SignificanceThis study expands our knowledge of protein hydration, which is highly related to the macromolecular antifreeze property of proteins. We examined a polypentagonal network formation of waters for a series of artificial variants of a 65-residue ice-binding protein. The polypentagonal waters were created solely on the surface of an activity-improved variant, which appeared to contain two sets of water clusters exhibiting a perfect position match to the waters constructing the first prism and pyramidal ice planes. These data suggest that a minute structural change in a protein organizes the surface waters into a polypentagonal arrangement, which merges with the intrinsically disordered ice surface and freezes to specific ice crystal planes.

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

T4 phage lysozyme is an inverting glycoside hydrolase that degrades the murein of bacterial cell walls by cleaving the β‐1,4‐glycosidic bond. The substitution of the catalytic Thr26 residue to a histidine converts the wild type from an inverting to a retaining enzyme, which implies that the original general acid Glu11 can also act as an acid/base catalyst in the hydrolysis. Here, we have determined the neutron structure of the perdeuterated T26H mutant to clarify the protonation states of Glu11 and the substituted His26, which are key in the retaining reaction. The 2.09‐Å resolution structure shows that the imidazole group of His26 is in its singly protonated form in the active site, suggesting that the deprotonated Nɛ2 atom of His26 can attack the anomeric carbon of bound substrate as a nucleophile. The carboxyl group of Glu11 is partially protonated and interacts with the unusual neutral state of the guanidine moiety of Arg145, as well as two heavy water molecules. Considering that one of the water‐binding sites has the potential to be occupied by a hydronium ion, the bulk solvent could be the source for the protonation of Glu11. The respective protonation states of Glu11 and His26 are consistent with the bond lengths determined by an unrestrained refinement of the high‐resolution X‐ray structure of T26H at 1.04‐Å resolution. The detail structural information, including the coordinates of the deuterium atoms in the active site, provides insight into the distinctively different catalytic activities of the mutant and wild type enzymes.

We constructed a time-resolved photovoltage measurement system and examined the photovoltage kinetics of wild-type bacteriorhodopsin, its D96N mutant, and halorhodopsins from Halobacterium salinarum and Natronobacterium pharaonis. Upon illumination with a laser flash, wild-type bacteriorhodopsin showed photovoltage generation with fast (10-100 micros range) and slow (ms range) components while D96N lacked the latter, as reported previously [Holz, M., Drachev, L.A., Mogi, T., Otto, H., Kaulen, A.D., Heyn, M.P., Skulachev, V.P., and Khorana, H.G. (1989) Proc. Natl. Acad. Sci. USA 86, 2167-2171]. In contrast, photovoltage generation in halorhodopsins from H. salinarum and N. pharaonis was significant only in the ms time range. On the basis of the photovoltage kinetics and photocycle, we conclude that major charge (chloride) movements within halorhodopsin occur during the formation and decay of the N intermediate in the ms range. These observations are discussed in terms of the "Energization-Relaxation Channel Model" [Muneyuki, E., Ikematsu, M., and Yoshida, M. (1996) J. Phys. Chem. 100, 19687-19691].

Neutron crystallography has been used to elucidate the protonation states for the enhanced green fluorescent protein, which has revolutionized imaging technologies. The structure has a deprotonated hydroxyl group in the fluorescent chromophore. Also, the protonation states of His148 and Thr203, as well as the orientation of a critical water molecule in direct contact with the chromophore, could be determined. The results demonstrate that the deprotonated hydroxyl group in the chromophore and the nitrogen atom ND1 in His148 are charged negatively and positively, respectively, forming an ion pair. The position of the two deuterium atoms in the critical water molecule appears to be displaced slightly toward the acceptor oxygen atoms according to their omit maps. This displacement implies the formation of an intriguing electrostatic potential realized inside of the protein. Our findings provide new insights into future protein design strategies along with developments in quantum chemical calculations.