Determination of Affinities for lck SH2 Binding Peptides Using a Sensitive Fluorescence Assay: Comparison between the pYEEIP and pYQPQP Consensus Sequences Reveals Context-Dependent Binding Spe

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• 作者：Ruby C. Cousins-Wasti ; Richard H. Ingraham ; Maurice M. Morelock ; and Christine A. Grygon
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：December 24, 1996
• 年：1996
• 卷：35
• 期：51
• 页码：16746 - 16752
• 全文大小：249K
• 年卷期：v.35,no.51(December 24, 1996)
• ISSN：1520-4995

文摘

The development of a sensitive fluorescence binding assay forevaluating the binding ofphosphotyrosyl (pY) peptides to the recombinant SH2 domain oflck in solution is described. Severalfluorescent peptides containing the consensus sequence of the viralhamster polyoma middle T antigen(pYEEI) were characterized. The peptides contained either theacetamido-anilino-naphthyl sulfonic acid(AANS), acrylodan, or dansyl groups as fluorophores. The spectralfeatures of these probes werecharacterized in the presence and absence of the lck SH2domain. The binding affinities (K_d) forthefluorescent peptides studied ranged from 40 to 500 nM. Thefluorescent peptide containing the sequenceFTATEC(AANS)QpYEEIP exhibited the highest binding affinity(K_d = 3.98 × 10^-8M) and largestchange in emission intensity (ages/entities/ap.gif">8.7-fold) upon binding the SH2 domain.This probe was subsequentlyused in competitive binding assays to study the interaction of thelck SH2 domain with a series ofphosphopeptides related to the pYEEIP and pYQPQP (the pY⁵⁰⁵C-terminal) consensus sequences. Theeffects of peptide length and substitutions of residues within thepYEEIP sequence are discussed in termsof binding affinities. Comparison between the two peptide seriesrevealed that the contributions ofindividual substitutions to binding affinity are context-dependent.The data also led to the conclusionthat the presence of P at +2 results in a functional "truncation"of the binding sequence; i.e., residues atpositions higher than +2 do not participate significantly in binding.This implicit truncation may actuallybe a desired property for the autoregulatory nature of the pYQPQPsequence, since it retains specificityfor the SH2 domain while adjusting the K_d to avalue appropriate for maintaining the delicate balance ofreceptor-ligand interactions that are involved in signal transductionevents.