Keywordshelical structures; hydrogen bonds; molecular recognition; peptidomimetics; protein-protein interactionsThe α helix plays a fundamental role in imparting specificity to protein-protein and proteinnucleic acid interactions. Molecules that can predictably and selectively disrupt these interactions would be invaluable as tools in molecular biology and, potentially, as leads in drug discovery.[1] We recently described a new strategy for the synthesis of artificial α helices in which one main-chain i to i + 4 hydrogen bond in the target α helix is replaced with a carboncarbon bond derived from a ring-closing metathesis reaction (Figure 1). [2] A key feature of this hydrogen-bond surrogate (HBS) approach is that the internal placement of the cross-link affords short helices with minimal perturbations to their molecular recognition surfaces. This method differs significantly from the commonly employed side-chain cross-linking method for helix stabilization. A limitation of the latter approach is that side-chain functionality must be sacrificed to nucleate stable helical conformations. The modified side chains are unavailable for molecular recognition; moreover, the resulting tether blocks at least one face of the putative helix. The HBS approach uniquely allows the synthesis of artificial helices with all side chains available for molecular recognition, and does not place any steric encumbrances on the helix surface. We believe that our artificial α helices have the potential to target protein receptors and regulate protein-protein interactions more successfully than helices with cross-linked side chains.Our initial studies demonstrated that the HBS approach affords highly stable α helices from alanine-rich peptide sequences. Herein, we show that this metathesis-based method can effectively stabilize α-helical conformations in biologically relevant sequences, that the resulting molecules resist proteolytic degradation as compared with the unconstrained analogues, and that the HBS helices can bind a protein target with high affinity. For these proofof-principle protein-binding studies with our artificial helices, we chose to target the extensively studied α helix binding protein, Bcl-xL.[3] Bcl-xL is an antiapoptotic protein that regulates cell death by binding the α-helical BH3 domain of a family of proapoptotic proteins (including Bak, Bad, Bid, and Bax).[4-6] NMR spectroscopic studies by Fesik and co-workers have shown that the 16-mer peptide 1 derived from the Bak BH3 domain adopts an α-helical conformation upon binding to Several methods that afford stabilized α helices or helix mimetics have already been used to target Bcl-xL, thus allowing us to directly compare the performance of our internally constrained artificial α helices. [8][9][10] Significantly, Huang and co-workers recently reported that Bak BH3 α helices stabilized by a lactam-based side-chain cross-linking strategy were unable to bind Bcl-xl.[11] The authors speculated that the lack of binding might be a result of steric clashes between the c...
The activation mechanism of glycosylasparaginase of Flavobacterium meningosepticum has been analyzed by site-directed mutagenesis and activation of purified precursors in vitro. Mutation of Thr-152 to Ser or Cys leads to gene products that are not activated in vivo but are activated in vitro because processing of the mutant precursors is inhibited by certain amino acids in the cell. Kinetic studies reveal that activation is an intramolecular autoproteolytic process. The involvement of His-150 and Thr/Ser/Cys-152 in activation suggests that autoproteolysis resembles proteolysis by serine/cysteine proteases. Multiple functions of the highly conserved active threonine residue are implicated.
[1] Observations of nitrous acid (HONO) by laser-induced fluorescence (LIF) at the South Pole taken during the Antarctic Troposphere Chemistry Investigation (ANTCI), which took place over the time period of Nov. 15, 2003 to Jan. 4, 2004, are presented here. The median observed mixing ratio of HONO 10 m above the snow was 5.8 pptv (mean value 6.3 pptv) with a maximum of 18.2 pptv on Nov 30th, Dec 1st, 3rd, 15th, 17th, 21st, 22nd, 25th, 27th and 28th. The measurement uncertainty is ±35%. The LIF HONO observations are compared to concurrent HONO observations pe rformed by mist chambe r/ion chromatography (MC/IC). The HONO levels reported by MC/IC are about 7.2 ± 2.3 times higher than those reported by LIF.
Dedicated to Professor Peter B. Dervan on the occasion of his 60th birthdayThe a helix plays a fundamental role in imparting specificity to protein-protein and protein-nucleic acid interactions. Molecules that can predictably and selectively disrupt these interactions would be invaluable as tools in molecular biology and, potentially, as leads in drug discovery.[1] We recently described a new strategy for the synthesis of artificial a helices in which one main-chain i to i + 4 hydrogen bond in the target a helix is replaced with a carbon-carbon bond derived from a ring-closing metathesis reaction (Figure 1). [2] A key feature of this hydrogen-bond surrogate (HBS) approach is that the internal placement of the cross-link affords short helices with minimal perturbations to their molecular recognition surfaces. This method differs significantly from the commonly employed side-chain cross-linking method for helix stabilization. A limitation of the latter approach is that side-chain functionality must be sacrificed to nucleate stable helical conformations. The modified side chains are unavailable for molecular recognition; moreover, the resulting tether blocks at least one face of the putative helix. The HBS approach uniquely allows the synthesis of artificial helices with all side chains available for molecular recognition, and does not place any steric encumbrances on the helix surface. We believe that our artificial a helices have the potential to target protein receptors and regulate proteinprotein interactions more successfully than helices with crosslinked side chains.Our initial studies demonstrated that the HBS approach affords highly stable a helices from alanine-rich peptide sequences. Herein, we show that this metathesis-based
Glycosylasparaginase is an N-terminal nucleophile hydrolase and is activated by intramolecular autoproteolytic processing. This cis-autoproteolysis possesses unique kinetics characterized by a reversible N-O acyl rearrangement step in the processing. Arg-180 and Asp-183, involved in binding of the substrate in the mature enzyme, are also involved in binding of free amino acids in the partially formed substrate pocket on certain mutant precursors. This binding site is sequestered in the wild-type precursor. Binding of free amino acids on mutant precursors can either inhibit or accelerate their processing, depending on the individual mutants and amino acids. The polypeptide sequence at the processing site, which is highly conserved, adopts a special conformation. Asp-151 is essential for maintaining this conformation, possibly by anchoring its side chain into the partially formed substrate pocket through interaction with Arg-180. The reactive nucleophile Thr-152 is activated not only by deprotonation by His-150 but also by interaction with Thr-170, suggesting a His-Thr-Thr active triad for the autoproteolysis.
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