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Hydrogen exchange-mass spectrometry measures stapled peptide conformational dynamics and predicts pharmacokinetic properties.

ABSTRACT: Peptide drugs have traditionally suffered from poor pharmacokinetic properties due to their conformational flexibility and the interaction of proteases with backbone amide bonds. "Stapled Peptides" are cyclized using an all-hydrocarbon cross-linking strategy to reinforce their ?-helical conformation, yielding improved protease resistance and drug-like properties. Here we demonstrate that hydrogen exchange-mass spectrometry (HX-MS) effectively probes the conformational dynamics of Stapled Peptides derived from the survivin-borealin protein-protein interface and predicts their susceptibility to proteolytic degradation. In Stapled Peptides, amide exchange was reduced by over five orders-of-magnitude versus the native peptide sequence depending on staple placement. Furthermore, deuteration kinetics correlated directly with rates of proteolysis to reveal the optimal staple placement for improved drug properties.

SUBMITTER: Shi XE

PROVIDER: S-EPMC3883098 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Hydrogen exchange-mass spectrometry measures stapled peptide conformational dynamics and predicts pharmacokinetic properties.

Shi Xiangguo Eric XE Wales Thomas E TE Elkin Carl C Kawahata Noriyuki N Engen John R JR Annis D Allen DA

Analytical chemistry 20131114 23

Peptide drugs have traditionally suffered from poor pharmacokinetic properties due to their conformational flexibility and the interaction of proteases with backbone amide bonds. "Stapled Peptides" are cyclized using an all-hydrocarbon cross-linking strategy to reinforce their α-helical conformation, yielding improved protease resistance and drug-like properties. Here we demonstrate that hydrogen exchange-mass spectrometry (HX-MS) effectively probes the conformational dynamics of Stapled Peptide ...[more]

PMID: 24215480

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Project description:Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38alpha MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38alpha are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38alpha alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38alpha and X-ray structures of phosphorylated p38gamma. Conformational differences account for altered HX within the activation lip, the P+1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (alphaF-alphaG, beta7-beta8), the C-terminal core (alphaE), and the N-terminal core region (beta4-beta5, alphaL16, alphaC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38alpha. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38alpha because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38alpha are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38alpha and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.

Dataset Information

Hydrogen exchange-mass spectrometry measures stapled peptide conformational dynamics and predicts pharmacokinetic properties.

Publications

Hydrogen exchange-mass spectrometry measures stapled peptide conformational dynamics and predicts pharmacokinetic properties.

Similar Datasets

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