Capturing membrane protein ribosome nascent-chain complexes in a native-like environment for co-translational studies

TY - JOUR

T1 - Capturing membrane protein ribosome nascent-chain complexes in a native-like environment for co-translational studies

AU - Pellowe, Grant A

AU - Findlay, Heather E

AU - Lee, Karen

AU - Gemeinhardt, Tim M

AU - Blackholly, Laura R

AU - Reading, Eamonn

AU - Booth, Paula J

PY - 2020/8/4

Y1 - 2020/8/4

N2 - Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo-generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene-maleic acid co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milieu.

AB - Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo-generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene-maleic acid co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milieu.

UR - http://www.scopus.com/inward/record.url?scp=85089607432&partnerID=8YFLogxK

U2 - 10.1021/acs.biochem.0c00423

DO - 10.1021/acs.biochem.0c00423

M3 - Article

C2 - 32627541

SN - 0006-2960

VL - 59

SP - 2764

EP - 2775

JO - Biochemistry

JF - Biochemistry

IS - 30

ER -