Figure 1. Key materials used in this protocol include Plasmid Bluescript-KS (VET1448) provided by Creative Biogene. (Bose HS, et al., 2025)
Determination of a protein molten globule state
Protocol for assessing mitochondrial cholesterol transport and protein molten globule state in steroidogenic and nonsteroidogenic systems
Paper
2025
STAR Protocols |
DOI
Background
This STAR Protocols article presents a detailed protocol for studying mitochondrial cholesterol import and protein molten-globule conformations in steroidogenic and nonsteroidogenic cells. It describes cell culture, Lipofectamine transfection with StAR and F2 constructs, radioimmunoassay-based quantitation of pregnenolone/progesterone, and assay of biosynthetic proteins using isolated mitochondria. The protocol also includes methods to detect the inactive dry molten-globule state of P450scc using urea, digitonin, or mitochondrial uncouplers, followed by SDS-PAGE/autoradiography, as well as 14C-cholesterol conversion and GC/MS steroid verification. Troubleshooting and quality-control tips ensure reproducible results.
Key Findings
The protocol establishes a quantitative method to measure mitochondrial cholesterol transport by determining pregnenolone/progesterone synthesis. Overexpression of StAR with the F2 vector (P450scc, ferredoxin reductase, ferredoxin) in nonsteroidogenic COS-1/HEK-293 cells produced 6- to 10-fold higher steroid output relative to controls. For import/folding studies, cell-free 35S-labeled P450scc is incubated with isolated mitochondria; urea, digitonin, or decouplers such as oligomycin and valinomycin trap processing at the 57-kDa intermediate, preventing formation of the active 51-kDa enzyme and thereby producing the inactive dry molten-globule state. Urea-containing media applied to living transfected cells similarly blocks final processing and eliminates steroidogenic activity. The protocol includes RIA standard-curve generation, western-blot expression validation, and use of 22R-hydroxycholesterol as positive control. Metabolic conversion with 14C-cholesterol followed by TLC and GC/MS confirms steroid identity. These steps allow reliable comparison of wild-type vs mutant proteins and assessment of folding states in live cells.
Contribution
Creative Biogene supplied the Plasmid Bluescript-KS (VET1448), which was used as the normalization vector in the transfection assay.
This STAR Protocols article presents a detailed protocol for studying mitochondrial cholesterol import and protein molten-globule conformations in steroidogenic and nonsteroidogenic cells. It describes cell culture, Lipofectamine transfection with StAR and F2 constructs, radioimmunoassay-based quantitation of pregnenolone/progesterone, and assay of biosynthetic proteins using isolated mitochondria. The protocol also includes methods to detect the inactive dry molten-globule state of P450scc using urea, digitonin, or mitochondrial uncouplers, followed by SDS-PAGE/autoradiography, as well as 14C-cholesterol conversion and GC/MS steroid verification. Troubleshooting and quality-control tips ensure reproducible results.
Key Findings
The protocol establishes a quantitative method to measure mitochondrial cholesterol transport by determining pregnenolone/progesterone synthesis. Overexpression of StAR with the F2 vector (P450scc, ferredoxin reductase, ferredoxin) in nonsteroidogenic COS-1/HEK-293 cells produced 6- to 10-fold higher steroid output relative to controls. For import/folding studies, cell-free 35S-labeled P450scc is incubated with isolated mitochondria; urea, digitonin, or decouplers such as oligomycin and valinomycin trap processing at the 57-kDa intermediate, preventing formation of the active 51-kDa enzyme and thereby producing the inactive dry molten-globule state. Urea-containing media applied to living transfected cells similarly blocks final processing and eliminates steroidogenic activity. The protocol includes RIA standard-curve generation, western-blot expression validation, and use of 22R-hydroxycholesterol as positive control. Metabolic conversion with 14C-cholesterol followed by TLC and GC/MS confirms steroid identity. These steps allow reliable comparison of wild-type vs mutant proteins and assessment of folding states in live cells.
Contribution
Creative Biogene supplied the Plasmid Bluescript-KS (VET1448), which was used as the normalization vector in the transfection assay.