A phage display-based method for determination of relative affinities of mutants. Application of the actin-binding motifs in thymosin beta 4 and the villin headpiece.
We propose phage display combined with enzyme-linked 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 beta4, 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 beta4 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.
Rossenu
Stefaan
S
Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, B-9000 Ghent, Belgium.
Leyman
Shirley
S
Dewitte
Daisy
D
Peelaers
Danny
D
Jonckheere
Veronique
V
Van Troys
Marleen
M
Vandekerckhove
Joel
J
Ampe
Christophe
C
D000199:Actins / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D019151:Peptide Library; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics; D013947:Thymosin / Q000235:genetics* / Q000378:metabolism; D000099201:Thymosin beta(4)
D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; D013947:Thymosin; D000099201:Thymosin beta(4); C026615:villin