is involved in their long-distance transport in neurons. For the molecular dissection of the TDP-43 and G4-RNA interaction, we analyzed it here in vitro and in cultured cells using a set of 10 mutant TDP-43 proteins from familial and sporadic ALS patients as well as using the TDP-43 C-terminal Gly-rich domain alone.

Protection of scallop sarcoplasmic reticulum ATPase from thermal inactivation by removal of calcium from high-affinity binding sites on the enzyme.

Sarcoplasmic reticulum (SR) vesicles were isolated from scallop muscle by the method of Abe et al. (J. Biochem.

Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c.

One unique aspect of cytochrome c folding concerns the involvement of the covalently attached heme group and its axial ligands. To elucidate the role of the ligands in stabilizing the native and molten globule states, we studied the conformational and thermodynamic features of the iron-free derivative of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV circular dichroism suggested that porphyrin-cytochrome c has native-like alpha-helices, whereas near-UV CD suggested that the side-chains are flexible.

Thermodynamic stability of the molten globule states of apomyoglobin.

Whereas horse apomyoglobin is fully unfolded at pH 2 in the absence of salt, addition of a salt such as sodium chloride or sodium trichloroacetate stabilizes the molten globule state. Thermal unfolding of the salt-stabilized molten globule states of horse apomyoglobin at pH 2 measured by far-UV circular dichroism occurs not only on heating (i.e.

Structural characterization of the molten globule and native states of apomyoglobin by solution X-ray scattering.

Compactness and shape are two of the critical properties that describe the degree of protein folding. Solution X-ray scattering is an effective technique for measuring these properties quantitatively. Structural characteristics of various conformational states of horse myoglobin were studied in terms of size and shape by solution X-ray scattering.

C-terminal truncated Escherichia coli RecA protein RecA5327 has enhanced binding affinities to single- and double-stranded DNAs.

RecA5327 is a truncated RecA protein that is lacking 25 amino acid residues from the C-terminal end. The expression of RecA5327 protein in the cell resulted in the constitutive induction of SOS functions without damage to the DNA. Purified RecA5327 protein effectively promoted the LexA repressor cleavage reaction and ATP hydrolysis at a lower concentration of single-stranded DNA than that required for wild-type RecA protein.

Regulation of SOS functions: purification of E. coli LexA protein and determination of its specific site cleaved by the RecA protein.

The LexA protein of Escherichia coli was purified to more than 96% purity from cells harboring a recombinant plasmid carrying the lexA gene with the lacZ promoter sequence. The amino acid composition of the LexA protein and its amino-terminal sequence were analyzed. The results are in agreement with the prediction from the nucleotide sequence of the lexA gene.