Systematic profiling of temperature- and retinal-sensitive rhodopsin variants by deep mutational scanning

• DOI: 10.1016/j.jbc.2021.101359
• Publication Date: 2021-10-29T05:53:35Z
• Authors: Andrew G. McKee; Charles P. Kuntz; Joseph T. Ortega; Hope Woods; Victoria Most; Francis J. Roushar; Jens Meiler; Beata Jastrzebska; Jonathan P. Schle...
• Keywords: 0301 basic medicine; Rhodopsin; 0303 health sciences; 03 medical and health sciences; HEK293 Cells; Mutation; Retinaldehyde; Temperature; Humans; Research Article

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The of "temperature-sensitive" is also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples temperature-sensitive rhodopsin variants, misfolding which associated molecular basis retinitis pigmentosa. In this work, we employ deep mutational scanning compare effects temperature 9-cis-retinal, an investigational corrector, on plasma membrane 700 in HEK293T cells. We find change temperatures correlates response 9-cis-retinal among bearing mutations within a hydrophobic transmembrane domain (TM2). most appear disrupt helical kink domain. By comparison, mutants alter structure polar (TM7) weaker responses retinal poorly correlated. Statistical analyses suggest observed insensitivity cannot be attributed single variable, but likely arises from composite energetics integration, conformation, integrity retinal-binding pocket. Finally, show characteristics purified temperature- retinal-sensitive proteostatic may manifested during translation cotranslational folding. Together, our findings highlight several biophysical constraints influence sensitivity genetic small-molecule correctors.