Molecular evolution of Cypridina noctiluca secretory luciferase for production of spectrum-shifted luminescence-emitting mutants and their application in nuclear receptor-reporter assays.

Luciferase is a popular enzyme used for biological analyses, such as reporter assays. In addition to a conventional reporter assay using a pair of firefly and Renilla luciferases, a simple multicolor reporter assay using multiple firefly or beetle luciferases emitting different color luminescence with a single substrate has been reported. Secretory luciferases have also been used for convenient sample preparation in reporter assays; however, reporter assay using secretory luciferase mutants that emit spectrum-shifted luminescence have not yet been reported. In this study, we generated blue- and red-shifted (-16 and 12 nm) luminescence-emitting Cypridina secretory luciferase (CLuc) mutants using multiple cycles of random and site-directed mutagenesis. Even for red-shifted CLuc mutant, which exhibited relatively low activity and stability, its enzymatic activity was sufficiently high for a luciferase assay (3.26 × 106 relative light unit/s), light emission was sufficiently prolonged (half-life is 3 min), and stability at 37°C was high. We independently determined the luminescence of these CLuc mutants using a luminometer with an optical filter. Finally, we replaced the commonly used reporters, firefly and Renilla luciferases used in a conventional nuclear receptor-reporter assay with these CLuc mutants and established a secretory luciferase-based single-substrate dual-color nuclear receptor-reporter assay.

Calcium-Binding Generates the Semi-Clathrate Waters on a Type II Antifreeze Protein to Adsorb onto an Ice Crystal Surface.

Hydration is crucial for a function and a ligand recognition of a protein. The hydration shell constructed on an antifreeze protein (AFP) contains many organized waters, through which AFP is thought to bind to specific ice crystal planes. For a Ca2+-dependent species of AFP, however, it has not been clarified how 1 mol of Ca2+-binding is related with the hydration and the ice-binding ability. Here we determined the X-ray crystal structure of a Ca2+-dependent AFP (jsAFP) from Japanese smelt, Hypomesus nipponensis, in both Ca2+-bound and -free states. Their overall structures were closely similar (Root mean square deviation (RMSD) of Cα = 0.31 Å), while they exhibited a significant difference around their Ca2+-binding site. Firstly, the side-chains of four of the five Ca2+-binding residues (Q92, D94 E99, D113, and D114) were oriented to be suitable for ice binding only in the Ca2+-bound state. Second, a Ca2+-binding loop consisting of a segment D94-E99 becomes less flexible by the Ca2+-binding. Third, the Ca2+-binding induces a generation of ice-like clathrate waters around the Ca2+-binding site, which show a perfect position-match to the waters constructing the first prism plane of a single ice crystal. These results suggest that generation of ice-like clathrate waters induced by Ca2+-binding enables the ice-binding of this protein.

Ice recrystallization is strongly inhibited when antifreeze proteins bind to multiple ice planes.

Ice recrystallization is a phenomenon observed as the increase in ice crystal size within an already frozen material. Antifreeze proteins (AFPs), a class of proteins capable of arresting ice crystal growth, are known to inhibit this phenomenon even at sub milli-molar concentrations. A tremendous range in the possible applications of AFPs is hence expected in both medical and industrial fields, while a key determinant of the ice recrystallization inhibition (IRI) is hardly understood. Here, IRI efficiency and ice plane affinity were examined for the wild-type AFPI-III, a defective AFPIII isoform, and a fungal AFP isoform. To simplify the IRI analysis using the formal representation of Ostwald-ripening (r3 = r03 + kt), we monitored specific ice grains exhibiting only uniform growth, for which maximum Feret diameter was measured. The cube of an ice grain's radius (r3) increased proportionately with time (t), and its slope gave the recrystallization rate (k). There was a significant difference in the IRI efficiency between the samples, and the fungal AFP possessing the activity with the smallest amount (0.27 µM) exhibited an affinity to multiple ice planes. These results suggest that the IRI efficiency is maximized when AFPs bind to a whole set of ice planes.