Obtaining
sequential and conformational information on proteins
is vital to understand their functions. Although the nanopore-based
electrical detection can sense single molecule (SM) protein and distinguish
among different amino acids, this approach still faces difficulties
in slowing down protein translocation and improving ionic current
signal-to-noise ratio. Here, we observe the unfolding and multistep
sequential translocation of SM cytochrome c (cyt c) through a surface
enhanced Raman scattering (SERS) active conical gold nanopore. High
bias voltage unfolds SM protein causing more exposure of amino acid
residues to the nanopore, which slows down the protein translocation.
Specific SERS traces of different SM cyt c segments are then recorded
sequentially when they pass through the hotspot inside the gold nanopore.
This study shows that the combination of SM SERS with a nanopore can
provide a direct insight into protein segments and expedite the development
of nanopore toward SM protein sequencing.
Direct structural and dynamic characterization of protein conformers in solution is highly desirable but currently impractical. Herein, we developed a single molecule gold plasmonic nanopore system for observation of protein allostery, enabling us to monitor translocation dynamics and conformation transition of proteins by ion current detection and SERS spectrum measurement, respectively. Allosteric transition of calmodulin (CaM) was elaborately probed by the nanopore system. Two conformers of CaM were well-resolved at a single-molecule level using both the ion current blockage signal and the SERS spectra. The collected SERS spectra provided structural evidence to confirm the interaction between CaM and the gold plasmonic nanopore, which was responsible for the different translocation behaviors of the two conformers. SERS spectra revealed the amino acid residues involved in the conformational change of CaM upon calcium binding. The results demonstrated that the excellent spectral characterization furnishes a single-molecule nanopore technique with an advanced capability of direct structure analysis.
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