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Initially our approach was to complete a detailed examination of bradykinin using two-dimensional
NMR methods in combination with empirical energy calculations [19]. Our strategy was derived on the
basis of spectral data, biological results from conformationally restricted analogs, as well as the
relationship between ordering in bradykinin and the dielectric environment of the solvent. Our guiding
hypothesis was that, although in aqueous solution bradykinin is conformationally random, the
biologically active form of the peptide is likely ordered and stabilized within the lipid bilayer of the cell
membrane prior to binding with its receptor. Alternatively, the receptor binding environment might also
be hydrophobic and thereby lead to similar conformational biases in the ligand. We presumed that an
appropriate solvent environment should be able to stimulate, at least in terms of hydrophobicity and
dielectric constant, the nature of a cell membrane, and a 90:10 d 8-dioxane-H 2O mixture was selected for
NMR experiments. It was anticipated that under these nonsolvating conditions the conformational
diversity of bradykinin might be severely restricted. The ultimate analysis of the two-dimensional NMR
data collected at 500 MHz supported a single major conformational species. There were five HN-CαH
connectivities, one for each amide. This was confirmed in the 13C NMR spectrum where only nine
carbonyl resonances, one for each amino acid, were present.
In order to provide a starting point for subsequent molecular dynamics simulations the assumption was
made, based on multiple observed long-range amide-to-amide nuclear overhauser effects (NOEs), that it
was indeed a single major conformational species. Although bradykinin contains three proline residues,
the absence of any strong CαH i-CαH j+, cross peaks in the nuclear overhauser enhancement
spectroscopy (NOESY) spectrum was taken as proof that all peptide bonds were trans. In total, 35
interproton distances were extracted from the NOESY spectrum and, whenever possible, stereospecific
assignments for pro-R and pro-S hydrogens were made explicitly. A temperature-dependent study of the
chemical shifts of the amide protons resulted in a near-linear dependence suggesting no major
conformational changes were coinciding with the temperature change and thereby allowing a
comparison of slopes (Δδ/Δt). The lowest values obtained for these slopes corresponded to Phe 8 and
Arg 9 suggesting solvent sequestering for these amides.
Given the high Chou and Fasman probability of β-turns in the sequences Pro 2-Pro 3-Gly 4-Phe 5 and Ser 6-
Pro 7-Phe 8-Arg 9 (3.79 × 10 -4 and 1.99 × 10 -4, respectively), the computational strategy employed was to
begin from two initial structures: (a) an extended β strand, and (b) a structure containing these two
predicted β turns. Utilizing custom routines written using the program CHARMm, version 21 [21], the
interproton distances were incorporated into the potential-energy expression in the form of an additional
potential-energy term. During the 3-ps heating step of the molecular dynamics, the temperature was
raised from 0K to 300K in steps of 20K every 0.2 ps. Since the target distances
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