Biogenesis, quality control, and structural dynamics of proteins as explored in living cells via site-directed photocrosslinking

Abstract

Protein biogenesis and quality control are essential to maintaining a functional pool of proteins and involve numerous protein factors that dynamically and transiently interact with each other and with the substrate proteins in living cells. Conventional methods are hardly effective for studying dynamic, transient, and weak protein-protein interactions that occur in cells. Herein, we review how the site-directed photocrosslinking approach, which relies on the genetic incorporation of a photoreactive unnatural amino acid into a protein of interest at selected individual amino acid residue positions and the covalent trapping of the interacting proteins upon ultraviolent irradiation, has become a highly efficient way to explore the aspects of protein contacts in living cells. For example, in the past decade, this approach has allowed the profiling of the in vivo substrate proteins of chaperones or proteases under both physiologically optimal and stressful (e.g., acidic) conditions, mapping residues located at protein interfaces, identifying new protein factors involved in the biogenesis of membrane proteins, trapping transiently formed protein complexes, and snapshotting different structural states of a protein. We anticipate that the site-directed photocrosslinking approach will play a fundamental role in dissecting the detailed mechanisms of protein biogenesis, quality control, and dynamics in the future.

Keywords: dynamics of proteins; in vivo protein photocrosslinking; membrane protein biogenesis; molecular chaperones; proteases; protein quality control.

Figure 1

The general procedure for site‐specific (a) or random (b) incorporation of unnatural amino acid into a selected target protein in living cells. (a) The unnatural amino acid (the red ball) enters the cell that produces an orthogonal aminoacyl‐tRNA synthetase and an orthogonal tRNA (being usually expressed from a plasmid, but is expressed from the modified genome of the LY928 bacterial strain21) and is covalently linked to the orthogonal tRNA (carrying 5′ CUA 3′ as the anticodon) upon the catalysis of the orthogonal aminoacyl tRNA synthetase (left). The synthesized aminoacyl tRNA is then delivered to the ribosome to pair with the amber codon (5′ UAG 3′) on the mRNA transcribed from the genetically modified target gene (by site‐directed mutagenesis) that is usually carried by a plasmid (middle). A variant target protein in which an unnatural amino acid is incorporated at a selected position is produced (right). (b) Briefly,22 the chloramphenicol‐resistant gene (chl^R) was randomly inserted into the target gene (usually carried by an expression plasmid) with the help of the MuA transposase. The plasmids were then digested with the restriction enzyme MlyI, which removes the chl^R gene fragment by generating two blunt ends. Such linear plasmids were subsequently ligated with a kanamycin‐resistant gene (kana^R) carrying a TAG codon (the amber codon for unnatural amino acid incorporation) at one end, digested again with the MlyI restriction enzyme before being religated to form sealed plasmids. These manipulations randomly replace each codon of the target gene by a TAG amber codon (as indicated by the red asterisk), generating a library of plasmids each expressing a variant target protein having an unnatural amino acid introduced at a different residue position. This library was then applied to functional screening and photocrosslinking analysis. The nature of a variant (i.e., which residue is replaced by an unnatural amino acid) that forms photocrosslinked products is eventually characterized by a simple determination of its encoding DNA sequence.

Figure 2

Chemical structures (a) and photocrosslinking mechanisms (b) of the common unnatural amino acids. (a) The common photoreactive unnatural amino acids that are designed mainly as analogues of phenylalanine (top) or pyrrolysine (bottom) are applied for site‐specific incorporations. The others (photo‐Leu, photo‐Ile, photo‐Met, and photo‐Lys) are used for nonspecific incorporations (right).27, 28 pBpa, p‐benzoyl‐l‐phenylalanine20; pAzpa, p‐azido‐l‐phenylalanine29; TmdPhe, 4′‐[3‐(trifluoromethyl)‐3H‐diazirin‐3‐yl]‐l‐phenylalanine30; Abk, 3′‐azibutyl‐N‐carbamoyllysine31; TmdZLys, N ^ε‐[((4‐(3‐(trifluoromethyl)‐3H‐diazirin‐3‐yl)‐benzyl)oxy)carbonyl]‐l‐lysine10; DiZPK, 3‐(3‐methyl‐3H‐diazirin‐3‐yl)‐propaminocarbonyl‐N ^ε‐l‐lysine32; DiZSek, N ^ε‐3‐(3‐methyl‐3H‐diazirin‐3‐yl)‐propaminocarbonyl‐γ‐seleno‐l‐lysine33; DiZHSeC, Se‐(N‐(3‐(3‐methyl‐3H‐diazirin‐3‐yl)propyl)propanamide)‐3‐ylhomoselenocysteine;34 and DiZASeC.35 Note that DiZSek, DiZHSeC, and DiZASeC (colored red) are all cleavable photocrosslinkers. (b) Shown here are the three common photoactivation mechanisms. A diazirine group incorporated into the target protein (X) is activated upon ultraviolet irradiation at 350 nm to form a carbine, which would be immediately covalently bonded with a neighboring XH group of the interacting protein (P), leading to an intermolecular covalent crosslinkage (left). The benzophenone will be activated to form a diradical (middle), while the azide group will be activated to form a nitrene (right) before covalently linked to an XH group nearby.

Figure 3

Major pathways of the biogenesis and quality control of proteins existing in Gram‐negative bacteria. They could be categorized into three major pathways according to the final subcellular destinations of the proteins. The first pathway deals with the secretory proteins. Their nascent polypeptides, after being synthesized by the cytosolic ribosomes, are destined to the protein‐conducting channels (the SecYEG or SecA^N complexes) on the inner membrane (IM) for translocation, whose driving force is apparently provided by the SecA motor. Among the secretory proteins, the periplasmic proteins fold into their native conformations (N) after being translocated across the IM, while the outer membrane β‐barrel proteins (OMPs) (with the outer membrane lipoproteins being omitted for simplicity) are protected by such chaperones as SurA and delivered to the β‐barrel assembly machinery (BAM) complex for the final folding and integration into the outer membrane. A supercomplex for the biogenesis of OMPs seems to be formed in living cells (as shown with a dashed frame). The second pathway deals with the IM (i.e., cytoplasmic membrane) proteins. Their nascent polypeptides are probably also delivered to the SecYEG channel as guided by the signal recognition particle before being inserted into the IM with the help of the insertase YidC. The third pathway deals with the cytosolic proteins. Their nascent polypeptides fold into their native conformations, either cotranslationally under the assistance of such chaperones as the trigger factors and/or DnaK/DnaJ, or posttranslationally within the “Anfinsen” cage formed by the GroEL/GroES complex. Upon stress (e.g., heat shock), the unfolded proteins tend to aggregate unless they are protected by chaperones such as small heat shock proteins or degraded by proteases such as Lon/ClpP. Primary protein factors and complexes involved in protein biogenesis are diagrammed, mainly by referring to earlier review papers40, 41, 42 and our recent studies.21, 43

Figure 4

Certain proteins possess a highly dynamic structure in living cells. A “forbidden” residue (red ball) far away from the subunit “interfaces” (as revealed by in vitro structure determination) may become “permissive” to mediate the interaction of the protein subunits in living cells.91 This might happen through subunit rotation/reorientation (left arrow) and/or conformational change (right arrow) in living cells.