Biology subjects

Nagashima, T.

Publications and source records attributed to Nagashima, T..

3 recordsLinked to original sources

bioRxiv2021

Exploration of novel αβ-protein folds through de novo design

Most naturally occurring protein folds have likely been discovered1-3. The question is whether natural evolution has exhaustively sampled almost all possible protein folds4, or whether a large fraction of the possible folds remains unexplored5-7. To address this question, we introduce a set of rules for {beta}-sheet topology to predict novel folds, and carry out the systematic de novo protein design for the novel folds predicted by the rules. The rules predicted eight novel {beta}-folds with a four-stranded {beta}-sheet, including a knot-forming one. We designed proteins for all the predicted {beta}-folds and found that all the designs are monomeric with high thermal stability and fold into the structures close to the design models, demonstrating the ability of the set of rules to predict novel {beta}-folds. The rules also predicted about twelve thousand novel {beta}-folds with five- to eight-stranded {beta}-sheets; the number is far exceeding the number of {beta}-folds observed so far. This result suggests that the enormous number of {beta}-folds are possible but have not emerged or become extinct due to evolutionary bias. The predicted novel folds should open up the possibility of designing functional proteins of our interests.

biophysics↗

bioRxiv2021

Design of complicated all-α protein structures

A wide range of de novo protein structure designs have been achieved, but the complexity of naturally occurring protein structures is still far beyond these designs. To expand the diversity and complexity of de novo designed protein structures, we sought to develop a method for designing "difficult-to-describe"-helical protein structures composed of irregularly aligned -helices like globins. Backbone structure libraries consisting of a myriad of -helical structures with 5- or 6-helices were generated by combining 18 helix-loop-helix motifs and canonical -helices, and five distinct topologies were selected for de novo design. The designs were found to be monomeric with high thermal stability in solution and fold into the target topologies with atomic accuracy. This study demonstrated that complicated -helical proteins are created using typical building blocks. The method we developed would enable us to explore the universe of protein structures for designing novel functional proteins.

biophysics↗

bioRxiv2021

Compartmentalized three-dimensional human neuromuscular tissue models fabricated on a well-plate-format microdevice

Engineered three-dimensional models of neuromuscular tissues are promising for use in mimicking their disorder states in vitro. Although several models have been developed, it is still challenging to mimic the physically separated structures of motor neurons (MNs) and skeletal muscle (SkM) fibers in the motor units in vivo. In this study, we aimed to develop microdevices for precisely compartmentalized coculturing of MNs and engineered SkM tissues. The developed microdevices, which fit a well of 24 well plates, had a chamber for MNs and chamber for SkM tissues. The two chambers were connected by microtunnels for axons, permissive to axons but not to cell bodies. Human iPSC (hiPSC)-derived MN spheroids in one chamber elongated their axons into microtunnels, which reached the tissue-engineered human SkM in the SkM chamber, and formed functional neuromuscular junctions with the muscle fibers. The cocultured SkM tissues with MNs on the device contracted spontaneously in response to spontaneous firing of MNs. The addition of a neurotransmitter, glutamate, into the MN chamber induced contraction of the cocultured SkM tissues. Selective addition of tetrodotoxin or vecuronium bromide into either chamber induced SkM tissue relaxation, which could be explained by the inhibitory mechanisms. We also demonstrated the application of chemical or mechanical stimuli to the middle of the axons of cocultured tissues on the device. Thus, compartmentalized neuromuscular tissue models fabricated on the device could be used for phenotypic screening to evaluate the cellular type specific efficacy of drug candidates and would be a useful tool in fundamental research and drug development for neuromuscular disorders.

bioengineering↗