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Limited information is available on inherent stabilities of four-chain-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain alpha-helical chains 21, 28, and 35 amino acids in length are 3.1 x 10(-12), 6.7 x 10(-23), and 1.0 x 10(-38) M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 microM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled-coil formation in light of the work presented here.

Syk is a cytoplasmic protein-tyrosine kinase containing two amino-terminal Src homology 2 domains that is activated following ligation of the B cell antigen receptor. Syk activation in B cells correlates with Syk tyrosine phosphorylation as well as with Syk SH2-mediated association with the tyrosine-phosphorylated Igα and Igβ B cell antigen receptor subunits. Tyrosine-phosphorylated peptide 20-mers representing Igα and Igβ immunoreceptor tyrosine activation motifs were synthesized and found to stimulate the specific activity of Syk by as much as 10-fold in vitro. Maximal phosphopeptide-induced Syk activation required both Syk SH2 domains and phosphorylation of both tyrosine residues present in the immunoreceptor tyrosine activation motif. The biochemical mechanism responsible for the phosphopeptide-induced Syk enzyme activation appears to be a function of Syk autophosphorylation. Our observations suggest the association of Syk tandem SH2 domains with the tyrosine-phosphorylated Igα and/or Igβ immunoreceptor tyrosine activation motifs in B cells stimulates Syk autophosphorylation leading to Syk enzyme activation.

Tryptophan alkylation by resin-derived carbocations and formation of diketopiperazine are two major yield reducing side reactions observed in Fmoc/t-Bu based solid-phase peptide synthesis. With the development of a versatile silicon-based linkage agent, «SAC», 4-[1-hydroxy-2-(trimethylsilyl)ethyl] benzoic acid (1), for the production of peptide C-terminal acids, these problems have been successfully solved. Demonstration of the advantages of using SAC linker was provided by synthesizing a C-terminal tryptophan-containing dodecapeptide and a C-terminal proline-containing undecapeptide. The pure peptides were isolated in 30-40% yields, a dramatic improvement compared to the syntheses using the conventional HMPA linker. SAC linker was prepared in two steps in 70% overall yield. Attachment of the Fmoc-amino acid-SAC linker derivatives to amino functionalized solid supports was best carried out using the performed 2,4-dichlorophenyl ester derivatives. Tha amino acid-SAC linkage was tested and found to be stable under typical peptide synthesis conditions. An additional feature of SAC linker is the ability to generate protected peptide fragments using either fluoride ions or 1% TFA/ CH 2 Cl 2 solution. The versatility of using SAC linker was demonstrated by preparation of several Boc- or Fmoc-protected tetrapeptides and a cyclic heptapeptide by either fluoridolysis methodology or treatment with dilute acid. Furthermore, formation of succinimide, a side reaction resulting from cyclization of β-tert-butyl ester protected aspartyl residues, under fluoridolysis conditions, was also investigated

A new phosphitylating agent, compatible with the Fmoc/t-butyl strategy for the production of phosphotyrosine containing peptides was synthesized. Our results demonstrated that high yields and high purities of phosphotyrosine peptides can be obtained using this phosphitylating agent.

Unhindered carboxylic acids were converted to their (±)-1-(4-methoxyphenyl)ethyl (Mpe) esters in good yields by condensation with (±)-1-(4-methoxyphenyl)ethanol under mild conditions. The Mpe esters were rapidly and quantitatively cleavable by either 1% TFA in CH 2 Cl 2 or 10% DCA in CH 2 Cl 2 , conditions which leave Boc, t-butyl ester and ether intact. An Mpe ester was removed by hydrogenolysis much more slowly than the analogous benzyl ester.

It is shown in this work that FMOC amino acid chlorides are also applicable to solid-phase syntheses by the simple expedient of in situ conversion to the appropriate N-hydroxybenzotriazole ester via reaction with a 1:1 mixture of HOBt and an appropriate tertiary amine. In cases where acid chlorides are inaccessible due to side-chain incompatibility, pentafluorophenyl esters or BOP-induced couplings represent convenient spot substitutes

Electrostatic interactions between charged amino acids often affect heterospecificity in coiled coils as evidenced by the interaction between the oncoproteins, fos and jun. Such interactions have been successfully exploited in the design of heteromeric coiled coils in a number of laboratories. It has been suggested that heterospecificity in these dimeric coiled-coil systems is driven not by specific electrostatic interactions in the heterodimers but rather by electrostatic repulsion acting to destabilize the homodimer state relative to the heterodimer state. We show that it is possible to design ion pair interactions that directly stabilize the heterotetrameric coiled-coil state. Synthetic peptides were used whose sequences are based on the C-terminal tetramerization domain of Lac repressor, as a model system for four-chain coiled coils (Fairman et al., 1995). These Lac-based peptides, containing either glutamic acid (Lac21E) or lysine (Lac21K) at all b and c heptad positions, only weakly self-associate but, when mixed, afford a highly stable heterotetramer. This study represents the first experimental evidence for the importance of the b and c heptad positions to the stability of coiled coils. Finally, pH dependence and NaCl dependence studies show that heterotetramer stability is driven by ion pair interactions between glutamate and lysine; these interactions contribute about 0.6 kcal/mol of stabilizing free energy for each potential glutamate-lysine pair.

Le substituant fluorenyl-9 methylene est etudie en tant que groupe N-protecteur d'α-aminoacides ou d'α-aminoesters permettant d'eviter la racemisation lors de synthese peptidique. La protection des α-aminoacides se fait par la reaction de ceux-ci avec l'[hydroxy methoxy methyl]-9 fluorene

Unhindered carboxylic acids were converted to their (±)-1-(4-methoxyphenyl)ethyl (Mpe) esters in good yields by condensation with (±)-1-(4-methoxyphenyl)ethanol under mild conditions. The Mpe esters were rapidly and quantitatively cleavable by either 1% TFA in CH 2 Cl 2 or 10% DCA in CH 2 Cl 2 , conditions which leave Boc, t-butyl ester and ether intact. An Mpe ester was removed by hydrogenolysis much more slowly than the analogous benzyl ester.

Pour prevenir la racemisation pendant la synthese peptidique, les auteurs ont essaye d'utiliser les groupes protecteurs p-phenylazobenzoyl et p-phenylazobenzoyloxycarbonyl pour la synthese de di- et tripeptides

Le substituant fluorenyl-9 methylene est etudie en tant que groupe N-protecteur d'α-aminoacides ou d'α-aminoesters permettant d'eviter la racemisation lors de synthese peptidique. La protection des α-aminoacides se fait par la reaction de ceux-ci avec l'[hydroxy methoxy methyl]-9 fluorene

Pour prevenir la racemisation pendant la synthese peptidique, les auteurs ont essaye d'utiliser les groupes protecteurs p-phenylazobenzoyl et p-phenylazobenzoyloxycarbonyl pour la synthese de di- et tripeptides