Single-molecule FRET and crosslinking studies in structural biology enabled by noncanonical amino acids

Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle

“…Thus, it is possible to use non-natural reaction handles, such as azF, to modulate a protein interaction network through different chemical routes. Given the current importance of optogenetics 53,54 and non-native PTM, 1,6 additional and potentially versatile approaches to regulating protein function that are understood at the molecule level would be welcome. By targeting induced structural changes (as opposed to simple active site blocking), allosteric and non-catalytic bioprocesses may become accessible to nnPTM events using ncAAs, so expanding its use.…”

“…5,6 In natural biological systems, PTM is pivotal for modulating and supplementing protein function; 7 among the most important are permanent covalent modifications with their impact ranging from the introduction of new non-proteinaceous chemical features to induced conformational change. Protein modification using genetically encoded non-native chemistry has been used primarily for passive labelling with suitable reporter adducts (see 5,8,9 for recent reviews) rather than to modulate function.…”

“…SPAAC is a biocompatible and bioorthogonal reaction that is quickly emerging as a powerful approach to label biomolecules, including proteins, with various adducts both in vitro and in vivo . 1,6,22,23 Genetic encoding of azF 13 allows highly precise placement of phenyl azide chemistry opening up the opportunity to actively modulate of protein function via these two novel approaches. While a few studies have emerged recently 17,18 concerning the molecular basis of photochemical control, little is known beyond theoretical modelling 24 how attachment of click reagents such as cyclooctynes influence protein structure; both aspects thus lack a detailed molecular basis of action that could potentially aid future application with regards to new and general approaches to protein functional modulation.…”

The use of unnatural amino acids to study and engineer protein function

“…Recent developments in the field of genetic code expansion have expanded this repertoire by a plethora of bioorthogonal functionalities (8)(9)(10)(11)(12) [40,41], facilitating a number of elegant studies.…”

Deciphering Complexity in Molecular Biophysics with Single-Molecule Resolution

“…Such efforts coupled with creation of improved dyes by rational (based on mechanistic photophysics) or high-throughput means are expected to improve resolution in multiple ways. Improved methods and orthogonal means for site-specific dye labeling will continue to be made, building on efforts such as unnatural aminoacid, enzymatic, ligation and synthetic schemes, thus increasing the scope of SMF methods in more complex systems and for multicolor smFRET [49–52]. These newer multicolor experiments can provide additional layers of information on correlations between changes in different parts of molecules or complexes, but would suffer dramatically from a lack of site-specificity, for example by commonly used cysteine chemistry.…”