Heat-induced structural and chemical changes to a computationally designed miniprotein.
capillary electrophoresis
deamidation
dynamics
mass spectrometry
miniproteins
nuclear magnetic resonance
protein design
Journal
Protein science : a publication of the Protein Society
ISSN: 1469-896X
Titre abrégé: Protein Sci
Pays: United States
ID NLM: 9211750
Informations de publication
Date de publication:
Jun 2024
Jun 2024
Historique:
revised:
22
03
2024
received:
11
12
2023
accepted:
28
03
2024
medline:
17
5
2024
pubmed:
17
5
2024
entrez:
17
5
2024
Statut:
ppublish
Résumé
The de novo design of miniprotein inhibitors has recently emerged as a new technology to create proteins that bind with high affinity to specific therapeutic targets. Their size, ease of expression, and apparent high stability makes them excellent candidates for a new class of protein drugs. However, beyond circular dichroism melts and hydrogen/deuterium exchange experiments, little is known about their dynamics, especially at the elevated temperatures they seemingly tolerate quite well. To address that and gain insight for future designs, we have focused on identifying unintended and previously overlooked heat-induced structural and chemical changes in a particularly stable model miniprotein, EHEE_rd2_0005. Nuclear magnetic resonance (NMR) studies suggest the presence of dynamics on multiple time and temperature scales. Transiently elevating the temperature results in spontaneous chemical deamidation visible in the NMR spectra, which we validate using both capillary electrophoresis and mass spectrometry (MS) experiments. High temperatures also result in greatly accelerated intrinsic rates of hydrogen exchange and signal loss in NMR heteronuclear single quantum coherence spectra from local unfolding. These losses are in excellent agreement with both room temperature hydrogen exchange experiments and hydrogen bond disruption in replica exchange molecular dynamics simulations. Our analysis reveals important principles for future miniprotein designs and the potential for high stability to result in long-lived alternate conformational states.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e4991Subventions
Organisme : NIH HHS
Pays : United States
Informations de copyright
© 2024 The Protein Society.
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