skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Preparation of carotenoid cleavage dioxygenases for X-ray crystallography
Carotenoid cleavage dioxygenases (CCDs) constitute a superfamily of enzymes that are found in all domains of life where they play key roles in the metabolism of carotenoids and apocarotenoids as well as certain phenylpropanoids such as resveratrol. Interest in these enzymes stems not only from their biological importance but also from their remarkable catalytic properties including their regioselectivity, their ability to accommo- date diverse substrates, and the additional activities (e.g., isomerase) that some of these enzyme possess. X-ray crystallography is a key experimental approach that has allowed detailed investigation into the structural basis behind the interesting biochem- ical features of these enzymes. Here, we describe approaches used by our lab that have proven successful in generating single crystals of these enzymes in resting or ligand-bound states for high-resolution X-ray diffraction analysis.  more » « less
Award ID(s):
2107713
PAR ID:
10347096
Author(s) / Creator(s):
Editor(s):
Eleanore T. Wurtzel
Date Published:
Journal Name:
Methods in enzymology
Volume:
671
ISSN:
0076-6879
Page Range / eLocation ID:
243-271
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. (, Crystals)
    Ever since the first structure of an enzyme, lysozyme, was solved, scientists have been eager to explore how these molecules perform their catalytic function. There has been an overwhelmingly large body of publications that report the X-ray structures of enzymes determined after substrate and ligand binding. None of them truly show the structures of an enzyme working freely through a sequence of events that range from the formation of the enzyme–substrate complex to the dissociation of the product. The technical difficulties were too severe. By 1969, Sluyterman and de Graaf had pointed out that there might be a way to start a reaction in an enzyme crystal by diffusion and following its catalytic cycle in its entirety with crystallographic methods. The crystal only has to be thin enough so that the diffusion is not rate limiting. Of course, the key questions are as follows: How thin should the crystal be? Will the existing X-ray sources be able to collect data from a thin enough crystal fast enough? This review shines light on these questions. 
    more » « less
  2. ; ; ; ; ; ; ; (, BioMed Research International)
    Ubiquitin and ubiquitin like proteins (UBLs) play key roles in eukaryotes. These proteins are attached to their target proteins through an E1-E2-E3 cascade and modify the functions of these proteins. Since the discovery of ubiquitin, several UBLs have been identified, including Nedd8, SUMO, ISG15, and Atg8. Ubiquitin and UBLs share a similar three-dimensional structure: β -grasp fold and an X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus. We have previously reported that ubiquitin, Nedd8, and SUMO mimicking peptides which all contain the conserved motif X-X-[R/A/E/K]-X-X-[G/X]-G still retained their reactivity toward their corresponding E1, E2, and E3 enzymes. In our current study, we investigated whether such C-terminal peptides could still be transferred onto related pathway enzymes to probe the function of these enzymes when they are fused with a protein. By bioinformatic search of protein databases, we selected eight proteins carrying the X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus of the β -grasp fold. We synthesized the C-terminal sequences of these candidates as short peptides and found that three of them showed significant reactivity with the ubiquitin E1 enzyme Ube1. We next fused the three reactive short peptides to three different protein frames, including their respective native protein frames, a ubiquitin frame and a peptidyl carrier protein (PCP) frame, and measured the reactivities of these peptide-fused proteins with Ube1. Peptide-fused proteins on ubiquitin and PCP frames showed obvious reactivity with Ube1. However, when we measured E2 UbcH7 transfer, we found that the PCP-peptide fusions lost their reactivity with UbcH7. Taken together, these results suggested that the recognition of E2 enzymes with peptide-fused proteins depended not only on the C-terminal sequences of the ubiquitin-mimicking peptides, but also on the overall structures of the protein frames. 
    more » « less
  3. ; ; ; ; ; ; ; ; (, Nature Communications)
    Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data. 
    more » « less
  4. ; ; ; ; ; ; (, ACS Synthetic Biology)
    Cell-free gene expression systems derived from bacterial lysates enable the expression of biosynthetic pathways from inexpensive and easily prepared DNA templates. These systems hold great promise for modular and on-demand bioproduction of valuable small molecules in resource-limited settings but are constrained in their long-term stability, reusability, and deployability. In this work, we demonstrate that multiple cell-free expressed enzymes can be co-immobilized in biocompatible hydrogels made from poly(ethylene glycol) diacrylate (PEGDA) with added glycerol for enhanced gel integrity. Using small-angle X-ray scattering (SAXS), we show that the mesh size of PEGDA-glycerol hydrogels is comparable to the globular sizes of many proteins and enzymes, which could be used for protein entrapment. We found that the combination between entrapment and chemical ligation of the enzymes was effective to retain proteins. By employing a method for direct fluorescence measurement from hydrogels, we found that proteins can be retained in PEGDA-glycerol for at least a week. By separating the cell-free enzyme expression from the immobilization step, we successfully fabricated enzyme-laden hydrogels with three heterologous cell-free enzymes for the bioconversion of pyruvic acid to malic acid, an industrially valuable and versatile precursor chemical. Both heterologous and endogenous enzymes from the lysate remain functional in photo-cross-linked hydrogels and can be reused for multiple biocatalytic cycles. Moreover, we also found that the immobilized enzymes exhibit up to 1.6-fold higher activity and 2-fold longer lifetimes than free enzymes in liquid reactions. These results could advance the deployment of cell-free synthetic biology because they show that reusable, stable, and durable multienzyme systems can be created using readily available materials and fabrication techniques. 
    more » « less
  5. ; ; ; ; ; ; ; ; ; ; et al (, IUCrJ)
    X-ray free-electron lasers (XFELs) can probe chemical and biological reactions as they unfold with unprecedented spatial and temporal resolution. A principal challenge in this pursuit involves the delivery of samples to the X-ray interaction point in such a way that produces data of the highest possible quality and with maximal efficiency. This is hampered by intrinsic constraints posed by the light source and operation within a beamline environment. For liquid samples, the solution typically involves some form of high-speed liquid jet, capable of keeping up with the rate of X-ray pulses. However, conventional jets are not ideal because of radiation-induced explosions of the jet, as well as their cylindrical geometry combined with the X-ray pointing instability of many beamlines which causes the interaction volume to differ for every pulse. This complicates data analysis and contributes to measurement errors. An alternative geometry is a liquid sheet jet which, with its constant thickness over large areas, eliminates the problems related to X-ray pointing. Since liquid sheets can be made very thin, the radiation-induced explosion is reduced, boosting their stability. These are especially attractive for experiments which benefit from small interaction volumes such as fluctuation X-ray scattering and several types of spectroscopy. Although their use has increased for soft X-ray applications in recent years, there has not yet been wide-scale adoption at XFELs. Here, gas-accelerated liquid sheet jet sample injection is demonstrated at the European XFEL SPB/SFX nano focus beamline. Its performance relative to a conventional liquid jet is evaluated and superior performance across several key factors has been found. This includes a thickness profile ranging from hundreds of nanometres to 60 nm, a fourfold increase in background stability and favorable radiation-induced explosion dynamics at high repetition rates up to 1.13 MHz. Its minute thickness also suggests that ultrafast single-particle solution scattering is a possibility. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email