Exploring proteins in living cells

Abstract

<p indent="0mm">Proteins have been studied for over <sc>200 years,</sc> but much of that work has been performed <italic>in vitro</italic>. This article briefly summarized the current status of protein exploration in living cells, and then presented some of my own representative findings in this field. First, I summarized the major methodologies hitherto developed for studying proteins <italic>in vivo</italic>, including visualizing a target protein by fusing it with the green fluorescent protein; investigating the tertiary structure of the target protein via in-cell nuclear magnetic resonance spectroscopy; and exploring the behaviors of a target protein by genetically labeling it with different types of unnatural amino acids. Next, I described how my laboratory applied and modified an <italic>in vivo</italic> methodology in which the genetic introduction of a photoactivatable, unnatural amino acid at a selected residue position in a target protein then mediates crosslinking of that protein with other proteins when the cells are irradiated by UV light. By improving that method, my laboratory contributed the following: (1) The creation of a bacterial strain with a genome that has a gene that encodes the orthogonal aminoacyl-tRNA synthetase and one that encodes the tRNA that are both needed to incorporate an unnatural amino acid (i.e., Bpa) into any target protein. (2) The development of a system in which the unnatural amino acid is randomly introduced at each of the target protein’s amino acid residue positions and, after detecting a meaningful interaction, DNA sequencing subsequently identifies that unnatural amino acid’s exact position. (3) The incorporation of the unnatural amino acid into a target protein by directly modifying the endogenous target gene in the genomic DNA so that the variant protein is produced spatiotemporally identical to that of the wild-type protein. (4) The development of a high-throughput polyacrylamide gel electrophoresis system in which 384 protein samples can be analyzed in a single gel. Finally, I described a few representative results from my lab by using <italic>in vivo</italic> protein photocrosslinking analysis: (1) The serendipitous discovery of a reversible subcellular structure (regrowth-delay body) that forms when a highly selected group of important proteins assembles only in non-dividing bacterial cells, and then dissolves when the cells resume growth. Their presence thus marks dormant persister bacterial cells that are known to tolerate antibiotics. (2) A revelation of a “gear shifting” mechanism that modulates bacterial ATP synthase activity via adaptation of its ε-subunit to the “inserted” or “non-inserted” conformation as the cell responds to a variety of favorable to unfavorable cellular conditions. (3) The identification of a supramolecular protein complex that is composed of proteins present in the cytoplasm, inner membrane, periplasm and outer membrane of Gram-negative bacteria, and that mediates the biogenesis of a group of outer membrane proteins. (4) The unveiling of a unique way that the acid stress chaperone HdeA acts. It exhibits biological activity while its tertiary structure becomes largely disordered, and then it protects other chaperones under acidic conditions. (5) A clarification that the long-designated bifunctional protein DegP acts primarily as a proteinase with little chaperone activity. (6) The discovery that small heat shock proteins are activated in a temperature-responsive, cascade manner that allows them to significantly increase their biological activity at high temperatures. I then conclude with a list of unresolved issues regarding protein exploration in living cells.