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Temperature-dependent regulation of ion channel activity is critical for a variety of physiological processes ranging from immune response to perception of noxious stimuli. Our understanding of the structural mechanisms that underlie temperature sensing remains limited, in part due to the difficulty of combining high-resolution structural analysis with temperature stimulus. Here, we use NMR to compare the temperature-dependent behavior of Shaker potassium channel voltage sensor domain (WT-VSD) to its engineered temperature sensitive (TS-VSD) variant. Further insight into the molecular basis for temperature-dependent behavior is obtained by analyzing the experimental results together with molecular dynamics simulations. Our studies reveal that the overall secondary structure of the engineered TS-VSD is identical to the wild-type channels except for local changes in backbone torsion angles near the site of substitution (V369S and F370S). Remarkably however, these structural differences result in increased hydration of the voltage-sensing arginines and the S4–S5 linker helix in the TS-VSD at higher temperatures, in contrast to the WT-VSD. These findings highlight how subtle differences in the primary structure can result in large-scale changes in solvation and thereby confer increased temperature-dependent activity beyond that predicted by linear summation of solvation energies of individual substituents.
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This content will become publicly available on May 1, 2026
A structural perspective on the temperature dependent activity of enzymes
Enzyme activity varies with temperature. Unlike small-molecule catalysts, the structural ensembles of enzymes can change substantially with temperature, but it is unclear how this modulates temperature dependent activity. Here, multi-temperature X-ray crystallography was used to record structural changes from -20C to 40C for a mesophilic enzyme in complex with inhibitors mimicking substrate-, intermediate-, and product-bound states, representative of major complexes on the reaction coordinate. Inhibitors, substrates and active site loops increasingly populated catalytically competent conformations as temperature increased. These changes occurred even in temperature ranges where kinetic measurements showed roughly linear Arrhenius/Eyring behavior, where parameters characterizing the system are assumed to be temperature independent. Simple analysis shows that linear Arrhenius/Eyring behavior can still be observed when the underlying activation energy/enthalpy values vary with temperature. Our results indicate a critical role for temperature dependent atomic-resolution structural data in interpreting temperature dependent kinetic data from enzymatic systems.
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- Award ID(s):
- 2210041
- PAR ID:
- 10613708
- Editor(s):
- Kuhnel, Karen
- Publisher / Repository:
- Cell Press
- Date Published:
- Journal Name:
- Structure
- Edition / Version:
- 1
- Volume:
- 33
- Issue:
- 5
- ISSN:
- 0969-2126
- Page Range / eLocation ID:
- 924 to 934
- Subject(s) / Keyword(s):
- enzymes multi-temperature crystallography enzymatic activity
- Format(s):
- Medium: X Size: 4MB Other: pdf
- Size(s):
- 4MB
- Sponsoring Org:
- National Science Foundation
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