Design and synthesis of a globin fold.

We propose a simple method to find an amino acid sequence that is foldable into a globular protein with a desired structure based on a knowledge-based 3D-1D compatibility function. An asymmetric alpha-helical single-domain structure of sperm whale myoglobin consisting of 153 amino acid residues was chosen for the design target. The optimal sequence to fit the main-chain framework has been searched by recursive generation of the protein 3D profile. The heme-binding site was designed by fixing His64 and His93 at the distal and proximal positions, respectively, and by penalizing residues that protrude into the space with a repulsive function. The apparent bumps among side chains in the computer model of the converged, self-consistent sequence were removed by replacing some of the bumping residues with smaller ones according to the final 3D profile. The finally obtained sequence shares 26% of sequence with the natural myoglobin. The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Escherichia coli. Analyses using size-exclusion chromatography, circular dichroism spectroscopy, and solution X-ray scattering showed that DG1 folds into a monomeric, compact, highly helical, and globular form with an overall molecular shape similar to the target structure in an aqueous solution. Furthermore, it binds a single heme per protein molecule, which exhibited well-defined spectroscopic properties. The radius of gyration of DG1 was determined to be 20.6 A, slightly larger than that of natural apoMb, and decreased to 19.5 A upon heme binding based on X-ray scattering analysis. However, the heme-bound DG1 did not stably bind molecular oxygen as natural globins do, possibly due to high conformational diversity of side-chain structures observed in the NMR and denaturation experiments. These results give insight into the relationship between the sequence selection and the structural uniqueness of natural proteins to achieve biological functions.     

Laboratory appraisal of optimal gaseous conditions for growth of zoonotic Helicobacter felis ATCC 49179

An attempt was made to assess the hitherto undescribed optimal gaseous conditions for growth of zoonotic Helicobacter felis, focusing on the ratio of spiral-forms amongst the whole cells examined. The largest mean colony diameter was obtained under the gaseous condition of O₂ 12% and CO₂ 10%. In analyzing the five day old colonies, the highest percentage of spiral forms (85.5%) was observed under the condition of O₂ 18% and CO₂ 5%. In contrast, the lowest percentage of spiral forms (2.3%) was demonstrated under the condition of O₂ 1% and CO₂ 10%. The condition of O₂ 12% and CO₂ 10% was concluded to be optimal for obtaining cells with the largest colony sizes, although colonies proliferated under such conditions definitely contain many more coccoid cells than spiral forms. In culturing H. felis strains, optimal gaseous conditions should be employed according to the purposes or preferences of study designs.     

Circular permutation of ligand‐binding module improves dynamic range of genetically encoded FRET‐based nanosensor

Quantitative measurement of small molecules with high spatiotemporal resolution provides a solid basis for correct understanding and accurate modeling of metabolic regulation. A promising approach toward this goal is the FLIP (fluorescent indicator protein) nanosensor based on bacterial periplasmic binding proteins (PBPs) and fluorescence resonance energy transfer (FRET) between the yellow and cyan variants of green fluorescent protein (GFP). Each FLIP has a PBP module that specifically binds its ligand to induce a conformation change, leading to a change in FRET between the two GFP variant modules attached to the N‐ and C‐termini of the PBP. The larger is the dynamic range the more reliable is the measurement. Thus, we attempted to expand the dynamic range of FLIP by introducing a circular permutation with a hinge loop deletion to the PBP module. All the six circularly permutated PBPs tested, including structurally distinct Type I and Type II PBPs, showed larger dynamic ranges than their respective native forms when used for FLIP. Notably, the circular permutation made three PBPs, which totally failed to show FRET change when used as their native forms, fully capable of functioning as a ligand binding module of FLIP. These FLIPs were successfully used for the determination of amino acid concentration in complex solutions as well as real‐time measurement of amino acid influx in living yeast cells. Thus, the circular permutation strategy would not only improve the performance of each nanosensor but also expand the repertoire of metabolites that can be measured by the FLIP nanosensor technology.