Visualization of the nanospring dynamics of the IκBα ankyrin repeat domain in real time

Lamboy, Jorge A.; Kim, Hajin; Lee, Kyung Suk; Ha, Taekjip; Komives, Elizabeth A.

doi:10.1073/pnas.1102226108

Cited by 46 publications

(69 citation statements)

References 39 publications

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“…Moreover, studies on the six ankyrin repeat protein IB␣ show it also has a weakly folded C terminus. Here, repeats five and six fold upon binding to their cognate partner (the transcription factor NF-B) (29,30). It has been shown that the function and lifetime within the cell of IB␣ is critically linked to whether this region is structured via ligand binding or not (31).…”

Section: Discussionmentioning

confidence: 99%

LcrH, a Class II Chaperone from the Type Three Secretion System, Has a Highly Flexible Native Structure

Singh

Boyle

Main

2013

Journal of Biological Chemistry

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Background: Bacterial pathogens using the type III secretion system (T3SS) require class II chaperones (LcrH) for infection. Results: The modular TPR fold of LcrH produces a weakly folded structure held together by its weak dimeric interface. Conclusion: Under physiological conditions, LcrH populates partially folded structures, thus suggesting a mechanism for cargo binding. Significance: This work is important for understanding one essential part of the T3SS.

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Section: Discussionmentioning

confidence: 99%

LcrH, a Class II Chaperone from the Type Three Secretion System, Has a Highly Flexible Native Structure

Singh

Boyle

Main

2013

Journal of Biological Chemistry

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“…The created sample chamber was then filled with an aqueous solution of 167 mM APS for 30 min and rinsed with DI water, resulting in a glass surface modified with primary amine groups. The APS coverslips were functionalized with a 1:100 Mal-PEG-SVA/mPEG-SVA molar mixture (31,32) by incubation for 1 h in 0.1 M sodium bicarbonate buffer, pH 8.5 (33). Bubbles were removed from the sodium bicarbonate buffer by vortexing and ultrasonication.…”

Section: Total Internal Reflection Fluorescence Microscopy Imagingmentioning

confidence: 99%

Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis

Krasnoslobodtsev

Zhang

et al. 2015

Biophysical Journal

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The aggregation of a-synuclein (a-Syn) is linked to Parkinson's disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of a-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free a-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) a-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 5 3 ms and 3334 5 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 5 14 ms) > E46K (292 5 5 ms) > A53T (226 5 6 ms) > WT (197 5 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of a-Syn dimers could lead to different aggregation pathways.

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“…Developments in optical and force spectroscopy techniques have enabled single-molecule studies on several aspects of protein dynamics, folding, and interactions (34, 35). Single-molecule techniques provide new types of information at very low sample concentrations without ensemble averaging (35)(36)(37)(38)(39)(40)(41)(42). Using the distance dependence of single-molecule Förster/fluorescence resonance energy transfer (smFRET) to probe protein dimensions, we studied the effect on α-synuclein of urea and TMAO, both as individual cosolvents and also in varying mixing ratios.…”

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confidence: 99%

Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape

Ferreon

Moosa

Gambin

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importance, detailed studies on the role of these chemical chaperones in modulating structure and dimensions of intrinsically disordered proteins have been limited. Here, we used single-molecule Förster resonance energy transfer to test the counteraction hypothesis of counterbalancing effects between the protecting osmolyte trimethylamine-N-oxide (TMAO) and denaturing osmolyte urea for the case of α-synuclein, a Parkinson's disease-linked protein whose monomer exhibits significant disorder. The single-molecule experiments, which avoid complications from protein aggregation, do not exhibit clear solvent-induced cooperative protein transitions for these osmolytes, unlike results from previous studies on globular proteins. Our data demonstrate the ability of TMAO and urea to shift α-synuclein structures towards either more compact or expanded average dimensions. Strikingly, the experiments directly reveal that a 2∶1 ½urea∶½TMAO ratio has a net neutral effect on the protein's dimensions, a result that holds regardless of the absolute osmolyte concentrations. Our findings shed light on a surprisingly simple aspect of the interplay between urea and TMAO on α-synuclein in the context of intrinsically disordered proteins, with potential implications for the biological roles of such chemical chaperones. The results also highlight the strengths of single-molecule experiments in directly probing the chemical physics of protein structure and disorder in more chemically complex environments.Parkinson's disease | protein folding | urea-TMAO counteraction | smFRET P roteins are dynamic entities that are in constant interaction with their environment. Several components of the protein environment can affect the folding landscape (1) and function, including solvents (2), osmolytes (3), crowding agents (4), and small-molecule and macromolecular ligands (5-7). Osmolytes are naturally occurring low-molecular weight compounds that are utilized by biological systems as chemical chaperones that counteract deleterious effects of extreme physical conditions such as high osmotic and hydrostatic pressures (8, 9), dehydration (10), and high or low temperatures (11, 12). Urea, a major metabolic byproduct, is known to be used as a balancing osmolyte by several marine vertebrates (8), air-breathing teleosts (13) and some amphibians (14, 15) to deal with osmotic stress. In mammalian kidneys, urea plays an important role in balancing the medullary osmotic gradient (16). However, even at physiologically relevant concentrations, urea shows a strong denaturing effect on proteins (8,17). This apparent paradox is solved by the activity of several protecting osmolytes (e.g., methylamines and polyhydric alcohols) found in these urea-rich biological systems (8,18,19).T...

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Visualization of the nanospring dynamics of the IκBα ankyrin repeat domain in real time

Cited by 46 publications

References 39 publications

LcrH, a Class II Chaperone from the Type Three Secretion System, Has a Highly Flexible Native Structure

LcrH, a Class II Chaperone from the Type Three Secretion System, Has a Highly Flexible Native Structure

Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis

Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape

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