Gating-related Structural Dynamics of the MgtE Magnesium Channel in Membrane-Mimetics Utilizing Site-Directed Tryptophan Fluorescence

Chatterjee, S. N.; Brahma, Rupasree; Raghuraman, H.

doi:10.1016/j.jmb.2020.10.025

Cited by 14 publications

(17 citation statements)

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“…Intrinsic tryptophan (Trp) fluorescence is a widely used tool to monitor conformational changes in proteins and to gain information regarding local structure and dynamics ( Biswas et al., 2020 ; Chatterjee et al., 2021 ). SDFL has been proved powerful in determining protein and other macromolecular structural information in complex biological systems ( Raghuraman et al., 2019 ).…”

Section: Before You Beginmentioning

confidence: 99%

“…This means that emission maximum is sensitive to the probe’s surrounding microenvironment. In this regard, intrinsic Trp fluorescence is the most widely used tool to monitor the changes in local structure and dynamics in proteins ( Vivian and Callis, 2001 ; Raghuraman and Chattopadhyay, 2003 ; Raghuraman et al., 2005 ; Callis and Tusell, 2014 ; Chatterjee et al., 2021 ). Further, the environment sensitivity of Trp fluorescence has been extensively used to monitor protein folding/unfolding transitions, membrane partitioning, lipid-protein interactions etc.…”

Section: Expected Outcomesmentioning

confidence: 99%

“…However, fluorophores that are membrane-bound or at buried sites exhibit a higher anisotropy/polarization value due to hindered rotational mobility of the probe. ( Raghuraman and Chattopadhyay, 2004c ; Das et al., 2020 ; Chatterjee et al., 2021 ). The limiting anisotropy value for Trp is 0.16 ( Eftink et al., 1990 ) and the corresponding value for NBD is 0.354 ( Mukherjee et al., 2004 ).…”

Section: Expected Outcomesmentioning

confidence: 99%

“…High K SV means increased accessibility by the quencher, which, in case of aqueous quenchers like acrylamide and iodide ions, directly implies increased water accessibility and vice versa . For example, the K SV is ∼18 M -1 for a fully exposed Trp to aqueous medium ( Raghuraman and Chattopadhyay, 2004a ), and this value dramatically reduces to as low as ∼2–7 M -1 when Trp residue(s) is partitioned in micelles or lipid bilayer ( Chatterjee et al., 2019 , 2021 ). Comparing results between samples only on K SV values needs caution, because these values are only relevant if the fluorescence lifetime does not change significantly between the samples.…”

Section: Expected Outcomesmentioning

confidence: 99%

“…For complete details on the use and execution of this protocol, please refer to Das et al. (2020) , Das and Raghuraman (2021) , and Chatterjee et al. (2021) .…”

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

2022

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Section: Before You Beginmentioning

confidence: 99%

Section: Expected Outcomesmentioning

confidence: 99%

Section: Expected Outcomesmentioning

confidence: 99%

Section: Expected Outcomesmentioning

confidence: 99%

“…For complete details on the use and execution of this protocol, please refer to Das et al. (2020) , Das and Raghuraman (2021) , and Chatterjee et al. (2021) .…”

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

2022

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

Brahma

Raghuraman

2022

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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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Peptide‐assistedsupramolecular polymerization of the anionic porphyrinmeso‐tetra(4‐sulfonatophenyl)porphine

et al. 2022

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Electronics made from biological materials are desirable for use in medical devices, biosensors, and soft robotics because they have properties inaccessible to current inorganic technologies. Here, a series of cationic peptides were developed to selfassemble with the anionic porphyrin meso-tetra(4-sulfonatophenyl)porphine (TPPS 4 ) into supramolecular light-harvesting wires known as J-aggregates were developed. In addition to quantifying the binding events between each peptide and TPPS 4 , we found co-incubation with mixed peptides allowed TPPS 4 to bridge two different peptides at their terminal porphyrin-binding regions. The J-aggregates formed on macroscopic scale in near quantitative yield within $3 h and readily interconverted with the soluble monomers upon cycling the pH; to our knowledge, this is the first report of a peptide-porphyrin system with such properties. This work has practical and conceptual relevance to the development of new bioelectronic materials.

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Gating-related Structural Dynamics of the MgtE Magnesium Channel in Membrane-Mimetics Utilizing Site-Directed Tryptophan Fluorescence

Cited by 14 publications

References 93 publications

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

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

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

Peptide‐assistedsupramolecular polymerization of the anionic porphyrinmeso‐tetra(4‐sulfonatophenyl)porphine

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