Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP.
We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.
Blond-Elguindi
S
S
Department of Biochemistry, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235.
Cwirla
S E
SE
Dower
W J
WJ
Lipshutz
R J
RJ
Sprang
S R
SR
Sambrook
J F
JF
Gething
M J
MJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D018833:Chaperonins; D016384:Consensus Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000378:metabolism*; D015723:Gene Library; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D017912:Inovirus / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009842:Oligopeptides / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C060153:KAR2 protein, yeast; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D011506:Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D018833:Chaperonins