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An 11-residue peptide with the sequence DSLEFIASKLA was identified from a genomic library of Bacillus subtilis by phage display as an efficient substrate for Sfp phosphopantetheinyl transferasecatalyzed protein labeling by small molecule-CoA conjugates. We name this peptide the ''ybbR tag,'' because part of its sequence is derived from the ybbR ORF in the B. subtilis genome. The site of Sfp-catalyzed ybbR tag labeling was mapped to the underlined Ser residue, and the ybbR tag was found to have a strong tendency for adopting an ␣-helical conformation in solution. Here we demonstrate that the ybbR tag can be fused to the N or C termini of target proteins or inserted in a flexible loop in the middle of a target protein for site-specific protein labeling by Sfp. The short size of the ybbR tag and its compatibility with various target proteins, the broad substrate specificity of Sfp for labeling the ybbR tag with small-molecule probes of diverse structures, and the high specificity and efficiency of the labeling reaction make Sfp-catalyzed ybbR tag labeling an attractive tool for expanding protein structural and functional diversities by posttranslational modification.coenzyme A ͉ posttranslational modification ͉ Bacillus subtillis ͉ phage display

natural product biosynthesis ͉ enzymology ͉ organohalogen

Sfp phosphopantetheinyl transferase covalently attaches small-molecule probes including biotin and various organic fluorophores to a specific serine residue in the peptidyl carrier protein (PCP) or a short 11-residue peptide tag ybbR through a phosphopantetheinyl linker. We describe here a protocol for site-specific protein labeling by Sfp-catalyzed protein post-translational modification that includes (i) expression and purification of Sfp, (ii) synthesis of small-molecule probe-CoA conjugates, (iii) construction of target protein fusions with PCP or the ybbR tag, (iv) labeling PCP- or ybbR-tagged target protein fusions in cell lysates and on live cell surfaces and (v) imaging fluorophore-labeled cell surface receptors by fluorescence microscopy. To follow this protocol, we advise that you allow 3 d for the expression and purification of Sfp phosphopantetheinyl transferase, 1 d for the synthesis and purification of the small-molecule probe-CoA conjugates as the substrates of Sfp, 3 d for the cloning of target protein genes as fusions to the PCP or the ybbR tag in the appropriate plasmids and another 3 d for transfecting cell lines with the plasmids and the expression of PCP- or ybbR-tagged proteins. Labeling of the PCP- or the ybbR-tagged proteins in cell lysates or on cell surfaces should require only 15-30 min.

Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small molecules of diverse structures to the target proteins with high efficiency and specificity.

We report here the development of a general strategy for site-specific labeling of proteins with small molecules by posttranslational modification enzyme, phosphopantetheinyl transferase Sfp. The target proteins are expressed as fusions to the peptide carrier protein (PCP) excised from nonribosomal peptide synthetase, and Sfp catalyzes the covalent modification of a specific serine residue on PCP by the small molecule-phosphopantetheinyl conjugate. The labeling reaction proceeds with high specificity and efficiency, targeting PCP fusion proteins in the cell lysate. The PCP tag has been shown to be compatible with various proteins, and Sfp-catalyzed PCP modification, compatible with various small-molecule probes conjugated to coenzyme A, highlighting the potential of the PCP tag for site-specific protein labeling with small molecules.

While it is accepted that protein flexibility plays a role in protein folding, catalysis, and molecular recognition, few techniques are capable of the rigorous measurement of protein motions required to quantify flexibility. Three-pulse photon echo shift spectroscopy can be used to measure the time scale of protein motions, and we have used this technique, along with steady-state spectroscopy and binding and structural data, to examine the immunological evolution of protein flexibility in an anti-fluorescein antibody. Two light chain somatic mutations increase affinity for fluorescein by 12-fold but also significantly affect flexibility. Specifically, a rigidification of the protein is seen in each of three observable motions; two slower motions undergo decreased amplitudes of displacement, by 3-and 20-fold, respectively, in response to an applied force, and the distribution associated with the amplitude of a faster motion is narrowed upon somatic mutation. The somatic mutations appear to rigidify the antibody-fluorescein complex by more strongly anchoring fluorescein to the protein and by more tightly packing the complex. The data demonstrate that in addition to affinity, antibody dynamics are systematically manipulated during affinity maturation, and they imply that the evolution of protein flexibility may be a central component of the immune response. The results also reflect the type of protein rigidification that may be important for other biological interactions, such as protein-protein, protein-ligand or protein-drug, and enzymesubstrate recognition. M odels of molecular recognition, based on conformational selection (1-3), induced-fit (4-6), or lock-and-key-type mechanisms (5, 7), are central to describing virtually all proteinprotein and protein-ligand interactions. However, these models have been difficult to test, because they differ only in the flexibility of the protein, and protein flexibility has been difficult to quantify (8). Thus, studies of molecular recognition have not focused directly on flexibility, but instead have searched for manifestations of it, such as changes in on or off rates, varying binding entropies, and structural rearrangements. Nowhere is molecular recognition more important than in the immune system, where a finite number of receptors [antibodies (Ab) and T cell receptors] must bind a virtually infinite range of foreign molecules and peptides (9-16).While it is apparent that during the initial stages of an immune response, there must be Ab present that have a broad range of specificities, Ab may also be isolated that are highly specific. These highly specific Ab are typically isolated in the later stages of an immune response and are usually highly mutated compared with their corresponding germ-line gene sequences (17). It has thus been argued that during affinity maturation, somatic mutations act to rigidify the Ab, leading to more specific epitope recognition (9,13,(18)(19)(20)(21)(22)(23)(24). In this model, the binding sites of germ-line Ab (Ab before somatic mutation) ...

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