Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells.

RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins.

Time Scales of Conformational Gating in a Lipid-Binding Protein.

Lipid-binding proteins sequester amphiphilic molecules in a large internal cavity occupied by ∼30 water molecules, some of which are displaced by the ligand. The role of these internal water molecules in lipid binding and release is not understood. We use magnetic relaxation dispersion (MRD) to directly monitor internal-water dynamics in apo and palmitate-bound rat intestinal fatty acid-binding protein (rIFABP).

Weak self-interactions of globular proteins studied by small-angle X-ray scattering and structure-based modeling.

We investigate protein-protein interactions in solution by small-angle X-ray scattering (SAXS) and theoretical modeling. The structure factor for solutions of bovine pancreatic trypsin inhibitor (BPTI), myoglobin (Mb), and intestinal fatty acid-binding protein (IFABP) is determined from SAXS measurements at multiple concentrations, from Monte Carlo simulations with a coarse-grained structure-based interaction model, and from analytic approximate solutions of two idealized colloidal interaction models without adjustable parameters. By combining these approaches, we find that the structure factor is essentially determined by hard-core and screened electrostatic interactions.

Structure and kinetics of chemically cross-linked protein gels from small-angle X-ray scattering.

Glutaraldehyde (GA) reacts with amino groups in proteins, forming intermolecular cross-links that, at sufficiently high protein concentration, can transform a protein solution into a gel. Although GA has been used as a cross-linking reagent for decades, neither the cross-linking chemistry nor the microstructure of the resulting protein gel have been clearly established. Here we use small-angle X-ray scattering (SAXS) to characterise the microstructure and structural kinetics of gels formed by cross-linking of pancreatic trypsin inhibitor, myoglobin or intestinal fatty acid-binding protein.

Mobility of core water in Bacillus subtilis spores by 2H NMR.

Bacterial spores in a metabolically dormant state can survive long periods without nutrients under extreme environmental conditions. The molecular basis of spore dormancy is not well understood, but the distribution and physical state of water within the spore is thought to play an important role. Two scenarios have been proposed for the spore's core region, containing the DNA and most enzymes.

Internal water and microsecond dynamics in myoglobin.

Myoglobin (Mb) binds diatomic ligands, like O2, CO, and NO, in a cavity that is only transiently accessible. Crystallography and molecular simulations show that the ligands can migrate through an extensive network of transiently connected cavities but disagree on the locations and occupancy of internal hydration sites. Here, we use water (2)H and (17)O magnetic relaxation dispersion (MRD) to characterize the internal water molecules in Mb under physiological conditions.

Structural basis of the recognition of the SAMP motif of adenomatous polyposis coli by the Src-homology 3 domain.

Elucidation of the basis of interactions between biological molecules is essential for the understanding of living systems. Src-homology 3 (SH3) domains play critical roles in interaction networks of proteins by recognizing a proline-rich sequence motif, PxxP. There are, however, several SH3 domains that specifically bind to polypeptide chains without the conventional recognition sequence.

Structure and functional characterization of Vibrio parahaemolyticus thermostable direct hemolysin.

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.

A chiral wedge molecule inhibits telomerase activity.

In addition to the Watson-Crick double helix, secondary DNA structures are thought to play important roles in a variety of biological processes. One important example is the G-quadruplex structure that is formed at the chromosome ends, which inhibits telomerase activity by blocking its access to telomeres. G-quadruplex structures represent a new class of molecular targets for DNA-interactive compounds that may be useful to target telomeres.

Identification of a link between the SAMP repeats of adenomatous polyposis coli tumor suppressor and the Src homology 3 domain of DDEF.

The adenomatous polyposis coli (APC) tumor suppressor protein is a multifunctional protein with a well characterized role in the Wnt signal transduction pathway and in cytoskeletal regulation. The SAMP repeats region of APC, an Axin-binding site, is known to be important for tumor suppression and for the developmental function of APC. We performed a yeast two-hybrid screening using the first SAMP motif-containing region of Xenopus APC as bait and obtained several SAMP binding candidates including DDEF2 (development and differentiation enhancing factor 2), which is an ADP-ribosylation factor (Arf) GTPase-activating protein (GAP (ArfGAP)) involved in the regulation of focal adhesions.