Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane

Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel J.

doi:10.7554/elife.12125

Cited by 71 publications

(122 citation statements)

References 67 publications

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“…each of the 20 amino acids at 27 positions across the membrane (21). The dsTβL scale was in good agreement with what theory and previous experiments suggested; for instance, the hydrophobicity at the membrane core was in line with biophysical measurements (22) and 3-4 times larger than measured with Lep (23).…”

supporting

confidence: 79%

“…S1 and Table S1). Specifically, in the original dsTβL report (21), Val, Leu, and Met showed slightly nonsymmetric profiles, whereas the other hydrophobics, Ile and Phe, were close to symmetric, as expected. We therefore changed the hydrophobic residues' profiles so that all were symmetric, and maintained the insertion energy at the membrane midplane as in the original dsTβL scales.…”

Section: Resultsmentioning

confidence: 73%

“…2A). This high accuracy is not surprising given that the dsTβL scale is based on experimental data on a single-span membrane protein (21). Multispan membrane proteins are accordingly predicted less accurately, and above four segments prediction accuracy drops to 46%; the overall prediction accuracy across the entire set is 78%.…”

Section: Resultsmentioning

confidence: 99%

“…We therefore eliminate all signal peptides predicted by TOPCONS (13) and any subsequence that is predicted to be nonhelical (27), as well as subsequences that contain several charged or polar residues (SI Methods). To the remaining segments we assign apparent insertion free energies according to the dsTβL scale (21) in each of the two orientations relative to the membrane (locating the C-terminus either in the cytoplasm or outside). Because segments vary in length, we estimate the location z of every amino acid position i in the segment relative to the membrane midplane:…”

Section: Resultsmentioning

confidence: 99%

“…The three TopGraph variants output apparent insertion free energies that are based on the dsTβL scale (21). Applied to the Reeb dataset (16), nearly all segments (99%) predicted using the purist TopGraph exhibit negative apparent insertion energies with a mean of −6.9 kcal/mol (Fig.…”

Section: Energy-based Prediction Of Protein Orientation With Respect mentioning

confidence: 99%

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Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology

Elazar

Weinstein

Prilusky

et al. 2016

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

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The energetics of membrane-protein interactions determine protein topology and structure: hydrophobicity drives the insertion of helical segments into the membrane, and positive charges orient the protein with respect to the membrane plane according to the positive-inside rule. Until recently, however, quantifying these contributions met with difficulty, precluding systematic analysis of the energetic basis for membrane-protein topology. We recently developed the dsTβL method, which uses deep sequencing and in vitro selection of segments inserted into the bacterial plasma membrane to infer insertion-energy profiles for each amino acid residue across the membrane, and quantified the insertion contribution from hydrophobicity and the positive-inside rule. Here, we present a topology-prediction algorithm called TopGraph, which is based on a sequence search for minimum dsTβL insertion energy. Whereas the average insertion energy assigned by previous experimental scales was positive (unfavorable), the average assigned by TopGraph in a nonredundant set is −6.9 kcal/mol. By quantifying contributions from both hydrophobicity and the positive-inside rule we further find that in about half of large membrane proteins polar segments are inserted into the membrane to position more positive charges in the cytoplasm, suggesting an interplay between these two energy contributions. Because membrane-embedded polar residues are crucial for substrate binding and conformational change, the results implicate the positive-inside rule in determining the architectures of membrane-protein functional sites. This insight may aid structure prediction, engineering, and design of membrane proteins. TopGraph is available online (topgraph.weizmann.ac.il).membrane insertion | topology prediction | positive-inside rule | Bellman-Ford search T he plasma membrane is a complex physical environment comprising a hydrophobic core and a polar exterior, which is more negatively charged on its cytoplasmic side (1). Two hallmarks of membrane proteins are hydrophobic segments that span the membrane core, and positive charges at the membrane-cytoplasm interface (the positive-inside rule; ref. 2); these features drive insertion and orient segments relative to the membrane plane, respectively. Furthermore, recent work has shown that positive charges placed close to engineered segments can drive membrane insertion even of marginally polar segments (3, 4), suggesting a role for the positive-inside rule in insertion, and emphasizing the importance of accurate models of membraneprotein energetics for protein engineering and for understanding the physical basis of membrane-protein topology.Topology prediction is a stringent test of our models of membraneprotein energetics. The most parsimonious membrane-topology predictor would locate the membrane-spanning segments and determine their orientations by a sequence search for minimum insertion energy. To achieve that, however, the insertion energy scale must at a minimum exhibit two properties: (i) to drive membrane in...

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supporting

confidence: 79%

Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Energy-based Prediction Of Protein Orientation With Respect mentioning

confidence: 99%

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Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology

Elazar

Weinstein

Prilusky

et al. 2016

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

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Modeling membrane geometries implicitly in Rosetta

Woods,

Leman,

Meiler

2024

Protein Science

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Interactions between membrane proteins (MPs) and lipid bilayers are critical for many cellular functions. In the Rosetta molecular modeling suite, the implicit membrane energy function is based on a “slab” model, which represent the membrane as a flat bilayer. However, in nature membranes often have a curvature that is important for function and/or stability. Even more prevalent, in structural biology research MPs are reconstituted in model membrane systems such as micelles, bicelles, nanodiscs, or liposomes. Thus, we have modified the existing membrane energy potentials within the RosettaMP framework to allow users to model MPs in different membrane geometries. We show that these modifications can be utilized in core applications within Rosetta such as structure refinement, protein–protein docking, and protein design. For MP structures found in curved membranes, refining these structures in curved, implicit membranes produces higher quality models with structures closer to experimentally determined structures. For MP systems embedded in multiple membranes, representing both membranes results in more favorable scores compared to only representing one of the membranes. Modeling MPs in geometries mimicking the membrane model system used in structure determination can improve model quality and model discrimination.

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One‐shot design elevates functional expression levels of a voltage‐gated potassium channel

Weinstein,

Saikia,

Karbat

et al. 2024

Protein Science

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Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web‐accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native‐state energy. As a stringent test, we apply mPROSS to the Kv1.2–Kv2.1 paddle chimera voltage‐gated potassium channel. Four designs, encoding 9–26 mutations relative to the parental channel, were functional and maintained potassium‐selective permeation and voltage dependence in Xenopus oocytes with up to 14‐fold increase in whole‐cell current densities. Additionally, single‐channel recordings reveal no significant change in the channel‐opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamic channels and receptors may be enhanced through one‐shot design calculations.

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Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane

Cited by 71 publications

References 67 publications

Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology

Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology

Modeling membrane geometries implicitly in Rosetta

One‐shot design elevates functional expression levels of a voltage‐gated potassium channel

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