Thermal destabilization of rhodopsin and opsin by proteolytic cleavage in bovine rod outer segment disk membranes.


Abstract

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein. Study holds ProTherm entries: 11676, 11677, 11678, 11679, 11680, 11681, 11682, 11683, 11684, 11685, 11686, 11687, 11688, 11689, 11690, 11691, 11692, 11693, 11694, 11695, 11696, 11697, 11698, 11699, 11700, 11701, 11702, 11703, 11704, 11705, 11706, 11707, 11708, 11709, 11710, 11711 Extra Details: DSC scan rate is 0.25 deg/min G-protein coupled receptor; transmembrane helices; lipid bilayer;,loop domains; noncovalent interactions

Submission Details

ID: surWq4G74

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:42 p.m.

Version: 1

Publication Details
Landin JS;Katragadda M;Albert AD,Biochemistry (2001) Thermal destabilization of rhodopsin and opsin by proteolytic cleavage in bovine rod outer segment disk membranes. PMID:11551216
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1LN6 2002-07-10 STRUCTURE OF BOVINE RHODOPSIN (Metarhodopsin II)
1VQX 2005-01-18 ARRESTIN-BOUND NMR STRUCTURES OF THE PHOSPHORYLATED CARBOXY-TERMINAL DOMAIN OF RHODOPSIN, REFINED
1NZS 2004-03-02 NMR structures of phosphorylated carboxy terminus of bovine rhodopsin in arrestin-bound state
1EDW 2000-08-09 SOLUTION STRUCTURE OF THIRD INTRADISKAL LOOP OF BOVINE RHODOPSIN (RESIDUES 268-293)
1EDS 2000-08-09 SOLUTION STRUCTURE OF INTRADISKAL LOOP 1 OF BOVINE RHODOPSIN (RHODOPSIN RESIDUES 92-123)
1EDX 2000-08-09 SOLUTION STRUCTURE OF AMINO TERMINUS OF BOVINE RHODOPSIN (RESIDUES 1-40)
1JFP 2001-10-05 Structure of bovine rhodopsin (dark adapted)
1EDV 2000-08-09 SOLUTION STRUCTURE OF 2ND INTRADISKAL LOOP OF BOVINE RHODOPSIN (RESIDUES 172-205)
1FDF 2000-07-27 HELIX 7 BOVINE RHODOPSIN
1RY1 2004-04-20 12.0 Structure of the signal recognition particle interacting with the elongation-arrested ribosome
1U19 2004-10-12 2.2 Crystal Structure of Bovine Rhodopsin at 2.2 Angstroms Resolution
4X1H 2015-11-04 2.29 Opsin/G(alpha) peptide complex stabilized by nonyl-glucoside
5DYS 2016-08-10 2.3 Crystal Structure of T94I rhodopsin mutant
6FK6 2018-04-04 2.36 Crystal structure of N2C/D282C stabilized opsin bound to RS01
6FKC 2018-04-04 2.46 Crystal structure of N2C/D282C stabilized opsin bound to RS15
6FKD 2018-04-04 2.49 Crystal structure of N2C/D282C stabilized opsin bound to RS16
1L9H 2002-05-15 2.6 Crystal structure of bovine rhodopsin at 2.6 angstroms RESOLUTION
3OAX 2011-01-19 2.6 Crystal structure of bovine rhodopsin with beta-ionone
2G87 2006-09-02 2.6 Crystallographic model of bathorhodopsin
6FK7 2018-04-04 2.62 Crystal structure of N2C/D282C stabilized opsin bound to RS06
6FK9 2018-04-04 2.63 Crystal structure of N2C/D282C stabilized opsin bound to RS09
4J4Q 2013-10-30 2.65 Crystal structure of active conformation of GPCR opsin stabilized by octylglucoside
3C9L 2008-08-05 2.65 Structure of ground-state bovine rhodospin in a hexagonal crystal form
1GZM 2003-11-20 2.65 Structure of Bovine Rhodopsin in a Trigonal Crystal Form
5TE3 2017-03-15 2.7 Crystal structure of Bos taurus opsin at 2.7 Angstrom
6FKA 2018-04-04 2.7 Crystal structure of N2C/D282C stabilized opsin bound to RS11
4PXF 2014-09-17 2.75 Crystal structure of the active G-protein-coupled receptor opsin in complex with the finger-loop peptide derived from the full-length arrestin-1
1HZX 2001-07-04 2.8 CRYSTAL STRUCTURE OF BOVINE RHODOPSIN
2HPY 2006-08-22 2.8 Crystallographic model of lumirhodopsin
1F88 2000-08-04 2.8 CRYSTAL STRUCTURE OF BOVINE RHODOPSIN
5EN0 2016-08-10 2.81 Crystal Structure of T94I rhodopsin mutant
3PQR 2011-03-09 2.85 Crystal structure of Metarhodopsin II in complex with a C-terminal peptide derived from the Galpha subunit of transducin
6FK8 2018-04-04 2.87 Crystal structure of N2C/D282C stabilized opsin bound to RS08
3CAP 2008-06-24 2.9 Crystal Structure of Native Opsin: the G Protein-Coupled Receptor Rhodopsin in its Ligand-free State
4BEY 2013-05-08 2.9 Night blindness causing G90D rhodopsin in complex with GaCT2 peptide
2PED 2007-10-30 2.95 Crystallographic model of 9-cis-rhodopsin
3PXO 2011-03-09 3.0 Crystal structure of Metarhodopsin II
2X72 2011-03-16 3.0 CRYSTAL STRUCTURE OF THE CONSTITUTIVELY ACTIVE E113Q,D2C,D282C RHODOPSIN MUTANT WITH BOUND GALPHACT PEPTIDE.
5W0P 2017-08-09 3.01 Crystal structure of rhodopsin bound to visual arrestin determined by X-ray free electron laser
6FKB 2018-04-04 3.03 Crystal structure of N2C/D282C stabilized opsin bound to RS13
6FUF 2018-10-03 3.12 Crystal structure of the rhodopsin-mini-Go complex
3DQB 2008-09-23 3.2 Crystal structure of the active G-protein-coupled receptor opsin in complex with a C-terminal peptide derived from the Galpha subunit of transducin
5WKT 2017-12-13 3.2 3.2-Angstrom In situ Mylar structure of bovine opsin at 100 K
4A4M 2012-01-25 3.3 Crystal structure of the light-activated constitutively active N2C, M257Y,D282C rhodopsin mutant in complex with a peptide resembling the C-terminus of the Galpha-protein subunit (GaCT)
4ZWJ 2015-07-29 3.3 Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser
4BEZ 2013-04-24 3.3 Night blindness causing G90D rhodopsin in the active conformation
3C9M 2008-08-05 3.4 Structure of a mutant bovine rhodopsin in hexagonal crystal form
2J4Y 2007-09-25 3.4 Crystal structure of a rhodopsin stabilizing mutant expressed in mammalian cells
2I35 2006-10-17 3.8 Crystal structure of rhombohedral crystal form of ground-state rhodopsin
5TE5 2017-03-15 4.01 Crystal structure of Bos taurus opsin regenerated with 6-carbon ring retinal chromophore
2I36 2006-10-17 4.1 Crystal structure of trigonal crystal form of ground-state rhodopsin
2I37 2006-10-17 4.15 Crystal structure of a photoactivated rhodopsin
6QNO 2019-07-10 4.38 Rhodopsin-Gi protein complex
6CMO 2018-06-20 4.5 Rhodopsin-Gi complex
5DGY 2016-03-23 7.7 Crystal structure of rhodopsin bound to visual arrestin
4UE5 2015-09-09 9.0 Structural basis for targeting and elongation arrest of Bacillus signal recognition particle
2J28 2006-11-08 9.5 MODEL OF E. COLI SRP BOUND TO 70S RNCS

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.8 Rhodopsin Q68J47 OPSD_LOXAF
90.5 Rhodopsin O62793 OPSD_MESBI
92.2 Rhodopsin P51489 OPSD_RAT
92.2 Rhodopsin P28681 OPSD_CRIGR
91.7 Rhodopsin O62792 OPSD_GLOME
92.2 Rhodopsin P15409 OPSD_MOUSE
91.7 Rhodopsin O62798 OPSD_TURTR
92.0 Rhodopsin O62791 OPSD_DELDE
92.2 Rhodopsin O62795 OPSD_PAGGO
91.7 Rhodopsin O62796 OPSD_TRIMA
92.0 Rhodopsin P49912 OPSD_RABIT
92.2 Rhodopsin Q6W3E1 OPSD_CALPD
92.5 Rhodopsin O62794 OPSD_PHOVI
92.5 Rhodopsin P08100 OPSD_HUMAN
92.8 Rhodopsin Q769E8 OPSD_OTOCR
93.4 Rhodopsin P32308 OPSD_CANLF
93.7 Rhodopsin O18766 OPSD_PIG
94.3 Rhodopsin Q95KU1 OPSD_FELCA
95.4 Rhodopsin P02700 OPSD_SHEEP
100.0 Rhodopsin P02699 OPSD_BOVIN