Ricin-membrane interaction: membrane penetration depth by fluorescence quenching and resonance energy transfer.
The entry of the plant toxin ricin and its A- and B-subunits in model membranes in the presence as well as absence of monosialoganglioside (GM1) has been studied. Dioleoylphosphatidylcholine and 5-, 10-, and 12-doxyl- or 9,10-dibromophosphatidylcholines serve as quenchers of intrinsic tryptophan fluorescence of the proteins. The parallax method of Chattopadhyay and London [(1987) Biochemistry 26, 39-45] has been employed to measure the average membrane penetration depth of tryptophans of ricin and its B-chain and the actual depth of the sole Trp 211 in the A-chain. The results indicate that both of the chains as well as intact ricin penetrate the membrane deeply and the C-terminal end of the A-chain is well inside the bilayer, especially at pH 4.5. An extrinsic probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS) has been attached to Cys 259 of the A-chain, and the kinetics of penetration has been followed by monitoring the increase in AEDANS fluorescence at 480 nm. The insertion follows first-order kinetics, and the rate constant is higher at a lower pH. The energy transfer distance analysis between Trp 211 and AEDANS points out that the conformation of the A-chain changes as it inserts into the membrane. CD studies indicate that the helicity of the proteins increases after penetration, which implies that some of the unordered structure in the native protein is converted to the ordered form during this process. Hydrophobic forces seem to be responsible for stabilizing a particular protein conformation inside the membrane.
Ramalingam
T S
TS
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore.
Das
P K
PK
Podder
S K
SK
D002942:Circular Dichroism; D003497:Cyclic N-Oxides / Q000737:chemistry / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004735:Energy Transfer; D005456:Fluorescent Dyes; D006531:HEPES / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008954:Models, Biological; D009282:Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012276:Ricin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013439:Sulfhydryl Reagents; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D009282:Naphthalenesulfonates; D010713:Phosphatidylcholines; D013113:Spin Labels; D013439:Sulfhydryl Reagents; C006422:1,8-I-AEDANS; D014364:Tryptophan; D012276:Ricin; C017251:1,2-oleoylphosphatidylcholine; D006531:HEPES; D004229:Dithiothreitol; C003959:TEMPO