Single-Molecule Electrical and Spectroscopic Profiling Protein Allostery Using a Gold Plasmonic Nanopore

Abstract

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.

Developing Solid-State Single-, Arrayed-, and Composite-Nanopore Sensors for Biochemical Sensing Applications

“…The spectral and ion-current signals of the analyte thus can be obtained at the same time, which greatly improves the analytical reliability. It is revealed in our previous work that, through assembling plasmonic metal structures (e.g., silver triangles and gold nanoplates) onto the tip of a nanopipette, surface enhanced Raman spectroscopy (SERS) can be used to directly recognize the structure of biomolecules that translocated through the pipette. , For example, a gold plasmonic nanopore was developed to monitor the translocation dynamics and conformation transition of calmodulin (CaM) using both electrochemical and SERS techniques (Figure a) . This allowed us to effectively differentiate between the two conformers of CaM at the single-molecule level.…”

“…(a) Schematic illustration of single-molecule sensing with a gold plasmonic nanopore. Reproduced with permission from ref . Copyright 2023 American Chemical Society.…”

Surface-Enhanced Raman Spectroscopy: Current Understanding, Challenges, and Opportunities

“…Integration of SERS and nanopore could directly facilitate further generation of sequencing and the development of molecular dynamic research and related fields, such as biocatalysts and drug development . However, the efficiency of plasmonic coupling of the nanopore structure are still not high enough to provide sufficient fingerprint information on molecules in the nanopore, − which demands rational design of nanopore structures …”

Iontronic Sensing Based on Confined Ion Transport

“…Iontronic sensing on the basis of confined ion transport has emerged as an effective platform for analyzing single molecules using the principle of RPS. The RPS mechanism allows the detection of single molecules, such as ions/molecules, , and macromolecules like DNA/RNA/proteins, ,− with extraordinary sensitivity, enabling the extraction of their structural information. As single-molecule detection methods gain increasing importance in diagnostic applications, iontronics play a significant role in advancing the capabilities of single-molecule sensing.…”