Fluorescence-based characterization of non-fluorescent transient states of tryptophan – prospects for protein conformation and interaction studies

Hevekerl, Heike; Tornmalm, Johan; Widengren, Jerker

doi:10.1038/srep35052

Cited by 18 publications

(41 citation statements)

References 63 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ZMW samples are rinsed with ultrapure water and isopropanol and then exposed to oxygen plasma for 5 minutes to remove any remaining organic residues and densify the oxide layer. To protect the aluminum surface and mitigate the corrosion effects, 46,47 together are abbreviated as PODCAT and play the role of an oxygen scavenger 51 that removes a significant fraction of oxygen from the solution and increases photostability and brightness of βgalactosidase, whereas ascorbic acid is introduced as a protein-friendly antioxidant to reduce pH of the solution for the measurement, 52 and help stabilize the aluminum nanostructures. 49 All the aforementioned chemicals are purchased from Sigma Aldrich.…”

Section: Surface Passivationmentioning

confidence: 99%

Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides

et al. 2019

View full text Add to dashboard Cite

Single molecule detection provides detailed information about molecular structures and functions, but it generally requires the presence of a fluorescent marker which can interfere with the activity of the target molecule or complicate the sample production. Detecting a single protein with its natural UV autofluorescence is an attractive approach to avoid all the issues related to fluorescence labelling.However, the UV autofluorescence signal from a single protein is generally extremely weak. Here, we use aluminum plasmonics to enhance the tryptophan autofluorescence emission of single proteins in the UV range. Zero-mode waveguides nanoapertures enable observing the UV fluorescence of single label-free β-galactosidase proteins with increased brightness, microsecond transit times and operation at micromolar concentrations. We demonstrate quantitative measurements of the local concentration, diffusion coefficient and hydrodynamic radius of the label-free protein over a broad range of zero-mode waveguide diameters. While the plasmonic fluorescence enhancement has generated a tremendous interest in the visible and near-infrared parts of the spectrum, this work pushes further the limits of plasmonic-enhanced single molecule detection into the UV range and constitutes a major step forward in our ability to interrogate single proteins in their native state at physiological concentrations.

show abstract

Section: Surface Passivationmentioning

confidence: 99%

Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Conversely, oxygen scavengers such as sodium azide or glucose oxidase 66 do not change significantly the behavior as compared to pure water (data not shown). Ascorbic acid is a good reducing agent, 67 but at pH 7 it does not influence significantly the photocorrosion of aluminum. However, at pH 4, ascorbic acid efficiently inhibits the photocorrosion (Fig.…”

Section: Resultsmentioning

confidence: 98%

Preventing Aluminum Photocorrosion for Ultraviolet Plasmonics

Barulin

Claude

Patra

et al. 2019

J. Phys. Chem. Lett.

View full text Add to dashboard Cite

Ultraviolet (UV) plasmonics aims at combining the strong absorption bands of molecules in the UV range with the intense electromagnetic fields of plasmonic nanostructures to promote surfaceenhanced spectroscopy and catalysis. Currently, aluminum is the most widely used metal for UV plasmonics, and is generally assumed to be remarkably stable thanks to its natural alumina layer passivating the metal surface. However, we find here that under 266 nm UV illumination, aluminum can undergo a dramatic photocorrosion in water within a few tens of seconds and even at low average UV powers. This aluminum instability in water environments critically limits the UV plasmonics applications. We show that the aluminum photocorrosion is related to the nonlinear absorption by water in the UV range leading to the production of hydroxyl radicals. Different corrosion protection approaches are tested using scavengers for reactive oxygen species and polymer layers deposited on top of the aluminum structures. Using optimized protection, we achieve a ten-fold increase in the available UV power range leading to no visible photocorrosion effects. This technique is crucial to achieve stable use of aluminum nanostructures for UV plasmonics in aqueous solutions.

show abstract

“…It is therefore convenient to determine this state using TRAST monitoring spectroscopy during aggregate formation because that method may be employed regardless of whether the molecules are mobile or immobile. Analyses of the transient state of tryptophan fluorescence using TRAST monitoring spectroscopy has been reported [ 112 ], and therefore the method might be used for determining the conformational changes of proteins. However, it is noticed that various verifications are necessary to link whether the transient state phenomena of the fluorophores are caused by the conformational change of labeling proteins.…”

Section: Fluorescence Fluctuation-based Spectroscopic Techniquesmentioning

confidence: 99%

State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation

Kitamura

Kinjo

2018

IJMS

View full text Add to dashboard Cite

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are devastating proteinopathies with misfolded protein aggregates accumulating in neuronal cells. Inclusion bodies of protein aggregates are frequently observed in the neuronal cells of patients. Investigation of the underlying causes of neurodegeneration requires the establishment and selection of appropriate methodologies for detailed investigation of the state and conformation of protein aggregates. In the current review, we present an overview of the principles and application of several methodologies used for the elucidation of protein aggregation, specifically ones based on determination of fluctuations of fluorescence. The discussed methods include fluorescence correlation spectroscopy (FCS), imaging FCS, image correlation spectroscopy (ICS), photobleaching ICS (pbICS), number and brightness (N&B) analysis, super-resolution optical fluctuation imaging (SOFI), and transient state (TRAST) monitoring spectroscopy. Some of these methodologies are classical protein aggregation analyses, while others are not yet widely used. Collectively, the methods presented here should help the future development of research not only into protein aggregation but also neurodegenerative diseases.

show abstract

Fluorescence-based characterization of non-fluorescent transient states of tryptophan – prospects for protein conformation and interaction studies

Cited by 18 publications

References 63 publications

Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides

Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides

Preventing Aluminum Photocorrosion for Ultraviolet Plasmonics

State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation

Contact Info

Product

Resources

About