Membrane proteins enter the fold

Marx, Dagan C.; Fleming, Karen G.

doi:10.1016/j.sbi.2021.03.006

Cited by 9 publications

(3 citation statements)

References 52 publications

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“…In such cases, mutations compromise membrane protein stability, resulting in aberrant protein function. Experimental and high-throughput measurements are key for understanding (i) the effects of genetic mutations on the thermostability of membrane proteins and (ii) how mutations reshape native lipid-protein interactions to drive disease states, which have been historically underappreciated 37,38 . Furthermore, our method will be used to test membrane protein design candidates in cases where the goal is to design a more thermostable protein to facilitate biochemical or structural studies.…”

Section: Resultsmentioning

confidence: 99%

Dark nanodiscs as a model membrane for evaluating membrane protein thermostability by differential scanning fluorimetry

Selvasingh,

McDonald,

Mckinney

et al. 2023

Preprint

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Measuring protein thermostability provides valuable information on the biophysical rules that govern structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently-developed non-fluorescent membrane scaffold protein (MSP) to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). This approach offers a label-free and direct measurement of the intrinsic tryptophan fluorescence of the membrane protein as it unfolds in solution without signal interference from the “dark” nanodisc. In this work, we demonstrate the application of this method using the disulfide bond formation protein B (DsbB) as a test membrane protein. NanoDSF measurements of DsbB reconstituted in dark nanodiscs show a complex biphasic thermal unfolding pattern in the presence of lipids with a minor unfolding transition followed by a major transition. The inflection points of the thermal denaturation curve reveal two distinct unfolding midpoint melting temperatures (Tm) of 70.5 °C and 77.5 °C, consistent with a three-state unfolding model. Further, we show that the catalytically conserved disulfide bond between residues C41 and C130 drives the intermediate state of the unfolding pathway for DsbB in a nanodisc. We introduce this method as a new tool that can be used to understand how compositionally, and biophysically complex lipid environments drive membrane protein stability.

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Section: Resultsmentioning

confidence: 99%

Dark nanodiscs as a model membrane for evaluating membrane protein thermostability by differential scanning fluorimetry

Selvasingh,

McDonald,

Mckinney

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Therefore, large-scale and easily accessible experimental data for benchmarking computational tools are sparse. Despite recent methodological advances ( Marx and Fleming, 2021 ), computational methods for membrane proteins are not as developed as those for soluble proteins. Thus, the application of computational analyses for examining a potential correlation between protein stability, cellular abundance, and function analogous to that for soluble proteins may be particularly challenging for membrane proteins.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, large-scale and easily accessible experimental data for benchmarking computational tools are sparse. Despite recent methodological advances (34), computational methods for membrane proteins are not as developed as those for soluble proteins. Furthermore, the diverse experimental studies measure different levels of unfolding, which further challenges computational method development.…”

Section: Introductionmentioning

confidence: 99%

Interpreting the molecular mechanisms of disease variants in human transmembrane proteins

Tiemann

Zschach

Lindorff‐Larsen

et al. 2022

Preprint

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Next-generation sequencing of human genomes reveals millions of missense variants, some of which may lead to loss of protein function and ultimately disease. We here investigate missense variants in membrane proteins -- key drivers in cell signaling and recognition. We find enrichment of pathogenic variants in the transmembrane region across 19,000 functionally classified variants in human membrane proteins. A key mechanism underlying pathogenicity in missense variants of soluble proteins has been shown to be loss of stability. We here perform structure-based estimations of changes in thermodynamic stability and evolutionary conservation analyses of 15 membrane proteins. We find evidence for loss of stability being the cause of pathogenicity in 59% of pathogenic variants, indicating that this is a driving factor also in membrane-protein-associated diseases. Our findings show how computational tools aid in gaining mechanistic insights into variant consequences for membrane proteins. To enable broader analyses of disease-related and population variants, we include variant mappings for the entire human proteome.

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Photoradical‐Mediated Catalyst‐Independent Protein Cross‐Link with Unusual Fluorescence Properties

Bora

Mahalakshmi

2023

ChemBioChem

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Photo‐actively modified natural amino acids have served as lucrative probes for precise mapping of the dynamics, interaction networks, and turnover of cytosolic proteins both in vivo and ex vivo. In our attempts to extend the utility of photoreactive reporters to map the molecular characteristics of vital membrane proteins, we carried out site‐selective incorporation of 7‐fluoro‐indole in the human mitochondrial outer membrane protein VDAC2 (voltage‐dependent anion channel isoform 2), with the aim of generating Trp−Phe/Tyr cross‐links. Prolonged irradiation at 282 nm provided us with a surprisingly unusual fluorophore that displayed sizably red‐shifted excitation (λex‐max=280 nm→360 nm) and emission (λem‐max=330 nm→430 nm) spectra that was reversible with organic solvents. By measuring the kinetics of the photo‐activated cross‐linking with a library of hVDAC2 variants, we demonstrate that formation of this unusual fluorophore is kinetically retarded, independent of tryptophan, and is site‐specific. Using other membrane (Tom40 and Sam50) and cytosolic (MscR and DNA Pol I) proteins, we additionally show that formation of this fluorophore is protein‐independent. Our findings reveal the photoradical‐mediated accumulation of reversible tyrosine cross‐links, with unusual fluorescent properties. Our findings have immediate applications in protein biochemistry and UV‐mediated protein aggregation and cellular damage, opening avenues for formulating therapeutics that prolong cell viability in humans.

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Membrane proteins enter the fold

Cited by 9 publications

References 52 publications

Dark nanodiscs as a model membrane for evaluating membrane protein thermostability by differential scanning fluorimetry

Dark nanodiscs as a model membrane for evaluating membrane protein thermostability by differential scanning fluorimetry

Interpreting the molecular mechanisms of disease variants in human transmembrane proteins

Photoradical‐Mediated Catalyst‐Independent Protein Cross‐Link with Unusual Fluorescence Properties

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