Formation of Disulfide Bonds in Synthetic Peptides and Proteins

Andreu, David; Alberício, Fernando; Solé, Núria A.; Munson, Mark; Ferrer, Marc; Bárány, George

doi:10.1385/0-89603-273-6:91

Cited by 145 publications

(140 citation statements)

References 51 publications

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“…In addition, the conditions for Acm removal do not impact antibody disulfide integrity (Figure S3), indicating that these conditions could be applied more generally for bio‐orthogonal protein modification. While the Acm protecting group has been used extensively in peptide synthesis and protein semi‐synthesis,13b, 14 to our knowledge this is the first use of an Acm‐masked Cys on a folded protein.…”

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confidence: 98%

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Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

et al. 2016

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A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site‐specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug‐linkers that have differing physiochemical properties and exert complementary anti‐cancer activities. Dual‐auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual‐drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site‐specific protein modification.

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confidence: 98%

“…Through this work, we identified aqueous conditions for the deprotection of acetamidomethyl (Acm)‐protected cysteines 13b. Specifically, maleimidocaproyl‐Cys(Acm) was synthesized and conjugated to the interchain cysteines of the CD30‐directed antibody cAC10 and subjected to potential deprotection conditions.…”

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confidence: 99%

Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

et al. 2016

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“…The TOAC positions on RyRp were chosen in locations where little or no contact with CaM is made, based on the Ca 2þ -bound crystal structure of the complex (15). For the TOAC-RyRp derivatives, Cys 22 was replaced with a-amino-n-butyric acid (Abu), which is isosteric with Cys (27,28), to prevent chemical artifacts during synthesis or disulfide-induced dimerization after synthesis (25,29. Mutation of Cys 3635 (Cys 22 in RyRp) to Ala in the intact receptor showed no effect on CaM binding (30).…”

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confidence: 99%

Calcium-Dependent Structural Dynamics of a Spin-Labeled RyR Peptide Bound to Calmodulin

Her

McCaffrey

Thomas

et al. 2016

Biophysical Journal

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We have used chemical synthesis, electron paramagnetic resonance (EPR), and circular dichroism to detect and analyze the structural dynamics of a ryanodine receptor (RyR) peptide bound to calmodulin (CaM). The skeletal muscle calcium release channel RyR1 is activated by Ca 2þ -free CaM and inhibited by Ca 2þ -bound CaM. To probe the structural mechanism for this regulation, wild-type RyRp and four spin-labeled derivatives were synthesized, each containing the nitroxide probe 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid substituted for a single amino acid. In 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid, the probe is rigidly and stereospecifically coupled to the a-carbon, enabling direct detection by EPR of peptide backbone structural dynamics. In the absence of CaM, circular dichroism indicates a complete lack of secondary structure, while 40% trifluoroethanol (TFE) induces >90% helicity and is unperturbed by the spin label. The EPR spectrum of each spin-labeled peptide indicates nanosecond dynamic disorder that is substantially reduced by TFE, but a significant gradient in dynamics is observed, decreasing from N-to C-terminus, both in the presence and absence of TFE. When bound to CaM, the probe nearest RyRp's N-terminus shows rapid rotational motion consistent with peptide backbone dynamics of a locally unfolded peptide, while the other three sites show substantial restriction of dynamics, consistent with helical folding. The two N-terminal sites, which bind to the C-lobe of CaM, do not show a significant Ca 2þ -dependence in mobility, while both C-terminal sites, which bind to the N-lobe of CaM, are significantly less mobile in the presence of bound Ca 2þ . These results support a model in which the interaction of RyR with CaM is nonuniform along the peptide, and the primary effect of Ca 2þ is to increase the interaction of the C-terminal portion of the peptide with the N-terminal lobe of CaM. These results provide, to our knowledge, new insight into the Ca 2þ -dependent regulation of RyR by CaM.

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“…Cyclized Monomer and Cross-linked Oligomers-Cysteine-containing peptides are prone to atmospheric oxidation (30), and ABri is known to aggregate (10,31). Thus to reduce any oxidized cysteines and to disassemble any pre-existing aggregates we incubated both Bri and ABri peptides in a buffer containing ␤ME and GdmHCl prior to analysis by size exclusion chromatography (Fig.…”

Section: Oxidation Of Bri Leads To Formation Of a Cyclized Monomer Wmentioning

confidence: 99%

The Familial British Dementia Mutation Promotes Formation of Neurotoxic Cystine Cross-linked Amyloid Bri (ABri) Oligomers

Cantlon

Frigerio

Freir

et al. 2015

Journal of Biological Chemistry

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Background: Familial British dementia is a disorder similar to Alzheimer disease and is believed to be caused by the ABri peptide. Results: Cystine-linked oligomers of ABri are toxic to neurons and block long-term potentiation. Conclusion:The type and toxicity of ABri assemblies depends on cystine bond formation. Significance: ABri offers a tractable system to study the structure of disease-causing oligomers.

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Formation of Disulfide Bonds in Synthetic Peptides and Proteins

Cited by 145 publications

References 51 publications

Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

Calcium-Dependent Structural Dynamics of a Spin-Labeled RyR Peptide Bound to Calmodulin

The Familial British Dementia Mutation Promotes Formation of Neurotoxic Cystine Cross-linked Amyloid Bri (ABri) Oligomers

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