TY - JOUR
T1 - One-shot design elevates functional expression levels of a voltage-gated potassium channel
AU - Weinstein, Jonathan Jacob
AU - Saikia, Chandamita
AU - Karbat, Izhar
AU - Goldenzweig, Adi
AU - Reuveny, Eitan
AU - Fleishman, Sarel Jacob
N1 - Publisher Copyright: © 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
PY - 2024/6
Y1 - 2024/6
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85193205171&partnerID=8YFLogxK
U2 - https://doi.org/10.1002/pro.4995
DO - https://doi.org/10.1002/pro.4995
M3 - مقالة
C2 - 38747377
SN - 0961-8368
VL - 33
JO - Protein Science
JF - Protein Science
IS - 6
M1 - e4995
ER -