Low Temperature Assembly of Functional 3D DNA-PNA-Protein Complexes

Proteins have evolved to carry out nearly all the work required of living organisms within complex inter- and intracellular environments. However, systematically investigating the range of interactions experienced by a protein that influence its function remains challenging. DNA nanostructures are emerging as a convenient method to arrange a broad range of guest molecules. However, flexible methods are needed for arranging proteins in more biologically relevant 3D geometries under mild conditions that preserve protein function. Here we demonstrate how peptide nucleic acid (PNA) can be used to control the assembly of cytochrome c (12.5 kDa, pI 10.5) and azurin (13.9 kDa, pI 5.7) proteins into separate 3D DNA nanocages, in a process that maintains protein function. Toehold-mediated DNA strand displacement is introduced as a method to purify PNA-protein conjugates. The PNA-proteins were assembled within 2 min at room temperature and within 4 min at 11 °C, and hybridize with even greater efficiency than PNA conjugated to a short peptide. Gel electrophoresis and steady state and time-resolved fluorescence spectroscopy were used to investigate the effect of protein surface charge on its interaction with the negatively charged DNA nanocage. These data were used to generate a model of the DNA-PNA-protein complexes that show the negatively charged azurin protein repelled away from the DNA nanocage while the positively charged cytochrome c protein remains within and closely interacts with the DNA nanocage. When conjugated to PNA and incorporated into the DNA nanocage, the cytochrome c secondary structure and catalytic activity were maintained, and its redox potential was reduced modestly by 20 mV possibly due to neutralization of some positive surface charges. This work demonstrates a flexible new approach for using 3D nucleic acid (PNA-DNA) nanostructures to control the assembly of functional proteins, and facilitates further investigation of protein interactions as well as engineer more elaborate 3D protein complexes

Purification and Biophysical Characterization of the CapA Membrane Protein FTT0807 from <i>Francisella tularensis</i>

The <i>capA</i> gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The <i>capA</i> gene was overexpressed in Escherichia coli as a C-terminal His₆-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3–9 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1 M resulted in a small increase of the α-helical content and a corresponding decrease in the random-coil content. Secondary-structure predictions on the basis of the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophilic domain is predicted to contain a long disordered region of 50–60 residues, suggesting that the increase of α-helical content at high ionic strength could arise because of electrostatic interactions involving the disordered region. CapA is shown to be an inner-membrane protein and is predicted to play a key cellular role in the assembly of polysaccharides

Additional file 1: of Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography

Figure S1. Schematics of the short-time-point mixing injector. Figure S2. Selected views of the CEF binding site in the BlaC shard crystals including simulated annealing omit maps. Figure S3. Structural details, and simulated annealing omit maps, shard crystal form, subunit B (stereo representation, from 30 ms to 2 s). Figure S4. Structural details and simulated annealing omit maps, shard crystal form, subunit D (stereo representation, from 30 ms to 2 s). Figure S5. Structural details, and simulated annealing omit maps, needle crystal form (stereo representation, from 30 ms to 2 s). Figure S6. Backside view of the catalytic cleft of BlaC in the shard crystal form, structural details and simulated annealing omit maps (stereo representation, selected time points). Figure S7. 2mFo-DFc electron density in the catalytic clefts of BlaC in the shard crystal form (stereo representation, from 30 ms to 2 s). Figure S8. 2mFo-DFc electron density and structural details in the catalytic clefts of BlaC in the needle crystal form (stereo representation from 30 ms to 2 s). Figure S9. Details in the catalytic cleft of subunit B in the shard crystal form at 500 ms including the stacked CEF, 2FoFc maps, and simulated annealing omit maps (stereo representation). Figure S10. The catalytic cleft of BlaC, further details, including a difference map between the 500 ms and 100 ms time points. Figure S11. Crystal packing in shards and needles. Figure S12. Dynamic light scattering results. Table S1. B-factors for CEF species observed in the shard crystals at different time delays. (PDF 1646 kb

Size measurements of Rice Dwarf Virus (RDV) in the liquid and gas phase

Size measurements of Rice Dwarf Virus (RDV) in the liquid and gas phase