No abstract

Single chain antibodies (scFvs) are engineered proteins composed of IgG variable heavy (V(H)) and variable light (V(L)) domains tethered together by a flexible peptide linker. We have characterized the individual V(H) or V(L) domain activities of several scFvs isolated from a yeast surface-display library for their ability to bind environmentally sensitive fluorogenic dyes causing them to fluoresce. For many of the scFvs, both V(H) and V(L) domains are required for dye binding and fluorescence. The analysis of other scFvs, however, revealed that either the V(H) or the V(L) domain alone is sufficient to cause the fluorogenic dye activation. Furthermore, the inactive complementary domains in the original scFvs either contribute nothing to, or actually inhibit the activity of these active single domains. We have explored the interactions between active variable domains and inactive complementary domains by extensive variable domain swapping through in vitro gene manipulations to create hybrid scFvs. In this study, we demonstrate that significant alteration of the fluorogenic dye activation by the active V(H) or V(L) domains can occur by partnering with different V(H) or V(L) complementary domains in the scFv format. Hybrid scFvs can be generated that have fluorogen-activating domains that are completely inhibited by interactions with other domains. Such hybrid scFvs are excellent platforms for the development of several types of genetically encoded, fluorescence-generating biosensors.

Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies.

Directed evolution is an exceptionally powerful tool that uses random mutant library generation and screening techniques to engineer or optimize functions of proteins. One class of proteins for which this process is particularly effective is antibodies, where properties such as antigen specificity and affinity can be selected to yield molecules with improved efficacy as molecular labels or in potential therapeutics. Typical antibody structure includes disulfide bonds that are required for stability and proper folding of the domains. However, these bonds are unable to form in the reducing environment of the cytoplasm, stymieing the effectiveness of optimized antibodies in many research applications. We have removed disulfide-forming cysteine residues in a single chain antibody fluorogen-activating protein (FAP), HL4, and employed directed evolution to select a derivative that is capable of activity in the cytoplasm. A subsequent round of directed evolution was targeted at increasing the overall brightness of the fluoromodule (FAP–fluorogen complex). Ultimately, this approach produced a novel FAP that exhibits strong activation of its cognate fluorogen in the reducing environment of the cytoplasm, significantly expanding the range of applications for which fluoromodule technology can be utilized.

Ubiquitin interacting motifs (UIMs) are short α-helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N-terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1-UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α-helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type.

Protein phosphorylation is the most abundant post-translational modification in cells. Src homology 2 (SH2) domains specifically recognize phosphorylated tyrosine (pTyr) residues to mediate signaling cascades. A conserved pocket in the SH2 domain binds the pTyr side chain and the EF and BG loops determine binding specificity. By using large phage-displayed libraries, we engineered the EF and BG loops of the Fyn SH2 domain to alter specificity. Engineered SH2 variants exhibited distinct specificity profiles and were able to bind pTyr sites on the epidermal growth factor receptor, which were not recognized by the wild-type Fyn SH2 domain. Furthermore, mass spectrometry showed that SH2 variants with additional mutations in the pTyr-binding pocket that enhanced affinity were highly effective for enrichment of diverse pTyr peptides within the human proteome. These results showed that engineering of the EF and BG loops could be used to tailor SH2 domain specificity, and SH2 variants with diverse specificities and high affinities for pTyr residues enabled more comprehensive analysis of the human phosphoproteome. Statement: Src Homology 2 (SH2) domains are modular domains that recognize phosphorylated tyrosine embedded in proteins, transducing these post-translational modifications into cellular responses.Here we used phage display to engineer hundreds of SH2 domain variants with altered binding specificities and enhanced affinities, which enabled efficient and differential enrichment of the human phosphoproteome for analysis by mass spectrometry. These engineered SH2 domain variants will be useful tools for elucidating the molecular determinants governing SH2 domains binding specificity and for enhancing analysis and understanding of the human phosphoproteome.

A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools used to enrich phosphotyrosine (pTyr) are limited in their applicability and scope. Here, we engineered new superbinder Src-Homology 2 (SH2) domains that enrich diverse sets of pTyr-peptides. We used phage display to select a Fes-SH2 domain variant (superFes; sFes1) with high affinity for pTyr and solved its structure bound to a pTyr-peptide. We performed systematic structure–function analyses of the superbinding mechanisms of sFes1 and superSrc-SH2 (sSrc1), another SH2 superbinder. We grafted the superbinder motifs from sFes1 and sSrc1 into 17 additional SH2 domains and confirmed increased binding affinity for specific pTyr-peptides. Using mass spectrometry (MS), we demonstrated that SH2 superbinders have distinct specificity profiles and superior capabilities to enrich pTyr-peptides. Finally, using combinations of SH2 superbinders as affinity purification (AP) tools we showed that unique subsets of pTyr-peptides can be enriched with unparalleled depth and coverage.

The 1029 series of mammary epithelial cell lines (D6, GP+E, r3 and r3T) are progressively more transformed: the latter two by val 12 ras. These cell lines respond to TGFβ by undergoing early events of epithelial-mesenchymal transition (EMT), including morphological changes and redistribution of E-cadherin. Tumors formed by r3T cells in the choroid of the eye express vimentin, a late marker of EMT, possibly in response to TGFβ. In vitro, vimentin expression is induced in all the cell lines by TGFβ treatment, while cytokeratin expression is only slightly affected. Surprisingly, ras transformation results in a 10-fold suppression of vimentin expression. Neither suppression of vimentin by ras transformation nor induction by TGFβ are mediated by the vimentin promoter in r3T cells. In transient transfection assays, several human vimentin promoter constructs are more active in the low-expressing r3T cell line than in the vimentin-expressing mesenchymal cell line NIH3T3. In the r3T cells, there is no effect of TGFβ treatment for 9 days on the activity of either promoter. Azacytidine treatment does not affect vimentin expression in either NIH3T3 or r3T, suggesting that promoter methylation is not the mechanism of suppression by ras. Finally, the half life of the vimentin mRNA is similar in both the r3T cells and NIH3T3 cells. We conclude that the suppression of vimentin expression by ras, and the relief of this suppression by TGFβ, occurs in a promoter-independent fashion, possibly through sequences in the first or second intron.