Fluorescence analysis of the size of a binding pocket of a peptide receptor at natural abundance.
We have studied the topography of interaction of a family of fluorescent formyl peptides containing four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys- fluorescein), and six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and CHO-Met-Leu-Phe-Phe-Phe-Lys- fluorescein) amino acids with their receptor using spectroscopic methods adapted to small sample volumes. Only the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, indicating physical contact of the chromophore with the receptor. In contrast, only the hexapeptides were accessible to antibodies to fluorescein. Taken together, these results suggest that the carboxy terminus of the tetrapeptide or the pentapeptide is protected in the receptor binding pocket while the fluorescein on the carboxy terminus of either hexapeptide is exposed and recognized by the antibody to fluorescein. These results indicate that the binding pocket accommodates at least five but no more than six amino acids.
Sklar
L A
LA
Scripps Clinic and Research Foundation, La Jolla, California 92037.
Fay
S P
SP
Seligmann
B E
BE
Freer
R J
RJ
Muthukumaraswamy
N
N
Mueller
H
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D019793:Fluorescein; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives*; D009504:Neutrophils / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013861:Thiocyanates; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016650:Fluorescein-5-isothiocyanate; D019793:Fluorescein