Genetic Perturbation Alters Functional Substates in Alkaline Phosphatase.

Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new functions. Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, we quantitatively analyzed functional substates in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase by employing a high-throughput single-molecule assay with a femtoliter reactor array device. Interestingly, many mutant enzymes exhibited significantly heterogeneous functional substates with various types, while the wild-type enzyme showed a highly homogeneous substate. We identified a correlation between the degree of functional substates and the level of improvement in promiscuous activities. Our work provides much comprehensive evidence that the functional substates can be easily altered by mutations, and the evolution toward a new catalytic activity may involve the modulation of the functional substates.

Multidimensional Digital Bioassay Platform Based on an Air-Sealed Femtoliter Reactor Array Device.

Single-molecule experiments have been helping us to get deeper inside biological phenomena by illuminating how individual molecules actually work. Digital bioassay, in which analyte molecules are individually confined in small compartments to be analyzed, is an emerging technology in single-molecule biology and applies to various biological entities (e.g., cells and virus particles). However, digital bioassay is not compatible with multiconditional and multiparametric assays, hindering in-depth understanding of analytes. This is because current digital bioassay lacks a repeatable solution-exchange system that keeps analytes inside compartments. To address this challenge, we developed a digital bioassay platform with easy solution exchanges, called multidimensional (MD) digital bioassay. We immobilized single analytes in arrayed femtoliter (10-15 L) reactors and sealed them with airflow. The solution in each reactor was stable and showed no cross-talk via solution leakage for more than 2 h, and over 30 rounds of perfect solution exchanges were successfully performed. With multiconditional assays based on our system, we could quantitatively determine inhibitor sensitivities of single influenza A virus particles and single alkaline phosphatase (ALP) molecules, which has never been achieved with conventional digital bioassays. Further, we demonstrated that ALPs from two origins can be precisely distinguished by a single-molecule multiparametric assay with our system, which was also difficult with conventional digital bioassays. Thus, MD digital bioassay is a versatile platform to gain in-depth insight into biological entities in unprecedented resolution.

Selection of cDNA candidates that induce oligomerization of NLRP3 using a chimeric receptor approach.

Since diverse cellular events are regulated by protein oligomerization, identification of molecules that affect oligomerization of a target protein is important for understanding cellular physiology and developing therapeutics. In this study, we aimed to screen cDNA candidates that induce oligomerization of NLRP3, which is one of the important inflammatory sensor proteins, in mammalian cytoplasm. In our screening method, the chimera composed of NLRP3 and the kinase domain of c-kit, one of the receptor tyrosine kinases (RTKs) activated by oligomerization, is expressed in cytoplasm of an IL-3-dependent mammalian cell line. The cells are then transduced with a cDNA library, and cultured in the absence of IL-3. If the transduced cDNA is a NLRP3 activator, the kinase domain of the NLRP3-c-kit chimera is activated by oligomerization, which induces cell growth even in the absence of IL-3. Using this system, constitutive oligomers of two NLRP3 variants were clearly detected by cell growth. Furthermore, cDNA screening resulted in identification of three distinct cDNAs that are potential candidates of NLRP3 activators. These results demonstrate the utility of our chimeric receptor-based system for screening candidates that induce oligomerization of a target protein.