Keisuke Motone

The ability to sequence single protein molecules in their native, full-length form would enable a more comprehensive understanding of proteomic diversity. Current technologies, however, are limited in achieving this goal1,2. Here, we establish a method for the long-range, single-molecule reading of intact protein strands on a commercial nanopore sensor array. By using the ClpX unfoldase to ratchet proteins through a CsgG nanopore3,4, we provide single-molecule evidence that ClpX translocates substrates in two-residue steps. This mechanism achieves sensitivity to single amino acids on synthetic protein strands hundreds of amino acids in length, enabling the sequencing of combinations of single-amino-acid substitutions and the mapping of post-translational modifications, such as phosphorylation. To enhance classification accuracy further, we demonstrate the ability to reread individual protein molecules multiple times, and we explore the potential for highly accurate protein barcode sequencing. Furthermore, we develop a biophysical model that can simulate raw nanopore signals a priori on the basis of residue volume and charge, enhancing the interpretation of raw signal data. Finally, we apply these methods to examine full-length, folded protein domains for complete end-to-end analysis. These results provide proof of concept for a platform that has the potential to identify and characterize full-length proteoforms at single-molecule resolution. A technique for threading long protein strands through a nanopore by electrophoresis and back using a protein unfoldase motor, ClpX, enables single protein molecules to be analyzed multiple times with single-amino-acid sensitivity.

Occupying less than 1% of the seas, coral reefs are estimated to harbor ∼25% of all marine species. However, the destruction of coral reefs has intensified in the face of global climate changes, such as rising seawater temperatures, which induce the overproduction of reactive oxygen species harmful to corals. Although reef-building corals form complex consortia with bacteria and photosynthetic endosymbiotic algae of the family Symbiodiniaceae , the functional roles of coral-associated bacteria remain largely elusive. By manipulating the Symbiodiniaceae bacterial community, we demonstrated that a bacterium that produces an antioxidant carotenoid could mitigate thermal and light stresses in cultured Symbiodiniaceae isolated from a reef-building coral. Therefore, this study illuminates the unexplored roles of coral-associated bacteria under stressful conditions.

Cis-regulatory elements (CREs) are one of the important factors in controlling gene expression and elucidation of their roles has been attracting great interest. We have developed an improved method for analyzing a large variety of mutant CRE sequences in a simple and high-throughput manner. In our approach, mutant CREs with unique barcode sequences were obtained by biased randomization in a single PCR amplification. The original T7 promoter sequence was randomized by biased randomization, and the target number of base substitutions was set to be within the range of 0 to 5. The DNA library and subsequent transcribed RNA library were sequenced by next generation sequencers (NGS) to quantify transcriptional activity of each mutant. We succeeded in producing a randomized T7 promoter library with high coverage rate at each target number of base substitutions. In a single NGS analysis, we quantified the transcriptional activity of 7847 T7 promoter variants. We confirmed that the bases from -9 to -7 play an important role in the transcriptional activity of the T7 promoter. This information coincides with the previous researches and demonstrated the validity of our methodology. Furthermore, using an in vitro transcription/translation system, we found that transcriptional activities of these T7 variants were well correlated with the resultant protein abundance. We demonstrate that our method enables simple and high-throughput analysis of the effects of various CRE mutations on transcriptional regulation.