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

AbstractProtein analysis has relied on electrophoresis, mass spectroscopy and immunoassay, which separate, detect and identify proteins based on the size, charge, mobility and binding to antibodies. However, measuring these quantities at the single molecule level has not been possible. We tether a protein to a surface with a flexible polymer, drive the protein into mechanical oscillation with an alternating electric field, and image the protein oscillation with a near field imaging method, from which we determine the size, charge, mobility of the protein. We also measure binding of antibodies to single proteins and ligand binding-induced conformational changes in single proteins. This work provides new capabilities for protein analysis and disease biomarker detection at the single molecule level.

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(Invited) Measure Single Protein Size and Binding Kinetics with Plasmonic Scattering Imaging

ECS Meeting Abstracts ◽

10.1149/ma2021-02561643mtgabs ◽

2021 ◽

Vol MA2021-02 (56) ◽

pp. 1643-1643

Author(s):

Pengfei Zhang ◽

Guangzhong Ma ◽

Zijian Wan ◽

Nongjian Tao ◽

Shaopeng Wang

Keyword(s):

Binding Kinetics ◽

Protein Size ◽

Single Protein

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Orientation of actin filaments during motion in motility assay

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136635 ◽

1995 ◽

Vol 53 ◽

pp. 52-53

Author(s):

J. Borejdo ◽

S. Burlacu

Keyword(s):

Actin Filaments ◽

Thin Filament ◽

Striated Muscle ◽

Myosin Head ◽

Classical Method ◽

Polarization Of Fluorescence ◽

Single Protein ◽

Intracellular Organelles ◽

Change Orientation ◽

Active Entity

Polarization of fluorescence is a classical method to assess orientation or mobility of macromolecules. It has been a common practice to measure polarization of fluorescence through a microscope to characterize orientation or mobility of intracellular organelles, for example anisotropic bands in striated muscle. Recently, we have extended this technique to characterize single protein molecules. The scientific question concerned the current problem in muscle motility: whether myosin heads or actin filaments change orientation during contraction. The classical view is that the force-generating step in muscle is caused by change in orientation of myosin head (subfragment-1 or SI) relative to the axis of thin filament. The molecular impeller which causes this change resides at the interface between actin and SI, but it is not clear whether only the myosin head or both SI and actin change orientation during contraction. Most studies assume that observed orientational change in myosin head is a reflection of the fact that myosin is an active entity and actin serves merely as a passive "rail" on which myosin moves.

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Five good reasons to use single protein production for membrane proteins

PSI Structural Genomics Knowledgebase ◽

10.1038/th_psisgkb.2009.60 ◽

2009 ◽

Author(s):

Maria Hodges

Keyword(s):

Membrane Proteins ◽

Protein Production ◽

Single Protein

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Native Ubiquitin Structural Changes Resulting from Complexation with β-methylamino-L-alanine (BMAA)

10.26434/chemrxiv.12996335.v1 ◽

2020 ◽

Author(s):

Katie Mae Wilson ◽

Aurora Burkus-Matesevac ◽

Samuel Maddox ◽

Christopher Chouinard

Keyword(s):

Structural Changes ◽

Noncovalent Complex ◽

Unfolded Protein ◽

Protein Size ◽

High Concentrations ◽

The Unfolded Protein Response ◽

Critical Binding ◽

Protein Response ◽

The Brain ◽

Lateral Sclerosis

β-methylamino-L-alanine (BMAA) has been linked to the development of neurodegenerative (ND) symptoms following chronic environmental exposure through water and dietary sources. The brains of those affected by this condition, often referred to as amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), have exhibited the presence of plaques and neurofibrillary tangles (NFTs) from protein aggregation. Although numerous studies have sought to better understand the correlation between BMAA exposure and onset of ND symptoms, no definitive link has been identified. One prevailing hypothesis is that BMAA acts a small molecule ligand, complexing with critical proteins in the brain and reducing their function. The objective of this research was to investigate the effects of BMAA exposure on the native structure of ubiquitin. We hypothesized that formation of a Ubiquitin+BMAA noncovalent complex would alter the protein’s structure and folding and ultimately affect the ubiquitinproteasome system (UPS) and the unfolded protein response (UPR). Ion mobility-mass spectrometry revealed that at sufficiently high concentrations BMAA did in fact form a noncovalent complex with ubiquitin, however similar complexes were identified for a range of additional amino acids. Collision induced unfolding (CIU) was used to interrogate the unfolding dynamics of native ubiquitin and these Ubq-amino acid complexes and it was determined that complexation with BMAA led to a significant alteration in native protein size and conformation, and this complex required considerably more energy to unfold. This indicates that the complex remains more stable under native conditions and this may indicate that BMAA has attached to a critical binding location.

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Native Ubiquitin Structural Changes Resulting from Complexation with β-methylamino-L-alanine (BMAA)

10.26434/chemrxiv.12996335 ◽

2020 ◽

Author(s):

Katie Mae Wilson ◽

Aurora Burkus-Matesevac ◽

Samuel Maddox ◽

Christopher Chouinard

Keyword(s):

Structural Changes ◽

Noncovalent Complex ◽

Unfolded Protein ◽

Protein Size ◽

High Concentrations ◽

The Unfolded Protein Response ◽

Critical Binding ◽

Protein Response ◽

The Brain ◽

Lateral Sclerosis

β-methylamino-L-alanine (BMAA) has been linked to the development of neurodegenerative (ND) symptoms following chronic environmental exposure through water and dietary sources. The brains of those affected by this condition, often referred to as amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), have exhibited the presence of plaques and neurofibrillary tangles (NFTs) from protein aggregation. Although numerous studies have sought to better understand the correlation between BMAA exposure and onset of ND symptoms, no definitive link has been identified. One prevailing hypothesis is that BMAA acts a small molecule ligand, complexing with critical proteins in the brain and reducing their function. The objective of this research was to investigate the effects of BMAA exposure on the native structure of ubiquitin. We hypothesized that formation of a Ubiquitin+BMAA noncovalent complex would alter the protein’s structure and folding and ultimately affect the ubiquitinproteasome system (UPS) and the unfolded protein response (UPR). Ion mobility-mass spectrometry revealed that at sufficiently high concentrations BMAA did in fact form a noncovalent complex with ubiquitin, however similar complexes were identified for a range of additional amino acids. Collision induced unfolding (CIU) was used to interrogate the unfolding dynamics of native ubiquitin and these Ubq-amino acid complexes and it was determined that complexation with BMAA led to a significant alteration in native protein size and conformation, and this complex required considerably more energy to unfold. This indicates that the complex remains more stable under native conditions and this may indicate that BMAA has attached to a critical binding location.

Download Full-text