Optical imaging of single-protein size, charge, mobility, and binding

Ma, Guangzhong; Wan, Zijian; Yang, Yuanyuan; Zhang, Pengfei; Wang, Shaopeng; Tao, Nongjian

doi:10.1038/s41467-020-18547-w

Cited by 34 publications

(49 citation statements)

References 55 publications

(77 reference statements)

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“…Then the N‐ and C‐lobes of Ca‐wtCaM collapse together to grip the M13 peptide (Figures 1 a and S1 f). All these state changes were well resolved by MspA nanopore trapping, indicating an improved sensing resolution compared with previous attempts using solid‐state nanopores [35] or surface plasmon resonance microscopy [36] . Different states of CaM were trapped in a label‐free fashion without translocation, providing sufficient retention time for analysis, and the current blocking features are clear and highly distinguishable.…”

Section: Introductionmentioning

confidence: 69%

Allosteric Switching of Calmodulin in a Mycobacterium smegmatis porin A (MspA) Nanopore‐Trap

et al. 2021

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Recent developments concerning large protein nanopores suggest a new approach to structure profiling of native folded proteins. In this work, the large vestibule of Mycobacterium smegmatis porin A (MspA) and calmodulin (CaM), a Ca2+‐binding protein, were used in the direct observation of the protein structure. Three conformers, including the Ca2+‐free, Ca2+‐bound, and target peptide‐bound states of CaM, were unambiguously distinguished. A disease related mutant, CaM D129G was also discriminated by MspA, revealing how a single amino acid replacement can interfere with the Ca2+‐binding capacity of the whole protein. The binding capacity and aggregation effect of CaM induced by different ions (Mg2+/Sr2+/Ba2+/Ca2+/Pb2+/Tb3+) were also investigated and the stability of MspA in extreme conditions was evaluated. This work demonstrates the most systematic single‐molecule investigation of different allosteric conformers of CaM, acknowledging the high sensing resolution offered by the MspA nanopore trap.

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Section: Introductionmentioning

confidence: 69%

Allosteric Switching of Calmodulin in a Mycobacterium smegmatis porin A (MspA) Nanopore‐Trap

et al. 2021

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“…We found many moving parabolic patterns inside live cells under CAR illumination, which are organelles such as mitochondria (Fig. 6a and Supplementary Movie 3) 35,36 . The parabolic shape arises from the interference between the evanescent wave and scattered light from the organelles 5,35 .…”

Section: Discussionmentioning

confidence: 97%

“…6a and Supplementary Movie 3) 35,36 . The parabolic shape arises from the interference between the evanescent wave and scattered light from the organelles 5,35 . However, such patterns did not appear under SPR illumination (Fig.…”

Section: Discussionmentioning

confidence: 99%

Critical angle reflection imaging for quantification of molecular interactions on glass surface

Liang

Wan

et al. 2021

Nat Commun

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Quantification of molecular interactions on a surface is typically achieved via label-free techniques such as surface plasmon resonance (SPR). The sensitivity of SPR originates from the characteristic that the SPR angle is sensitive to the surface refractive index change. Analogously, in another interfacial optical phenomenon, total internal reflection, the critical angle is also refractive index dependent. Therefore, surface refractive index change can also be quantified by measuring the reflectivity near the critical angle. Based on this concept, we develop a method called critical angle reflection (CAR) imaging to quantify molecular interactions on glass surface. CAR imaging can be performed on SPR imaging setups. Through a side-by-side comparison, we show that CAR is capable of most molecular interaction measurements that SPR performs, including proteins, nucleic acids and cell-based detections. In addition, we show that CAR can detect small molecule bindings and intracellular signals beyond SPR sensing range. CAR exhibits several distinct characteristics, including tunable sensitivity and dynamic range, deeper vertical sensing range, fluorescence compatibility, broader wavelength and polarization of light selection, and glass surface chemistry. We anticipate CAR can expand SPR′s capability in small molecule detection, whole cell-based detection, simultaneous fluorescence imaging, and broader conjugation chemistry.

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“…Find the ∆ (∆ 0 = 63 nm) value for each individual protein using the intensity vs. applied potential plots ( Figure 5). Determine I for each protein using equation [1].…”

Section: E Single Protein Size Analysismentioning

confidence: 99%

“…We thank financial support from National Institute of Health (R44GM126720). This protocol is based on our work published in Nature Communication (Ma et al, 2020).…”

Section: Acknowledgmentsmentioning

confidence: 99%

Simultaneous Imaging of Single Protein Size, Charge, and Binding Using A Protein Oscillation Approach

Ma¹,

Wan²,

Wang³

2021

BIO-PROTOCOL

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Electrophoresis and Western blot are important tools in protein research for detection and identification of proteins. These traditional techniques separate the proteins based on size and charge differences and identify the proteins by antibody binding. Over the past decade, the emergence of singlemolecule techniques has shown great potential in improving the resolution of the traditional protein analysis methods to the single-molecule level. However, such single-molecule techniques measure either size or charge, and it is challenging to measure both at the same time. Recently, we have developed a single-molecule approach to address this problem. We tether the single proteins to a surface with a polymer linker and drive them into oscillation with an electric field. By tracking the electromechanical response of the proteins to the field using an optical imaging method, the size and charge can be obtained simultaneously. Binding of antibodies or ions to the tethered protein also changes the size and charge, which allows us to probe the interactions. This protocol includes fabrication of protein oscillators, configuration of the optical detection system, and analysis of the oscillation signal for quantification of protein size and charge. We wish this protocol will enable researchers to perform comprehensive single-protein analysis on a single platform.

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Optical imaging of single-protein size, charge, mobility, and binding

Cited by 34 publications

References 55 publications

Allosteric Switching of Calmodulin in a Mycobacterium smegmatis porin A (MspA) Nanopore‐Trap

Allosteric Switching of Calmodulin in a Mycobacterium smegmatis porin A (MspA) Nanopore‐Trap

Critical angle reflection imaging for quantification of molecular interactions on glass surface

Simultaneous Imaging of Single Protein Size, Charge, and Binding Using A Protein Oscillation Approach

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