We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable new approaches to biological and medical challenges. Here we describe a novel motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1,000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of complexes containing designed and evolved proteins reveal binding is entirely through the designed interface, making use of specific designed interactions. Surprisingly, in the evolved complex one of the partners is rotated 180 degrees relative to the design model. This work demonstrates that current understanding of protein-protein interfaces is sufficient to rationally design interfaces de novo, and underscores remaining challenges.

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Escherichia coli biotin ligase (BirA) has been harnessed for numerous biotechnological applications, including protein labeling,1-3 purification,4,5 and immobilization.6 BirA catalyzes the ATP-dependent covalent ligation of biotin to a lysine sidechain of a 15-amino acid recognition sequence called the "acceptor peptide" (AP).7 Previously, we showed that BirA could be applied to site-specific protein labeling on the surface of living cells by making use of a ketone isostere of biotin that could be ligated to AP fusion proteins, then derivatized with hydrazide or hydroxylamine probes.8 Since that work, we have focused on expanding the small-molecule specificity of BirA to incorporate other useful functional groups, such as azides and alkynes, which can be derivatized with even more chemoselective reactions than the ketone.9 Because we found that BirA does not ligate diverse structural analogs of biotin to the AP, in this study, we explored the activities of biotin ligases from nine other organisms. We discovered that yeast biotin ligase accepts an alkyne derivative of biotin, while Pyrococcus horikoshii biotin ligase utilizes both alkyne and azide biotin analogs. These new ligation reactions demonstrate the differential substrate specificities of ligases from different species, and open the door to novel protein labeling applications.Due to our interest in novel methodologies for targeting chemical probes to proteins in the cellular context, and the previous work of our lab in exploiting E. coli BirA for this purpose, 1,8 we synthesized or purchased each of the biotin analogs shown in Figure 1. Desthiobiotin azide (DTB-Az) 2 and propargyl biotin (PB) 3, whose syntheses are shown in Figure 2, contain unique functional group handles. Azides are naturally absent from cells and can be selectively derivatized with strained alkynes or phosphines under physiological conditions,9 while alkynes, also absent from cells, can be selectively derivatized with azides via [3+2] cycloaddition in the presence of a copper catalyst.10 Iminobiotin 4 and diaminobiotin 5 exhibit pH-dependent binding to streptavidin and can be used for protein purification.11 Nitrobenzoxadiazole gamma-amino butyric acid 6 is a fluorophore, and iodouracil and thiouracil valeric acid probes (7 and 8) are photoactivatable crosslinkers.12We found that wild-type BirA does not ligate analogs 2-8 to the AP (data not shown). Because precedent exists for differential substrate specificity among homologous enzymes from different species,13,14 we decided to clone, express, and evaluate biotin ligases from nine other species. All organisms express one or two biotin ligases,15,16 which attach biotin to protein domains involved in the transfer of carboxyl groups. In some organisms, the ligase plays an additional role in repressing transcription at the biotin biosynthetic operon or other biotin-sensitive genes.17 To select our panel of novel biotin ligases, we first noted that biochemical and/or structural data were available for the human,18 S. cerevisiae (yeast),...

Site-specific protein labeling with Escherichia coli biotin ligase (BirA) has been used to introduce fluorophores, quantum dots (QDs), and photocross-linkers onto recombinant proteins fused to a 15-amino acid acceptor peptide (AP) substrate for BirA and expressed on the surface of living mammalian cells. Here, we used phage display to engineer a new and orthogonal biotin ligase-AP pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not recognize the AP, but we discovered a new 15-amino acid substrate for yBL called the yeast acceptor peptide (yAP), using two generations of phage display selection from 15-mer peptide libraries. The yAP is not recognized by BirA, and thus, we were able to specifically label AP and yAP fusion proteins coexpressed in the same cell with differently colored QDs. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is extremely sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to twocolor imaging and yeast-based applications.