Neutron crystallography enables direct observation of hydrogen atoms which play crucial roles in the physiological functions of enzymes, including molecular recognition through hydrogen bonding and catalytic reactions involving proton-coupled electron transfer. Now neutron crystallography is a limited method for protein structure determination, but steadily catholicizes with an operation of diffractometers for bio-macromolecules at neutron facilities and accumulated techniques for sample preparation. In this article, we give a commentary on the current status of neutron crystallography for bio-macromolecules in the world, and illustrate our recent results, neutron structural analyses of copper amine oxidase and copper-containing nitrite reductase, which provide in-depth understandings of the enzymatic reaction mechanism.

Voltage-dependent Ca²⁺ channels (Cavs) are indispensable for coupling action potentials with Ca²⁺ signaling. We report the first identification of a native prokaryotic Cav, CavMr, whose selectivity filter contains a smaller number of negatively charged residues than that of artificial prokaryotic Cavs. A relative mutant whose selectivity filter was replaced with that of CavMr exhibits high Ca²⁺ selectivity. The glycine residue of the CavMr selectivity filter is a determinant for Ca²⁺ selectivity. This glycine residue is well conserved among subdomains I and III of eukaryotic Cavs, which provide new insight into Ca²⁺ selectivity mechanism conserved from prokaryotes to eukaryotes.

The Escherichia coli UvrD protein is a superfamily 1, non-hexameric DNA helicase that plays a crucial role in repair mechanisms. Previous studies suggested that wild-type UvrD has optimal activity in its oligomeric form. Nevertheless, a conflicting monomer model was proposed using a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C). Here, single-molecule direct visualization of UvrDΔ40C revealed that two or three UvrDΔ40C molecules were simultaneously involved in DNA unwinding, presumably in an oligomeric form, similar to that with wild-type UvrD. Thus, single-molecule direct visualization of nucleic acid-binding proteins provides quantitative and kinetic information to address their fundamental mechanisms.

How life emerged on the ancient earth is one of the biggest questions in biology. RNA has a strong advantage as a candidate for the first polymer of life because it can simultaneously achieve information storage and enzymatic functions. However, RNA is difficult to be synthesized prebiotically and also chemically unstable. Recent studies have shown that positively charged peptides can support RNA to overcome such weaknesses. Such peptides also stimulate activities of ribozymes by assisting interactions between negatively charged RNA molecules. The primitive relationship between RNA and peptide is probably still surviving in modern enzymes like ribosome and RNA polymerase.

Understanding the mechanism of the amyloid fibril formation is crucial for the development of therapeutic methods of amyloidoses and neurodegenerative diseases. We focused on the role of prefibrillar intermediates that accumulate prior to the fibril formation by using a model peptide derived from insulin. Using circular dichroism spectroscopy, nuclear magnetic resonance, dynamic light scattering, and small-angle X-ray scattering, we found that the fibril formation occurred via specific prefibrillar intermediates. We also found that a plasma protein, fibrinogen, prohibited the fibril formation. It was suggested that the inhibition was exhibited by the specific and strong interaction of fibrinogen with the surface of the prefibrillar intermediates.

How can evolution shape a diverse biological world from a lifeless molecular world? In vitro evolution of artificial molecular replication systems is an attractive approach to investigate possible pathways of a simple molecular system developing towards biological complexity. We describe the progress of experimental investigation to test evolvability of molecules from the pioneering Spiegelman’s work to our studies on an artificial RNA replication system. Especially, we found that coevolution with parasitic molecules is a key to extend evolvability for emergence of diversity and continuous evolution.