A Phage Display-based Method for Determination of Relative Affinities of Mutants

Abstract

We propose phage display combined with enzymelinked immunosorbent assay as a tool for the systematic analysis of protein-protein interactions by investigating the binding behavior of variants to a partner protein. Via enzyme-linked immunosorbent assay we determine both the amount of fusion protein presented at the phage surface and the amount of complex formed, the ratio of which is proportional to the affinity. Hence this method enables us to calculate the relative affinities of a large number of mutants. As model systems, we investigated actin-binding motifs conserved in a number of proteins binding monomeric or filamentous actin. The hexapeptide motifs LKKTET, present in thymosin ␤4, and LKKEKG, present in the villin headpiece, were mutated, and the variants were analyzed. Study of the positional tolerance allows postulating that the motifs, although similar in primary structures adopt different conformations when bound to actin. In addition, our data show that the second and the fourth amino acid of the thymosin ␤4 motif and the first three residues of the villin headpiece motif are most important for actin binding. The latter result challenges the charged crown hypothesis for the villin headpiece filamentous actin interaction.As a consequence of various genome-sequencing projects, novel proteins are being identified, many of which may take part in new interactions. Efforts are going on to tackle the discovery of protein-protein interactions globally (1), but there is also need for novel methods for the analysis of these interactions, preferably easy, reliable, and high through-put techniques. Several means to probe amino acids that contribute to the binding of two proteins have been developed. X-ray crystallography and NMR may yield superior information about the interface of proteins at atomic resolution (2, 3). However, both methods are intrinsically difficult (even for noncomplexed proteins) and time-consuming and require expensive and sophisticated equipment, as well as large amounts of biological material. Cross-linking of interacting proteins followed by mass spectroscopy or conventional sequence determination of covalently coupled peptide fragments has limited application (4 -6). Probing the accessible surface in protein complexes by deuterium exchange has also been employed (7). But by far the two most popular methods are the use of peptide mimetics either in solution (8) or on membranes (9, 10) and especially site-directed mutagenesis (11, 12). However, in the latter powerful technique to study functions of proteins or interactions between proteins, one often faces the difficulty of choosing the position and type of amino acid exchange to be introduced. To circumvent this, one may perform a saturation mutagenesis at several positions thought to be important for the interaction. Obviously this creates a new problem, i.e. screening a large number of mutants in a systematic way.We propose to combine saturation mutagenesis, phage display and ELISA 1 for the systematic screening and quantification of pr...

Glutamate provides a key structural contact between reticulon-4 (Nogo-66) and phosphocholine

“…As shown previously [27], the ratio of the Nogo-66 binding levels (A) to display levels (A o ) has a linear correlation to the K A for the interaction, provided the concentration of the target protein (NgR) greatly exceeds the concentration of the displayed protein. This is a reasonable assumption for a phage-displayed 7.5 kD protein.…”

Dissecting binding of a β-barrel membrane protein by phage display

“…SI 5A and Fig. SI 5B); the modest degree of variablilty was accounted for by normalization as described previously 31 . The results confirm all loop variants used for this study are displayed on the bacteriophage with similar levels.…”

The Scope of Phage Display for Membrane Proteins

“…As shown previously, the ratio of the protein display levels (A o ) to binding levels (A) has a linear correlation to the K D values for the interactions. 29 The linear correlation requires the concentration of the target protein (f-actin) to exceed the concentration of the displayed protein, which is a reasonable assumption for a phage-displayed protein. The constitutively active neuromodulin (S41D) bound to f-actin with 4-fold higher affinity than wild-type, a measurement within the range of previous measurements made without phage display 27 (Fig.…”