Total Chemical Synthesis of a Unique Transcription Factor-Related Protein: cMyc-Max

Abstract

cMyc and Max are proteins that regulate gene expression by associating to form noncovalent dimers that bind to specific regions of double-stranded DNA, thus activating or inhibiting mRNA transcription. Each of these transcription factor proteins contains an -90 amino acid residue basichelix-loop-helidzipper (b/HLWZ) domain responsible for formation of the protein dimers and for DNA binding. Noncovalent heterodimers of the cMyc and Max b/HLWZ domains are believed to be functionally important in vivo but have not been accessible by conventional means (recDNA expression). Here we report the total chemical synthesis of novel covalently-linked dimers of the b/HLWZ domains of these transcription factors by a modular strategy based on the convergent chemical ligation of several unprotected peptide segments. The resulting -20 kDa synthetic proteins were purified to homogeneity and characterized by electrospray mass spectrometry. The synthetic covalent cMyc-Max heterodimer and the covalent Max homodimer control were shown to bind DNA specifically at the region involved in the regulation of mRNA transcription. Access to defined heterodimeric forms of the b/HLWZ transcription factors will allow the structural and functional properties of these important protein regulators of gene expression to be studied.

Nucleophilic Catalysis of Oxime Ligation

“…[1][2][3][4][5][6][7][8] Aldehydes and ketones can be readily introduced into biomolecules and are virtually inert towards reaction with other functional groups in these molecules. Under acidic conditions the carbonyl group reacts with primary amines to form a reversible imine (Scheme 1 a), and the equilibrium favors the free carbonyl.…”

“…However, when a-effect nitrogens [9] such as aminooxy groups and hydrazides are used, the equilibrium favors the imine. [1][2][3][4][5][6][7][8] Oxime ligations are often called upon to link complex and precious macromolecules. [7,8] The oxime bond is stable under physiological conditions, whereas more dynamic imines, such as hydrazones, are often reduced to obtain a stable linkage.…”

“…Furthermore, aniline is soluble in a range of solvents and can be used as a convenient aqueous buffer with acetic acid, and both its solubility and nucleophilicity can be tuned through the introduction of substituents on the aromatic ring. Aniline catalysis will have a major impact on the numerous applications of imine chemistry including bioconjugation, [1][2][3][4][5][6][7][8] cellular labeling, [18,19] surface immobilization, [20] and dynamic combinatorial libraries. [21,22] .…”

Polypeptide functionalized gold nanoparticles for bioanalytical applications

“…This problem can however be circumvented by employing ligation techniques that allow for conjugation of separate peptide segments into full length proteins. [77][78] In SPPS polystyrene beads cross-linked with a low degree of divinylbenzene is often employed as the solid support. 72 The beads are further functionalized with linkers that can anchor the C-terminal amino acid to the support.…”

Synthesis of an Analog of the Thyroid Hormone-binding Protein Transthyretin via Regioselective Chemical Ligation

“…These include the synthesis of linked heterodimeric basic helix-loop-helix transcription factors (22) and Cpn10 (23), respectively. Recently, techniques for forming a native amide bond have been applied to a range of protein types including serine protease inhibitors (24 -26), human II secretory phospholipase A2 (27), and barnase (28,29).…”