Statement of Purpose Hybrid nanoparticles in which a polymer is used to stabilize the secondary structure of enzyme provide a means to preserve its activity in non-native environments. This approach is illustrated here with horseradish peroxidase (HRP), an important heme enzyme used in medical diagnostic, biosensing, and biotechnological applications. Polymer chaperones in these polymer-enzyme complex (PEC) nanoparticles can enhance the utility of enzymes in unfavorable environments. Structural analysis of the PECs is a crucial link in the machine-learning driven iterative optimization cycle of polymer synthesis and testing. Here, we discuss the utility of small-angle X-ray scattering (SAXS) and quartz crystal microbalance with dissipation (QCMD) for evaluating PECs. Materials and Methods Six polymers were synthesized by automated photoinduced electron/energy transfer-reversible addition-fragmentation chain-transfer (PET-RAFT) polymerization directly in 96-well plates.1 Multiple molar ratios of enzyme:polymer (1:1, 1:5, 1:10, and 1:50) were characterized. HRP was mixed with the polymer and heated to 65 °C for 1 hr to form PECs. Enzyme assay and circular dichroism measurements were performed along with SAXS and QCMD to understand polymer-protein interactions. SAXS data were obtained at NSLS-II beamline 16-ID. Results and Discussion SAXS data were analyzed to determine the radius of gyration (Rg), Porod exponent and pair distancemore »
An integrative NMR-SAXS approach for structural determination of large RNAs defines the substrate-free state of a trans -cleaving Neurospora Varkud Satellite ribozyme
The divide-and-conquer strategy is commonly used for protein structure determination, but its applications to high-resolution structure determination of RNAs have been limited. Here, we introduce an integrative approach based on the divide-and-conquer strategy that was undertaken to determine the solution structure of an RNA model system, the Neurospora VS ribozyme. NMR and SAXS studies were conducted on a minimal trans VS ribozyme as well as several isolated subdomains. A multi-step procedure was used for structure determination that first involved pairing refined NMR structures with SAXS data to obtain structural subensembles of the various subdomains. These subdomain structures were then assembled to build a large set of structural models of the ribozyme, which was subsequently filtered using SAXS data. The resulting NMR-SAXS structural ensemble shares several similarities with the reported crystal structures of the VS ribozyme. However, a local structural difference is observed that affects the global fold by shifting the relative orientation of the two three-way junctions. Thus, this finding highlights a global conformational change associated with substrate binding in the VS ribozyme that is likely critical for its enzymatic activity. Structural studies of other large RNAs should benefit from similar integrative approaches that allow conformational sampling of assembled more »
- Publication Date:
- NSF-PAR ID:
- 10306124
- Journal Name:
- Nucleic Acids Research
- Volume:
- 49
- Issue:
- 20
- Page Range or eLocation-ID:
- p. 11959-11973
- ISSN:
- 0305-1048
- Publisher:
- Oxford University Press
- Sponsoring Org:
- National Science Foundation
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