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Protein Sci
2024 Jun 01;336:e4995. doi: 10.1002/pro.4995.
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One-shot design elevates functional expression levels of a voltage-gated potassium channel.
Weinstein JJ
,
Saikia C
,
Karbat I
,
Goldenzweig A
,
Reuveny E
,
Fleishman SJ
.
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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.
Larry Marks Center for Brain Disorders, Wilner Family Fund, donation in memory of Sam Switzer, Dr. Barry Sherman Institute for Medicinal Chemistry, 815379 European Research Council through a Consolidator Award, 1844 Israel Science Foundation, 349/22 Israel Science Foundation
FIGURE 1. Steps in applying mPROSS to the Kv channel. (a) Scheme of the mPROSS algorithm. mPROSS starts with a phylogenetic analysis of homologs of the target protein and inserts the protein into a virtual membrane. The structure is minimized and subjected to combinatorial design calculations, generating designs with different mutational loads for experimental analysis. (b) Overview of the paddle chimera structure, including the β‐2 soluble subunits responsible for attenuating channel dynamics (PDB entry: 2R9R). The selectivity filter is indicated in orange, and the voltage sensor is in red. The left‐hand side shows the tetrameric arrangement of the channel looking from the extracellular domain toward the fourfold symmetry axis of the channel. Pink spheres indicate K+ ions. The right‐hand side shows a side view of one protomer, and a single β‐2 subunit is shown as a molecular surface.
FIGURE 2. mPROSS introduces mutations that increase lipophilicity and the “positive‐inside” rule. For each of the 20 proteins in the benchmark, we analyzed a single mPROSS design that incorporates approximately 15% mutations, the maximal fraction of mutations we recommend for testing. (a) The number of hydrophobic‐to‐hydrophobic mutations within the membrane domain. (b) Normalized distributions of mutations across the membrane. The horizontal axis represents the distance to the membrane midplane, with negative and positive values representing the intra‐ and extracellular domains, respectively. Numbers denote the number of mutations in each distribution. (c) Fraction of mutations in the membrane or soluble domain in each protein. The number of mutations is noted above the bars, and Protein Data Bank (PDB) entries are noted below the bars. (d) Energies of refined and designed proteins. Line marks x = y. R.e.u./AA denotes the total Rosetta energy divided by protein length.
FIGURE 3. Designed mutations improve protein lipophilicity and native‐state molecular interactions. mPROSS designs both the membrane and cytosolic domains. Mutations increase the lipophilicity of membrane‐exposed positions, add positively charged residues in the inner leaflet (following the “positive‐inside” rule (von Heijne, 1989)), rigidify loops, and improve interhelix packing. The protein mainchain is shown in gray, and mutations are shown in thick yellow sticks or spheres. Only one protomer is shown in cartoon representation, and the other three are represented as colored molecular surfaces. The β‐2 subunits are shown in lighter colors (top).
FIGURE 4. Current–voltage relationship of the Kv1.2–Kv2.1 paddle chimera and its designs. (a) Currents of the paddle chimera (PC) and designs are plotted against varying test voltages. Each curve represents recordings from n ≥ 8 oocytes. Error bars represent standard deviations. (Inset) Ratios of currents observed for D1 and D2 at voltages in the range 5–25 mV. (b) Average currents (paddle chimera: 0.91 ± 0.19 μA; D1: 4.65 ± 1.52 μA; D2: 12.50 ± 1.63 μA). (c) Western blot analysis of total membrane expression. The blots were labeled with anti‐FLAG (top) or anti‐GIRK2 (as control; bottom) antibodies. (d) Normalized blot intensities. The experiment was performed in triplicate. Error bars represent the standard deviation. (e) Normalized tail currents were recorded at −50 mV as a function of the test pulse voltage (see inset). Measured tail current amplitudes were fitted with a single‐component Boltzmann equation (solid lines), from which V
1/2 of activation was inferred. (f) Inferred V
1/2 values (paddle chimera: 16.10 ± 5.39 mV; D1: −8.33 ± 3.49 mV; and D2: −16.61 ± 2.75 mV).
FIGURE 5. Design D2 behaves similarly to the parental channel at the single‐channel level. (a) Representative current traces were elicited at +40 mV from a holding potential of −80 mV under cell‐attached configuration in Xenopus oocytes for the parental channel chimera and D2. The upward deflection indicates the open state. (b) Individual single‐channel amplitudes at different potentials from all recorded patches. (c) Channel conductance derived from the current to voltage slopes shown in (b).
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