Abstract
Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of “temperature-sensitive” variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples of temperature-sensitive rhodopsin variants, the misfolding of which is associated with the molecular basis of retinitis pigmentosa. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and 9-cis-retinal, an investigational corrector, on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures correlates with the response to 9-cis-retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants appear to disrupt a native helical kink within this transmembrane domain. By comparison, mutants that alter the structure of a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest that this observed insensitivity cannot be attributed to a single variable, but likely arises from the composite effects of mutations on the energetics of membrane integration, the stability of the native conformation, and the integrity of the retinal-binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings highlight several biophysical constraints that appear to influence the sensitivity of genetic variants to temperature and small-molecule correctors.
Highlights
Eukaryotic membrane proteins are prone to misfolding, and mutations that enhance their propensity to misfold are associated with a myriad of diseases of aberrant protein homeostasis.[1]
Temperature-sensitive expression is an emergent property of class II mutations that serves as a marker for correctable disease variants within integral membrane proteins
We find that G90M and basis of these phenotypes are needed to
Summary
Eukaryotic membrane proteins are prone to misfolding, and mutations that enhance their propensity to misfold are associated with a myriad of diseases of aberrant protein homeostasis.[1]. We find fewer temperature-sensitve mutations within TM7, and show that temperature-sensitivity does not coincide with retinal-sensitivity within this region Statistical trends within these data suggest this disconnect arises from the differential effects of these mutations on the stability of the native conformation, the fidelity of cotranslational folding, and/ or the integrity of the retinal binding pocket. We first hypothesized that certain mutations in TM7 render rhodopsin expression insensitive to changes in temperature and/ or retinal by enhancing the stability of the native conformation and increasing the fraction of folded apoprotein To test this hypothesis, we Across all TM2 variants, the change in analyzed mutant sensitivity profiles in relation to expression in the presence of 9-cis-retinal is their predicted impacts on conformational statistically correlated with the change in stability. This observed instability of the regenerated pigments suggests the temperature-sensitivity of these variants is unlikely to arise from an enhanced conformational stability and/ or binding affinity in these full-length variants
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