RT Journal Article
SR Electronic
T1 Systematic Profiling of Temperature- and Retinal-Sensitive Rhodopsin Variants by Deep Mutational Scanning
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.03.26.437229
DO 10.1101/2021.03.26.437229
A1 Andrew G. McKee
A1 Charles P. Kuntz
A1 Joseph T. Ortega
A1 Hope Woods
A1 Francis J. Roushar
A1 Jens Meiler
A1 Beata Jastrzebska
A1 Jonathan P. Schlebach
YR 2021
UL http://biorxiv.org/content/early/2021/03/26/2021.03.26.437229.abstract
AB 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. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and an investigational corrector (9-cis-retinal) on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures is correlated with the response to retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants within this helix appear to disrupt a network of hydrogen bonds that stabilizes a native helical kink. By comparison, mutants that alter a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest this insensitivity primarily arises from an abundance of mutations that enhance its membrane integration, stabilize its native conformation, and/ or perturb 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 elucidate various factors that mediate the sensitivity of genetic variants to temperature and to small molecule correctors.Competing Interest StatementThe authors have declared no competing interest.