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


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 UniProtKB Entries

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