In situ neutralization in Boc chemistry SPPS: High yield assembly of difficult sequences

Schnölzer, Martina; Alewood, Paul F.; Jones, Ashley; Kent, Stephen B. H.

doi:10.1007/978-94-011-2264-1_248

Cited by 235 publications

(368 citation statements)

References 8 publications

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“…Peptides were manually synthesized by standard solid phase Boc chemistry using previously described protocols (Reymond et al, 1992;Schnolzer et al, 1992). Peptides were blocked at the N-terminus with an acetyl group and at the C-terminus with a primary amide group.…”

Section: Peptide Synthesis and Purijkationmentioning

confidence: 99%

Folding propensities of peptide fragments of myoglobin

et al. 1997

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Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthesized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformational preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the a region of (c#J,$) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A-and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate p-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, earlier studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.Keywords: myoglobin; nuclear magnetic resonance; peptide conformation; protein folding; reverse turn; secondary structureThe mechanism by which proteins fold into their native conformations remains one of the central unsolved problems in molecular biology. One view is that folding occurs along defined pathways, often with structured intermediates (for reviews, see Creighton,

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Section: Peptide Synthesis and Purijkationmentioning

confidence: 99%

Folding propensities of peptide fragments of myoglobin

et al. 1997

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“…A stock solution of [Aba67,95]HIV-1 protease (Schnolzer et al, 1992) was similarly prepared, and subsequent dilutions of these solutions, containing equal concentrations of enzyme by active site titration, were used for comparison of BTD and non-BTD HIV-1 protease kinetics.…”

Section: Product Characterization and Enzyme Assaysmentioning

confidence: 99%

Structural engineering of the HIV‐1 protease molecule with a β‐turn mimic of fixed geometry

1993

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An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type 11' 0-turn mimic of predetermined three-dimensional structure.The mimic &turn replaced residues GlyI6,l7 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' 0-turns in the native, unliganded enzyme. The 0-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the GlyI67l7-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the @turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid 0-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.

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“…The α-conotoxins Vc1.1, AuIB and MII were synthesised as previously described (Schnolzer et al 1992;Cartier et al 1996;Luo et al 1998;Clark et al 2006). Briefly, Boc solid phase chemistry was used to synthesise, deprotect and cleave from the resin all peptides as described (Schnolzer et al 1992).…”

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confidence: 99%

“…Briefly, Boc solid phase chemistry was used to synthesise, deprotect and cleave from the resin all peptides as described (Schnolzer et al 1992). 100 mM ammonium bicarbonate (pH 7.5-8.2) was used to oxidise reduced HPLC-purified peptides at room temperature with constant stirring.…”

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confidence: 99%

Intrathecal α-conotoxins Vc1.1, AuIB and MII acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain

et al. 2012

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The large diversity of peptides from venomous creatures with high affinity for molecules involved in the development and maintenance of neuropathic pain has led to a surge in venom-derived analgesic research. Some members of the α-conotoxin family from Conus snails which specifically target subtypes of nicotinic acetylcholine receptors (nAChR) have been shown to be effective at reducing mechanical allodynia in neuropathic pain models. We sought to determine if three such peptides, Vc1.1, AuIB and MII were effective following intrathecal administration in a rat neuropathic pain model because they exhibit different affinities for the major putative pain relieving targets of α-conotoxins. Intrathecal administration of α-conotoxins, Vc1.1, AuIB and MII into neuropathic rats reduced mechanical allodynia for up to 6 hours without significant side effects. In vitro patch-clamp electrophysiology of primary afferent synaptic transmission revealed the mode of action of these toxins was not via a GABA B -dependant mechanism, and is more likely related to their action at nAChRs containing combinations of α3, α7 or other subunits. Intrathecal nAChR subunitselective conotoxins are therefore promising tools for the effective treatment of neuropathic pain. AbstractThe large diversity of peptides from venomous creatures with high affinity for molecules involved in the development and maintenance of neuropathic pain has led to a surge in venom-derived analgesic research. Some members of the α-conotoxin family from Conus snails which specifically target subtypes of nicotinic acetylcholine receptors (nAChR) have been shown to be effective at reducing mechanical allodynia in neuropathic pain models. We sought to determine if three such peptides, Vc1.1, AuIB and MII were effective following intrathecal administration in a rat neuropathic pain model because they exhibit different affinities for the major putative pain relieving targets of α-conotoxins. Intrathecal administration of α-conotoxins, Vc1.1, AuIB and MII into neuropathic rats reduced mechanical allodynia for up to 6 hours without significant side effects. In vitro patch-clamp electrophysiology of primary afferent synaptic transmission revealed the mode of action of these toxins was not via a GABA B -dependant mechanism, and is more likely related to their action at nAChRs containing combinations of α3, α7 or other subunits. Intrathecal nAChR subunitselective conotoxins are therefore promising tools for the effective treatment of neuropathic pain.3

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In situ neutralization in Boc chemistry SPPS: High yield assembly of difficult sequences

Cited by 235 publications

References 8 publications

Folding propensities of peptide fragments of myoglobin

Folding propensities of peptide fragments of myoglobin

Structural engineering of the HIV‐1 protease molecule with a β‐turn mimic of fixed geometry

Intrathecal α-conotoxins Vc1.1, AuIB and MII acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain

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