Monitoring gramicidin conformations in membranes: a fluorescence approach.
We have monitored the membrane-bound channel and nonchannel conformations of gramicidin utilizing red-edge excitation shift (REES), and related fluorescence parameters. In particular, we have used fluorescence lifetime, polarization, quenching, chemical modification, and membrane penetration depth analysis in addition to REES measurements to distinguish these two conformations. Our results show that REES of gramicidin tryptophans can be effectively used to distinguish conformations of membrane-bound gramicidin. The interfacially localized tryptophans in the channel conformation display REES of 7 nm whereas the tryptophans in the nonchannel conformation exhibit REES of 2 nm which highlights the difference in their average environments in terms of localization in the membrane. This is supported by tryptophan penetration depth measurements using the parallax method and fluorescence lifetime and polarization measurements. Further differences in the average tryptophan microenvironments in the two conformations are brought out by fluorescence quenching experiments using acrylamide and chemical modification of the tryptophans by N-bromosuccinimide. In summary, we report novel fluorescence-based approaches to monitor conformations of this important ion channel peptide. Our results offer vital information on the organization and dynamics of the functionally important tryptophan residues in gramicidin.
Centre for Cellular and Molecular Biology, Hyderabad, India.
Satinder S
SS
Rawat
Devaki A
DA
Kelkar
Amitabha
A
Chattopadhyay
D006096:Gramicidin / Q000737:chemistry*; D007473:Ion Channels / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008567:Membranes, Artificial; D010713:Phosphatidylcholines / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry*
D007473:Ion Channels; D008081:Liposomes; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D006096:Gramicidin; D014364:Tryptophan; C028694:1-palmitoyl-2-oleoylphosphatidylcholine