Proteins are the workhorses of the cell. The shape that a protein molecule adopts enables it to carry out its role. However, a protein’s shape, or 'conformation', is not static. Instead, a protein can shift between different conformations. This is particularly true for enzymes – the proteins that catalyze chemical reactions. The region of an enzyme where the chemical reaction happens, known as the active site, often has to change its conformation to allow catalysis to proceed. Changes in temperature can also make a protein shift between alternative conformations. Understanding how a protein shifts between conformations gives insight into how it works. A common method for studying protein conformation is X-ray crystallography. This technique uses a beam of X-rays to figure out where the atoms of the protein are inside a crystal made of millions of copies of that protein. At room temperature or biological temperature, X-rays can rapidly damage the protein. Because of this, most crystal structures are determined at very low temperatures to minimize damage. But cooling to low temperatures changes the conformations that the protein adopts, and usually causes fewer conformations to be present. Keedy, Kenner, Warkentin, Woldeyes et al. have used X-ray crystallography from a very low temperature (-173°C or 100 K) to above room temperature (up to 27°C or 300 K) to explore the alternative conformations of an enzyme called cyclophilin A. These alternative conformations include those that have previously been linked to this enzyme’s activity. Starting at a low temperature, parts of the enzyme were seen to shift from having a single conformation to many conformations above a threshold temperature. Unexpectedly, different parts of the enzyme have different threshold temperatures, suggesting that there isn’t a single transition across the whole protein. Instead, it appears the way a protein’s conformation changes in response to temperature is more complex than was previously realized. This result suggests that conformations in different parts of a protein are coupled to each other in complex ways. Keedy, Kenner, Warkentin, Woldeyes et al. then performed X-ray crystallography at room temperature using an X-ray free-electron laser (XFEL). This technique can capture the protein’s structure before radiation damage occurs, and confirmed that the alternative conformations observed were not affected by radiation damage. The combination of X-ray crystallography at multiple temperatures, new analysis methods for identifying and measuring alternative conformations, and XFEL crystallography should help future studies to characterize conformational changes in other proteins.
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Electrochemical Zero-Mode Waveguide Studies of Single Enzyme Reactions
Because electron transfer reactions are fundamental to life processes, such as respiration, vision, and energy catabolism, it is critically important to understand the relationship between functional states of individual redox enzymes and the macroscopically observed phenotype, which results from averaging over all copies of the same enzyme. To address this problem, we have developed a new technology, based on a bifunctional nanoelectrochemical-nanophotonic architecture - the electrochemical zero mode waveguide (E-ZMW) - that can couple biological electron transfer reactions to luminescence, making it possible to observe single electron transfer events in redox enzymes. Here we describe E-ZMW architectures capable of supporting potential-controlled redox reactions with single copies of the oxidoreductase enzyme, glutathione reductase, GR, and extend these capabilities to electron transfer events where reactive oxygen species are synthesized within the 100 zL volume of the nanopore.
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- Award ID(s):
- 1404744
- NSF-PAR ID:
- 10221839
- Date Published:
- Journal Name:
- 2018 IEEE 18th International Conference on Nanotechnology (IEEE-NANO)
- Page Range / eLocation ID:
- 1 to 2
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1109/NANO.2018.8626402
