Method and apparatus for measuring tightness of vessels

Abstract

The segment, YSDELRQRLAARLEALKENG, corresponding to residues 166 to 185 of human serum apolipoprotein A-I, was studied by circular dichroism and NMR spectroscopy in sodium dodecyl sulfate and dodecylphosphocholine micelles. 2-Dimensional NOESY, TOCSY and DQF-COSY spectra of apoA-I(166–185) in perdeuterated sodium dodecyl sulfate (SDS-d25) and dodecylphosphocholine (DPC-d38) micelles were collected at a peptide/SDS (DPC) ratio of 1:40. Similar CD spectra and NOE connectivity patterns were observed for apoA-I(166–185) in SDS and DPC, indicating a similar helical conformation in both. Conformations of apoA-I(166–185) in DPC-d38 micelles, and in SDS-d25 micelles at two pH values, 6.6 and 3.7, were determined using distance geometry calculations. Backbone superposition (N,Cα,CO) for an ensemble of twenty-nine structures in DPC at pH 6.0 gave a RMSD of 0.45 ± 0.09 Å for the region D168 to K182, while for all atoms it was 1.60 ± 0.17 Å. In SDS, the ensemble of nineteen structures each at pH 6.6 and 3.7 gave RMSDs of 0.28 ± 0.07 Åand 0.35 ± 0.10 Å, respectively, for the region D168 to K182. RMSD for superposition of all atoms was 1.36 ± 0.10 Åand 1.38 ± 0.21 Å at the respective pH values. In all cases a highly defined class A amphipathic helical structure was found for the region R171 to K182. Since the same structure occurs in micelles with either negatively charged or zwitterionic head groups it strongly suggests a dominant role for hydrophobic interactions in stabilizing the complex. The Y166 aromatic ring is bent back upon the helix axis at the lower pH. NMR determination of pKa values for D168, E169, E179 and E183 in the presence of SDS or DPC indicated a micro-pH at the micellar surface approximately one pH unit higher than the normal residue pKa. SDS interactions with the peptide were examined by collecting 1H NOESY spectra in the presence of protiated SDS. Residues R171, R173, R177, as well as the aromatic ring of Y166, were shown by intermolecular NOE measurements to interact with SDS, hence a key interaction in stabilizing the complex appears to be between interfacial basic side-chains and SDS alkyl chains.