The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.

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Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.

https://science.sciencemag.org/content/sci/296/5567/503.full.pdf

Multicolor and Electron Microscopic Imaging of Connexin Trafficking

The investigation of biological processes by chemical methods, commonly referred to as chemical biology, often requires chemical access to biologically relevant macromolecules such as peptides and proteins. Building upon solid-phase peptide synthesis, investigations have focused on the development of chemoselective ligation and modification strategies to link synthetic peptides or other functional units to larger synthetic and biologically relevant macromolecules. This Review summarizes recent developments in the field of chemoselective ligation and modification strategies and illustrates their application, with examples ranging from the total synthesis of proteins to the semisynthesis of naturally modified proteins.

Chemoselective Ligation and Modification Strategies for Peptides and Proteins

The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.

Process of Protein Transport by the Type III Secretion System

Cell biology is being transformed by the use of fluorescent proteins as fusion tags to track protein behaviour in living cells. Here, we discuss the techniques of photobleaching and photoactivation, which can reveal the location and movement of proteins. Widespread applications of these fluorescent-based methods are revealing new aspects of protein dynamics and the biological processes that they regulate.

Photobleaching and photoactivation: following protein dynamics in living cells.

Publisher Summary FlAsH labeling of recombinant proteins for cellular localization studies can be considered an alternative to the popular method using green fluorescent protein (GFP) fusions, with the FlAsH method having some advantages. The size of the fluorescent tag is considerably smaller: bound FlAsH has a molecular weight of less than 600 and the addition of a FlAsH target site can be as small as four introduced cysteines with an appended peptide adding less than 2 kDa. This compares with a molecular weight of 30,000 for GFP, which is more likely to perturb the native structure and function of the tagged protein. Both FlAsH and GFP tagging generate a fluorescent protein with similar brightness. However, multiple FlAsH sites can be introduced into a protein so that considerably brighter labeling aid in detecting low-abundance proteins. A major advantage of the FlAsH system is the comparative ease of chemical modification of FlAsH. Coupled with the synthetic versatility of organic chemistry, this enables the incorporation of functionalities other than fluorescence into targeted proteins or peptides.

Adams

Papers

Fluorescent labeling of recombinant proteins in living cells with FlAsH.