In This Issue

287 Examining post-translational modification-mediated protein–protein interactions using a chemical proteomics approach

Xiang Li, Emily A. Foley, Shigehiro A. Kawashima, Kelly R. Molloy, Yinyin Li, Brian T. Chait and Tarun M. Kapoor

Regulating interactions between proteins is an important function of posttranslational modifications (PTMs) across diverse biological processes, such as cell division, transcription and neuronal function. While a wide-range of PTMs have been characterized, identifying PTM-dependent protein-protein interactions remains challenging as PTMs can be dynamic and mediate relatively weak interactions. In this article, the authors develop and apply a robust method named CLASPI, which combines photo-cross-linking with quantitative mass spectrometry, to profile in native proteomes interactions mediated by PTMs, such as lysine trimethylation and threonine phosphorylation. This chemical proteomics approach is broadly applicable and can identify weak but selective PTM-dependent protein-protein interactions. 1

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258 Exploring the binding diversity of intrinsically disordered proteins involved in one-to-many binding

Wei-Lun Hsu, Christopher J. Oldfield, Bin Xue, Jingwei Meng, Fei Huang, Pedro Romero, Vladimir N. Uversky and A. Keith Dunker

Hsu etal. report that intrinsically disordered proteins utilize their flexibility to recognize diverse partners. Multiple short disordered regions of essentially identical sequence were observed to associate with dissimilar partners, some of which were similarly-folded and others of which were differently-folded. Backbone and side chain torsion angle rotations were observed to enable close fits between the partners. Alternative splicing and posttranslational modifications were also found to contribute to distinct partner binding, indicating that inherent flexibility, alternative splicing and post-translational surface modifications may collaborate to enable the same disordered segment to selectively associate with multiple partners. The small number of key residues in the disordered partner may simplify the evolvability of signaling network diversity. 2

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274 Protein prosthesis: β-peptides as reverse-turn surrogates

Ulrich Arnold, Bayard R. Huck, Samuel H. Gellman and Ronald T. Raines

One way to confer desirable attributes upon a protein is to remodel its framework. For example, oligomers of β-amino acids (“β-peptides”) are inert to proteolysis and can have high conformational stability. But, which β-peptides are truly useful as prosthetic modules? To begin to answer that question, expressed protein ligation was used to replace a reverse turn in a model protein with two distinct β-dipeptides. Enhanced conformational stability was conferred by a module that maintains a proper hydrogen-bonding pattern but not one that recapitulates hydrogen-bonded ring size. These findings highlight a benefit of well-chosen nonnatural structural elements. 3

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327 Protein lysine-Nζ alkylation and O-phosphorylation mediated by DTT-generated reactive oxygen species

Nigam Kumar, Hans Ippel, Christian Weber, Tilman Hackeng and Kevin H. Mayo

Under reducing conditions with dithiothreitol (DTT), proteins become Schiff base-alkylated at lysine-Nζ groups and O-phosphorylated at serine and threonine residues. Chemical adduction is mediated by DTT-generated reactive oxygen species (ROS) in the form of superoxide, as well as hydroxyl and phosphoryl radicals. These ROS-mediated reactions are promoted in the context of a well-folded protein with a structural signature involving clusters of positively charged and other polar groups, as illustrated in the accompanying figure that shows the most reactive lysine residues in the protein ubiquitin. Our findings stand as a cautionary note to investigators who use DTT in their research studies. 4

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