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Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR.

Lindfors HE, de Koning PE, Drijfhout JW, Venezia B, Ubbink M - J. Biomol. NMR (2008)

Bottom Line: Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially.Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined.It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

View Article: PubMed Central - PubMed

Affiliation: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands.

ABSTRACT
Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

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Structure of the nitroxide radical-containing amino acid 2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC)
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Fig1: Structure of the nitroxide radical-containing amino acid 2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC)

Mentions: For the study of peptide-protein interactions, labelling with 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC, Fig. 1) provides an alternative. TOAC is an amino acid with a stable nitroxide radical and a reduced mobility, due to its rigid structure. It can be incorporated directly into peptides via solid-phase synthesis (Marchetto et al. 1993; Toniolo et al. 1995; Martin et al. 2001) and recent advances in chemical protein synthesis (Nilsson et al. 2005) may also enable the incorporation of TOAC into proteins for paramagnetic NMR protein interaction studies. TOAC-containing peptides have been used extensively for EPR studies (Smythe et al. 1995; Pertinhez et al. 1997; Toniolo et al. 1998; Barbosa et al. 1999; McNulty et al. 2000; Victor and Cafiso 2001; Bettio et al. 2002; Nakaie et al. 2002; D’Amore et al. 2003; Schreier et al. 2004; Karim et al. 2004; Marsh 2006; de Deus Teixeira et al. 2007; Inbaraj et al. 2007). Despite the wide-spread use of TOAC for EPR, to our knowledge it has not been employed for structural studies using paramagnetic NMR. Here, we use PRE NMR spectroscopy to study the structure and dynamics of TOAC-labelled peptides binding to the Src homology 3 (SH3) domain of Src kinase. SH3 domains are ubiquitous interaction domains involved in a vast number of signal transduction pathways. These modular domains generally recognize and bind to proline-rich regions that can form polyproline type II helices, with a core motif of the form PxxP (Li 2005). The Src SH3 domain has been shown to bind to peptides derived from a region in focal adhesion kinase (FAK) with a sequence RALPSIPKL (Thomas et al. 1998). Using TOAC-labelled peptides derived from this region of FAK, we find that although the peptide-protein interaction is of a weak and transient kind, the peptides bind in a well-defined position relative to the protein.Fig. 1


Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR.

Lindfors HE, de Koning PE, Drijfhout JW, Venezia B, Ubbink M - J. Biomol. NMR (2008)

Structure of the nitroxide radical-containing amino acid 2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2480485&req=5

Fig1: Structure of the nitroxide radical-containing amino acid 2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC)
Mentions: For the study of peptide-protein interactions, labelling with 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC, Fig. 1) provides an alternative. TOAC is an amino acid with a stable nitroxide radical and a reduced mobility, due to its rigid structure. It can be incorporated directly into peptides via solid-phase synthesis (Marchetto et al. 1993; Toniolo et al. 1995; Martin et al. 2001) and recent advances in chemical protein synthesis (Nilsson et al. 2005) may also enable the incorporation of TOAC into proteins for paramagnetic NMR protein interaction studies. TOAC-containing peptides have been used extensively for EPR studies (Smythe et al. 1995; Pertinhez et al. 1997; Toniolo et al. 1998; Barbosa et al. 1999; McNulty et al. 2000; Victor and Cafiso 2001; Bettio et al. 2002; Nakaie et al. 2002; D’Amore et al. 2003; Schreier et al. 2004; Karim et al. 2004; Marsh 2006; de Deus Teixeira et al. 2007; Inbaraj et al. 2007). Despite the wide-spread use of TOAC for EPR, to our knowledge it has not been employed for structural studies using paramagnetic NMR. Here, we use PRE NMR spectroscopy to study the structure and dynamics of TOAC-labelled peptides binding to the Src homology 3 (SH3) domain of Src kinase. SH3 domains are ubiquitous interaction domains involved in a vast number of signal transduction pathways. These modular domains generally recognize and bind to proline-rich regions that can form polyproline type II helices, with a core motif of the form PxxP (Li 2005). The Src SH3 domain has been shown to bind to peptides derived from a region in focal adhesion kinase (FAK) with a sequence RALPSIPKL (Thomas et al. 1998). Using TOAC-labelled peptides derived from this region of FAK, we find that although the peptide-protein interaction is of a weak and transient kind, the peptides bind in a well-defined position relative to the protein.Fig. 1

Bottom Line: Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially.Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined.It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

View Article: PubMed Central - PubMed

Affiliation: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands.

ABSTRACT
Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

Show MeSH
Related in: MedlinePlus