| First Author | Krieger CC | Year | 2011 |
| Journal | Proc Natl Acad Sci U S A | Volume | 108 |
| Issue | 20 | Pages | 8269-74 |
| PubMed ID | 21527722 | Mgi Jnum | J:172788 |
| Mgi Id | MGI:5009054 | Doi | 10.1073/pnas.1018887108 |
| Citation | Krieger CC, et al. (2011) Cysteine shotgun-mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells. Proc Natl Acad Sci U S A 108(20):8269-74 |
| abstractText | Questions of if and when protein structures change within cells pervade biology and include questions of how the cytoskeleton sustains stresses on cells-particularly in mutant versus normal cells. Cysteine shotgun labeling with fluorophores is analyzed here with mass spectrometry of the spectrin-actin membrane skeleton in sheared red blood cell ghosts from normal and diseased mice. Sheared samples are compared to static samples at 37 degrees C in terms of cell membrane intensity in fluorescence microscopy, separated protein fluorescence, and tryptic peptide modification in liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spectrin labeling proves to be the most sensitive to shear, whereas binding partners ankyrin and actin exhibit shear thresholds in labeling and both the ankyrin-binding membrane protein band 3 and the spectrin-actin stabilizer 4.1R show minimal differential labeling. Cells from 4.1R-null mice differ significantly from normal in the shear-dependent labeling of spectrin, ankyrin, and band 3: Decreased labeling of spectrin reveals less stress on the mutant network as spectrin dissociates from actin. Mapping the stress-dependent labeling kinetics of alpha- and beta-spectrin by LC-MS/MS identifies Cys in these antiparallel chains that are either force-enhanced or force-independent in labeling, with structural analyses indicating the force-enhanced sites are sequestered either in spectrin's triple-helical domains or in interactions with actin or ankyrin. Shear-sensitive sites identified comprehensively here in both spectrin and ankyrin appear consistent with stress relief through forced unfolding followed by cytoskeletal disruption. |
