Carbene footprinting accurately maps binding sites in protein–ligand and protein–protein interactions

Abstract

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

“…As we noted previously and confirmed recently by Oldham et al [29], freezing increases the yield of insertion. We observed this to be true for all three reagents.…”

“…More recently, diazirine precursors have been incorporated into cross-linkers [27,28] and covalent labeling agents [23,29,30]. BCarbene footprinting^has been successfully demonstrated using gaseous diazirine [30], simple aliphatic diazirines [23,31,32], and most recently aromatic diazirine derivatives [29]. Carbene labeling appears to offer the hallmarks of an excellent, adaptable labeling tool.…”

Laser‐Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass‐Spectrometry‐Based Protein Footprinting

“…Reactive-species footprinting (prototype is •OH) provides general and fast reactions. [8] Fast photochemical oxidation of proteins (FPOP) (Fig.S1, SI p 3) affords a ready opportunity for broad-based implementation especially if the number of reactive species can be expanded to give broad coverage. [9] …”

Mass spectrometry-enabled structural biology of membrane proteins

“…Such methods have enabled MP folding to be monitored [215]. These fast reacting functional groups have recently been extended further to include diazirines which are also activated by laser irradiation [230,231]. This approach has the advantage that the diazirine probes used are membrane permeable, as shown by a recent study in which the majority of modifications were shown to be located in the transmembrane region of the MP, OmpF [231].…”

“…Recent work has revealed that a small hydrophobic, trifluoromethyl aryldiazirine probe could be used to footprint the membrane spanning regions of trimeric E. coli OmpF, with the trimerisation interface remaining relatively unlabeled when compared with the remainder of OMP [230,231]. A complementary approach to label proteins with trifluoromethyl radicals ( Å CF 3 ) has also been described recently [232].…”