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Toward New Designed Proteins Derived from Bovine Pancreatic Trypsin Inhibitor (BPTI): Covalent Cross-Linking of Two ‘Core Modules' by Oxime-Forming Ligation
journal contribution
posted on 2001-08-31, 00:00 authored by Natàlia Carulla, Clare Woodward, George BaranyA 25-residue disulfide-cross-linked peptide, termed ‘oxidized core module' (OxCM), that includes
essentially all of the secondary structural elements of bovine pancreatic trypsin inhibitor (BPTI) most
refractory to hydrogen exchange, was shown previously to favor nativelike β-sheet structure [Carulla,
N., Woodward, C., and Barany, G. (2000) Synthesis and Characterization of a β-Hairpin Peptide That
Represents a ‘Core Module' of Bovine Pancreatic Trypsin Inhibitor (BPTI). Biochemistry 39, 7927−7937]. The present work prepares to explore the hypothesis that the energies of nativelike
conformations, relative to other possible conformations, could be decreased further by covalent linkage
of two OxCMs. Optimized syntheses of six ∼50-residue OxCM dimers are reported herein, featuring
appropriate monomer modifications followed by oxime-forming ligation chemistry to create covalent
cross-links at various positions and with differing lengths. Several side reactions were recognized
through this work, and modified procedures to lessen or mitigate their occurrence were developed.
Particularly noteworthy, guanidine or urea denaturants that were included as peptide-solubilizing
components for some reaction mixtures were proven to form adducts with glyoxylyl moieties, thus
affecting rates and outcomes. All six synthetic OxCM dimers were characterized by 1D 1H NMR;
three of them showed considerable chemical shift dispersion suggestive of self-association and mutual
stabilization between the monomer units.
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nativelike conformationsBPTIOxCM dimersProteins Derivedresidueurea denaturantscore modulechemical shift dispersion1 D 1 H NMRglyoxylyl moietiesCorehydrogen exchangeform adductsBovine Pancreatic Trypsin Inhibitorreaction mixturesOptimized synthesesBiochemistry 39covalent linkagemonomer unitsSeveral side reactionsmonomer modificationsModulepancreatic trypsin inhibitor
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