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Building the Outer Membrane Protein G (OmpG) Nanopore Library: From the Discrimination of Biotin-Binding Proteins in Serum to Resolving Human Carbonic Anhydrase From Human Red Blood Cells
The use of pore-forming proteins (PFPs) in nanopore sensing has been fruitful largely due to their nanoscale size and the ease with which protein nanopores can be manipulated and consistently reproduced at a large scale. Nanopore sensing relies heavily on a steady ionic current afforded by rigid nanopores, as the change in current is indicative of analyte detection, revealing characteristics of the analyte such as its relative size, concentration, and charge, as well as the nanopore:analyte interaction. Rigid PFPs have been used in applications such as DNA sequencing, kinetic studies, analyte discrimination, and protein conformation dynamics at the single-molecule level. The work in this dissertation details a different approach to nanopore sensing using a non-rigid PFP, the monomeric outer membrane protein G (OmpG), which exhibits intrinsic current fluctuations called gating. We exploit the gating of OmpG for the effective detection and discrimination of protein homologues and isoforms, showing that OmpG gating is particularly useful in selectively detecting targets and probing nanopore:analyte interactions. Further, we demonstrate the ability of OmpG to retain its sensing capabilities in complex mixtures of serum and human cell lysate
Tuning the Selectivity and Sensitivity of an OmpG Nanopore Sensor by Adjusting Ligand Tether Length
We
have previously shown that a biotin ligand tethered to the rim
of an OmpG nanopore can be used to detect biotin-binding proteins.
Here, we investigate the effect of the length of the polyethylene
glycol tether on the nanopore’s sensitivity and selectivity.
When the tether length was increased from 2 to 45 ethylene repeats,
sensitivity decreased substantially for a neutral protein streptavidin
and slightly for a positively charged protein (avidin). In addition,
we found that two distinct avidin binding conformations were possible
when using a long tether. These conformations were sensitive to the
salt concentration and applied voltage. Finally, a longer tether resulted
in reduced sensitivity due to slower association for a monoclonal
antibiotin antibody. Our results highlight the importance of electrostatic,
electroosmotic, and electrophoretic forces on nanopore binding kinetics
and sensor readout
Selective Detection of Protein Homologues in Serum Using an OmpG Nanopore
Outer
membrane protein G is a monomeric β-barrel porin that
has seven flexible loops on its extracellular side. Conformational
changes of these labile loops induce gating spikes in current recordings
that we exploited as the prime sensing element for protein detection.
The gating characteristics, open probability, frequency, and current
decrease, provide rich information for analyte identification. Here,
we show that two antibiotin antibodies each induced a distinct gating
pattern, which allowed them to be readily detected and simultaneously
discriminated by a single OmpG nanopore in the presence of fetal bovine
serum. Our results demonstrate the feasibility of directly profiling
proteins in real-world samples with minimal or no sample pretreatment
\u3ci\u3eDrosophila\u3c/i\u3e Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution
The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu