Probing Protein–Protein Interactions with a Genetically Encoded Photo‐crosslinking Amino Acid

Ai, Hui‐wang; Shen, Weijun; Sagi, Amit; Chen, Peng R.; Schultz, Peter G.

doi:10.1002/cbic.201100194

Cited by 113 publications

(99 citation statements)

References 39 publications

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“…Because EGS can cross-link multiple members of protein complexes, we sought to confirm that RcsF interactions with OmpA, C, and F and BamA are direct. To confirm the interactions and to identify specific sites of RcsF interaction with the OMPs, we used sitespecific photo-activated cross-linking with a genetically encoded pBPA amino acid (28).…”

Section: Resultsmentioning

confidence: 99%

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

Konovalova

Perlman

Cowles

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

114

173

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RcsF (regulator of capsule synthesis) is an outer membrane (OM) lipoprotein that functions to sense defects such as changes in LPS. However, LPS is found in the outer leaflet, and RcsF was thought to be tethered to the inner leaflet by its lipidated N terminus, raising the question of how it monitors LPS. We show that RcsF has a transmembrane topology with the lipidated N terminus on the cell surface and the C-terminal signaling domain in the periplasm. Strikingly, the short, unstructured, charged transmembrane domain is threaded through the lumen of β-barrel OM proteins where it is protected from the hydrophobic membrane interior. We present evidence that these unusual complexes, which contain one protein inside another, are formed by the Bam complex that assembles all β-barrel proteins in the OM. The ability of the Bam complex to expose lipoproteins at the cell surface underscores the mechanistic versatility of the β-barrel assembly machine.membrane biogenesis | protein folding | signal transduction | envelope stress response

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Section: Resultsmentioning

confidence: 99%

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

Konovalova

Perlman

Cowles

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

114

173

View full text Add to dashboard Cite

show abstract

“…More recently, Schultz et al [104] confirmed the interaction between cyclin-dependent kinase 5 (Cdk5) and its substrate using the photocross-linkable 3 -azibutyl-N-carbamoyllysine (AbK). An MbPylRS library was developed by randomizing the residues Leu270, Tyr271, Leu274, Cys313, and an additional Tyr349Phe [105] to obtain a mutant that could effectively charge AbK (Table 12.2).…”

Section: Applicationsmentioning

confidence: 88%

“…The RacGTP-dependent kinase-catalyzed phosphorylation of Pak1 by Cdk5 and its activator p35 was evaluated by coexpressing the Rac mutant, p35, Pak1, and Cdk5 with Asp144TAG in the presence of AbK. After the expression of the proteins and exposure to UV light, Cdk5 and Pak1 were cross-linked, confirming their interactions [104].…”

Section: Applicationsmentioning

confidence: 90%

Posttranslational Modification of Proteins Incorporating Nonnatural Amino Acids

Yang

Montclare

2012

Functional Polymers by Post‐Polymerization Modification

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“…2 (39)) has been genetically introduced into the active site of the kinase CDK5 (cyclin-dependent kinase 5) [125]. The downstream kinase PAK1 was identified by crosslinking.…”

Section: Applications In Mass Spectrometrymentioning

confidence: 99%

Biochemical analysis with the expanded genetic lexicon

2012

Anal Bioanal Chem

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The information used to build proteins is stored in the genetic material of every organism. In nature, ribosomes use 20 native amino acids to synthesize proteins in most circumstances. However, laboratory efforts to expand the genetic repertoire of living cells and organisms have successfully encoded more than 80 nonnative amino acids in E. coli, yeast, and other eukaryotic systems. The selectivity, fidelity, and site-specificity provided by the technology have enabled unprecedented flexibility in manipulating protein sequences and functions in cells. Various biophysical probes can be chemically conjugated or directly incorporated at specific residues in proteins, and corresponding analytical techniques can then be used to answer diverse biological questions. This review summarizes the methodology of genetic code expansion and its recent progress, and discusses the applications of commonly used analytical methods.

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Probing Protein–Protein Interactions with a Genetically Encoded Photo‐crosslinking Amino Acid

Cited by 113 publications

References 39 publications

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

Posttranslational Modification of Proteins Incorporating Nonnatural Amino Acids

Biochemical analysis with the expanded genetic lexicon

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