Design, total chemical synthesis, and binding properties of a [Leu-91-N1-methyl-7-azaTrp]Ras-binding domain of c-Raf-1

被引:16
作者
Sydor, JR
Herrmann, C
Kent, SBH
Goody, RS
Engelhard, M
机构
[1] Max Planck Inst Mol Physiol, Phys Biochem Abt, D-44227 Dortmund, Germany
[2] Max Planck Inst Mol Physiol, Abt Strukturelle Biol, D-44227 Dortmund, Germany
[3] Gryphon Sci, San Francisco, CA 94080 USA
关键词
D O I
10.1073/pnas.96.14.7865
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The Res-binding domain (RBD) of c-Raf-1 has been synthesized chemically, taking advantage of the chemical ligation of two peptide fragments of the protein. This procedure allowed incorporation of an unnatural amino acid (N-1-methyl-7-azatryptophan) at position 91 of RED, producing a protein with fluorescent properties distinct from and distinguishable from those of proteins containing the natural fluorophore tryptophan. The resulting protein was shown to interact with Ras in a manner that was almost indistinguishable from that of unmodified RED based on transient kinetic monitoring of the binding event. Modified RED containing the L-isomer of the unnatural amino acid or its racemic D,L mixture appeared to interact identically with Ras. The approach demonstrates a general procedure for the introduction of unnatural amino acids that can be used for monitoring protein-protein interactions and for the introduction of an unnatural backbone structure at strategic positions.
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页码:7865 / 7870
页数:6
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