Detecting the conformation of individual proteins in live cells

Sakon, John J.; Weninger, Keith

doi:10.1038/nmeth.1421

Cited by 146 publications

(120 citation statements)

References 27 publications

Supporting

Mentioning

120

Contrasting

Order By: Relevance

“…Flory-Huggins theories as used here might thus provide novel insights into the demixing of multicomponent polymeric systems (41). An interesting next step will be a direct comparison of experiments in vitro with intracellular measurements (14,26), and the required quantitative tools are beginning to emerge (54)(55)(56).…”

Section: Discussionmentioning

confidence: 99%

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments

Soranno

Koenig

Borgia

et al. 2014

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

228

307

View full text Add to dashboard Cite

Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-molecule Förster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers. We observe a compaction of IDPs not only with increasing concentration, but also with increasing size of the crowding agents, at variance with the predictions from scaled-particle theory, the prevalent paradigm in the field. However, the observed behavior can be explained quantitatively if the polymeric nature of both the IDPs and the crowding molecules is taken into account explicitly. Our results suggest that excluded volume interactions between overlapping biopolymers and the resulting criticality of the system can be essential contributions to the physics governing the crowded cellular milieu. A surprisingly large number of eukaryotic proteins either contain substantial unstructured regions or are entirely unfolded under physiological conditions (1, 2). These "intrinsically disordered proteins" (IDPs) are involved in many crucial cellular processes, such as transcription, translation, and signal transduction; their functional and conformational properties are thus of great interest for a wide range of biological questions. Important advances in understanding the structures of IDPs have been made over the past decade, especially with spectroscopic techniques, e.g., NMR (3, 4), single-molecule fluorescence (5-7), and with atomistic and coarse-grained molecular simulations (8-10). In contrast with the stable folded structures we are familiar with from 50 y of structural biology, IDPs comprise highly heterogeneous and dynamic ensembles of conformations, which either lack stable tertiary structure altogether or fold only on binding their cellular targets (4). Important components of the cellular environment that affect IDPs include not only specific cellular ligands, but also pH and the concentration of salts (11,12). An additional contribution that has been difficult to investigate experimentally comes from the large number of different solutes present in a cell that do not interact with an IDP specifically, but result in an environment that is densely filled with macromolecules and metabolites (12)(13)(14). Given their lack of persistent structure, the conformations of IDPs are expected to be particularly sensitive to the effects of such molecular crowding. Indeed, first experiments indicate that some IDPs gain structure upon crowding (15), whereas others do not (16-18), but may change their dimensions (19)(20)(21). The question of how the conformational distributions of IDPs respond to crowded environments is of particular current interest because IDPs have a vital role in cellular compartments and regions with very high local concentrations of proteins and RNA, such as RNA granules and nuclear pore complexes (22)(23)(24)(25). Howeve...

show abstract

Section: Discussionmentioning

confidence: 99%

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments

Soranno

Koenig

Borgia

et al. 2014

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

228

307

View full text Add to dashboard Cite

show abstract

“…[43][44][45] Following the rapid conceptual transition provoked by expanding evidence protein disorder, one major recent challenge in the field is to characterize the structure (structural ensemble) of IDPs/IDRs by a combination of highly advanced structural techniques, 46,47 and interpret their function in terms of their heterogeneous and highly dynamic structural state. Current efforts also aim to provide insight into their structure either in vivo 48,49 or at the single-molecule level. 50,51 As outlined next, their basic functional modes are entirely relevant with the novel type of allostery we aim to describe.…”

Section: Structural Disordermentioning

confidence: 99%

“…Singlemolecule fluorescence resonance energy transfer (smFRET), for example, demonstrated multiple interactions in p53, between its N-terminal domain and DNA binding domains, 51 and it even enabled the characterization of the dynamics of soluble NSF attachment protein receptor (SNARE) complexes in live cells. 48 It also enabled to address the "supertertiary" structure of the complex regulatory protein PSD95. 164 Atomic-force microscopy (AFM) has also been recently applied for studying the structure and structural changes of the complex motor protein myosin V with disordered linkers 165 and the structural ensemble and unfolding patterns of α-synuclein.…”

Section: Structural Characterizationmentioning

confidence: 99%

Multisteric Regulation by Structural Disorder in Modular Signaling Proteins: An Extension of the Concept of Allostery

2013

View full text Add to dashboard Cite

“…To determine whether a conformational change in SNAREs is linked to fusion, the synaptosomal-associated protein 25 (SNAP25) mutant SCORE (SNARE COmplex REporter) has been developed (7), which contains two fluorescent proteins, CFP as a fluorescence resonance energy transfer (FRET) donor and Venus as a FRET acceptor. SCORE and constructs like it (5,(7)(8)(9) have the advantage that donor and acceptor exist at fixed stoichiometry, facilitating the analysis and interpretation of the measurements. Like SNAP25, SCORE forms SNARE complexes with syntaxin and vesicle-associated membrane protein (VAMP)/synaptobrevin (7,8), and when endogenous SNAP25 in chromaffin cells is cleaved by botulinum toxin E, a toxin-resistant SCORE rescues exocytosis (8).…”

mentioning

confidence: 99%

Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells

Zhao

Fang

Herbst

et al. 2013

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

View full text Add to dashboard Cite

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.TIR-FRET imaging | electrochemical imaging | time superresolution microscopy | image analysis | transmitter release N eurotransmitters, hormones, and many other mediators are stored in secretory vesicles, and their release occurs by the mechanism of exocytosis that begins with formation of a narrow fusion pore (1). Fusion-pore formation in neurosecretory vesicles is stimulated by an increase of intracellular [Ca 2+ ] and is thought to be induced by a large conformational change in the SNARE complex (2-4). Such changes may be involved in various steps from preparing vesicles for fusion (5) to fusion-pore dilation (6). To determine whether a conformational change in SNAREs is linked to fusion, the synaptosomal-associated protein 25 (SNAP25) mutant SCORE (SNARE COmplex REporter) has been developed (7), which contains two fluorescent proteins, CFP as a fluorescence resonance energy transfer (FRET) donor and Venus as a FRET acceptor. SCORE and constructs like it (5, 7-9) have the advantage that donor and acceptor exist at fixed stoichiometry, facilitating the analysis and interpretation of the measurements. Like SNAP25, SCORE forms SNARE complexes with syntaxin and vesicle-associated membrane protein (VAMP)/synaptobrevin (7,8), and when endogenous SNAP25 in chromaffin cells is cleaved by botulinum toxin E, a toxin-resistant SCORE rescues exocytosis (8). In PC12 cells expressing SCORE, a FRET change was evoked by high [K + ] stimulation, but with this method the time scale was tens of seconds (7). This FRET change was abolished in the absence of extracellular Ca 2+ , indicating that it is dependent on Ca 2+ entry. In contrast, the FRET change was not affected by tetanus neurotoxin treatment (7), which causes the blockade of exocytosis (10). In beta cells, a SCORE-like construct indicated a FRET chan...

show abstract

Detecting the conformation of individual proteins in live cells

Cited by 146 publications

References 27 publications

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments

Multisteric Regulation by Structural Disorder in Modular Signaling Proteins: An Extension of the Concept of Allostery

Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells

Contact Info

Product

Resources

About