| Chapter title |
Determining the Topology of Membrane-Bound Proteins Using PEGylation
|
|---|---|
| Chapter number | 15 |
| Book title |
Cholesterol Homeostasis
|
| Published in |
Methods in molecular biology, February 2017
|
| DOI | 10.1007/978-1-4939-6875-6_15 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6873-2, 978-1-4939-6875-6
|
| Authors |
Vicky Howe B.Sc. (Hons), Andrew J. Brown Ph.D., Howe, Vicky, Brown, Andrew J., Vicky Howe, Andrew J. Brown |
| Editors |
Ingrid C. Gelissen, Andrew J. Brown |
| Abstract |
Biochemical methods can help elucidate the membrane topology of hydrophobic membrane proteins where X-ray crystallography is difficult or impractical, providing important structural data. Here, we describe the method of PEGylation, which uses a cysteine-reactive molecule, maleimide polyethylene glycol (mPEG), to determine the cytosolic accessibility of introduced cysteine residues. This accessibility is visualized using Western blotting to detect a band shift that indicates cysteine labeling by mPEG. Using scanning cysteine mutagenesis, followed by PEGylation, one can map the accessibility of the introduced cysteines, hence inferring the membrane topology of the protein.We used PEGylation to determine the membrane topology of the sterol regulatory domain of a cholesterol synthesis enzyme, squalene monooxygenase, identifying that it is anchored to the membrane via a re-entrant loop. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 5 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 2 | 40% |
| Student > Bachelor | 1 | 20% |
| Unknown | 2 | 40% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 2 | 40% |
| Chemistry | 1 | 20% |
| Unknown | 2 | 40% |