Search

Naturally occurring mutations are useful in identifying domains that are important for protein function. We studied a mutation in the elastin gene, 800−3G>C, a common disease allele for SVAS (supravalvular aortic stenosis). We showed in primary skin fibroblasts from two different SVAS families that this mutation causes skipping of exons 16–17 and results in a stable mRNA. Tropoelastin lacking domains 16–17 (Δ16–17) was synthesized efficiently and secreted by transfected retinal pigment epithelium cells, but showed the deficient deposition into the extracellular matrix compared with normal as demonstrated by immunofluorescent staining and desmosine assays. Solid-phase binding assays indicated normal molecular interaction of Δ16–17 with fibrillin-1 and fibulin-5. However, self-association of Δ16–17 was diminished as shown by an elevated coacervation temperature. Moreover, negative staining electron microscopy confirmed that Δ16–17 was deficient in forming fibrillar polymers. Domain 16 has high homology with domain 30, which can form a β-sheet structure facilitating fibre formation. Taken together, we conclude that domains 16–17 are important for self-association of tropoelastin and elastic fibre formation. This study is the first to discover that domains of elastin play an essential role in elastic fibre formation by facilitating homotypic interactions. [ABSTRACT FROM AUTHOR]

Using molecular dynamics (MD) simulation, we simulate the structural change of a telomeric DNA by β-decay of substituted tritium to helium-3. The configuration of the telomeric DNA is obtained by removing TRF2 protein from the TRF2-Dbd-DNA complex (Protein Data Bank ID is 3SJM). We assume that hydrogens (H) of guanines in the telomeric DNA are replaced to helium-3. Since this replacement of the H atoms to the 3He atoms changes the charge distribution significantly, the charge distribution used in the MD simulation for the modified guanine is obtained by the density functional theory calculations. We adopt, as the MD simulation, nanoscale molecular dynamics code with CHARMM36 force field using Langevin thermostat and Nosé–Hoover Langevin piston to control the temperature and pressure of the system, respectively. Moreover, changing both the number of replaced guanine N and the temperature of the system T, we calculate the root mean square deviation RMSD to quantify the dependence of the durability of the telomeric DNA on the β-decays. From the MD simulation, it is found that as N or T becomes larger, the RMSD of the DNA becomes also larger. Namely, it denotes that as the intensity of the β-decays becomes larger or as the temperature is increased, the DNA structure becomes more fragile. [ABSTRACT FROM AUTHOR]

The observation of biological materials by transmission electron microscopy (TEM) frequently requires the use of negative staining and/or immuno-gold labeling to visualize or identify the specimen, but these techniques are limited. In contrast, genetic labeling with green fluorescent protein (GFP) and its homologs have led to rapid advances in the observation of proteins of interest in cells by light microscopy (LM). These fluorescent tags allow for the visualization of dynamic processes in live cells without the use of secondary reagents. Here, we report the development of an artificial metalloprotein fusion protein expressed in Escherichia coli grown in Cd2+-containing medium that allows for efficient protein detection by TEM without additional staining steps. We linked the subunits of the bacterial 14-mer protein GroEL with three repeats of metallothionein (3MT). The 3MT-fused GroEL (GroEL-14(3MT)) was successfully expressed in E. coli, and the purified protein included 250 cadmium atoms per molecule on average. Cd2+-bound GroEL-14(3MT) was detected by TEM in the absence of negative staining on a carbon grid, and the particle densities of GroEL-14(3MT) were much greater than those of untagged GroEL in vitreous ice. Taken together, our data indicate that the 3MT tag provides a promising means of allowing the identification of oligomeric proteins isolated from cells in the absence of other detection techniques. [ABSTRACT FROM AUTHOR]