Site-directed fluorescence approaches to monitor the structural dynamics of proteins using intrinsic Trp and labeled with extrinsic fluorophores

Brahma, Rupasree; Das, Anindita; Raghuraman, H.

doi:10.1016/j.xpro.2022.101200

Cited by 11 publications

(24 citation statements)

References 65 publications

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“…[24][25][26] The biomolecular structural dynamics can be monitored through several spectroscopic methods; among them, uorescence spectroscopy is the most adopted as it is very sensitive to minimal structural perturbations. [27][28][29][30] This technique is not limited to the size of biomolecules and provides real-time information from several microseconds to hundreds of femtoseconds. It also helps to study biomolecules at a single molecular level, imaging their interactions in vivo and in vitro.…”

Section: Introductionmentioning

confidence: 99%

Critical assessment of selenourea as an efficient small molecule fluorescence quenching probe to monitor protein dynamics

Jena,

Tulsiyan,

Sahoo

et al. 2023

Chem. Sci.

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Section: Introductionmentioning

confidence: 99%

Critical assessment of selenourea as an efficient small molecule fluorescence quenching probe to monitor protein dynamics

Jena,

Tulsiyan,

Sahoo

et al. 2023

Chem. Sci.

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“…Thus, the main feature (and advantage) of intrinsic fluorescent probes is their ability to be introduced selectively at certain sites at proteins. [18][19][20][21] In contrast, we define "extrinsic" probes as those not attached to biological macromolecules covalently, but associate with the target non-selectively via hydrophobic or electrostatic interactions. [22] These dyes can be applied for measuring surface hydrophobicity, probing active sites of enzymes, monitoring protein-ligand interactions, and detecting protein aggregation or fibrillation.…”

Section: Applications Of Intrinsic and Extrinsic Environment-sensitiv...mentioning

confidence: 99%

“…Site-directed fluorescence labeling of protein by organic dyes appears to be a promising alternative to intrinsic protein fluorescence. [10,19,21,53,65] Selective protein labeling with thiol-reactive fluorescent probes, such as Bimane, BODIPY, NBD and AEDANS (Scheme 2), revealed increasing promise and is widely used. [10,20,50,[66][67][68][69] The common site-specific labeling protocols utilize commercially available fluorophores with a succinimide ester or maleimide reactive group that targets the primary amino or thiol groups.…”

Section: Thiol-reactive Probes For Site-selective Protein Labelingmentioning

confidence: 99%

Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein‐Lipid Interactions

Kyrychenko,

Ladokhin

2023

The Chemical Record

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Fluorescence spectroscopy provides numerous methodological tools for structural and functional studies of biological macromolecules and their complexes. All fluorescence‐based approaches require either existence of an intrinsic probe or an introduction of an extrinsic one. Moreover, studies of complex systems often require an additional introduction of a specific quencher molecule acting in combination with a fluorophore to provide structural or thermodynamic information. Here, we review the fundamentals and summarize the latest progress in applications of different classes of fluorescent probes and their specific quenchers, aimed at studies of protein folding and protein‐membrane interactions. Specifically, we discuss various environment‐sensitive dyes, FRET probes, probes for short‐distance measurements, and several probe‐quencher pairs for studies of membrane penetration of proteins and peptides. The goals of this review are: (a) to familiarize the readership with the general concept that complex biological systems often require both a probe and a quencher to decipher mechanistic details of functioning and (b) to provide example of the immediate applications of the described methods.

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“…This makes membrane protein purification very expensive, with most of the cost in the selection of detergents suitable for extraction, purification, and crystallization. Any improvement in the extraction of pure, stable, and functional membrane proteins in a cost‐effective manner is highly useful to perform sophisticated structural (Moraes et al., 2014; Prive, 2007) and site‐directed spectroscopic studies (Brahma, Das, & Raghuraman, 2022; Brahma & Raghuraman, 2021, 2022; Raghuraman, Chatterjee, & Das, 2019; Raghuraman, Islam, Mukherjee, Roux, & Perozo, 2014).…”

Section: Commentarymentioning

confidence: 99%

Cost‐effective Purification of Membrane Proteins using a Dual‐detergent Strategy

Brahma

Raghuraman

2022

Current Protocols

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Understanding the mechanisms of membrane protein function is critical for biomedical research and drug discovery as membrane proteins constitute ∼30% of the proteins encoded by the genomes of both lower and higher organisms and are targets for two-thirds of approved drugs worldwide. Significant progress has been made in engineering host expression systems for large-scale production of membrane proteins and in determining their three-dimensional high-resolution structures. Despite these efforts, the study of membrane proteins at the atomic level is challenging due to poor expression and extraction, low yields of functional protein, and the complexity and heterogeneity of source membranes. Structural and spectroscopic studies of any membrane protein require that the protein be extracted from its native membranes into a membrane-mimetic stable environment, which is often achieved by the use of detergents. Unfortunately, there is no magic detergent that can extract all membrane proteins and successful extraction often requires a thorough screen of detergents. Furthermore, membrane protein purification in general and the detergents used are very expensive, which puts a financial constraint on sophisticated membrane protein studies. To overcome this hurdle, a dual-detergent strategy has recently been developed and successfully applied to purify various classes of pure, stable, and functionally relevant membrane proteins in a cost-effective manner. This strategy uses an inexpensive detergent for solubilization of the desired protein from membranes and a second detergent during protein purification. In the Basic Protocol, we describe the dual-detergent strategy to significantly reduce the overall purification cost of a bacterial membrane protein using the magnesium ion channel MgtE as an example. Support Protocols are also provided for selecting a suitable E. coli strain for protein expression and the optimal detergent(s) for membrane protein solubilization.

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Site-directed fluorescence approaches to monitor the structural dynamics of proteins using intrinsic Trp and labeled with extrinsic fluorophores

Cited by 11 publications

References 65 publications

Critical assessment of selenourea as an efficient small molecule fluorescence quenching probe to monitor protein dynamics

Critical assessment of selenourea as an efficient small molecule fluorescence quenching probe to monitor protein dynamics

Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein‐Lipid Interactions

Cost‐effective Purification of Membrane Proteins using a Dual‐detergent Strategy

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