Interpretation of Seemingly Contradictory Data: Low NMR S2 Order Parameters Observed in Helices and High NMR S2 Order Parameters in Disordered Loops of the Protein hGH at Low pH.

At low pH, human growth hormone (hGH) adopts a partially folded state, in which the native helices are maintained, but the long loop regions and side-chain packing become disordered. Some of the S2 order parameters for backbone N-H vectors derived from NMR relaxation measurements on hGH at low pH initially seem contradictory. Three isolated residues (15, 20, and 171) in helices A and D exhibit low order parameter values (<0.5) indicating flexibility, whereas residue 143 in the centre of a long flexible loop region has a high order parameter (0.82). Using S2 order parameter restraining MD simulations, this paradox has been resolved. Low S2 values in helices are due to the presence of a mixture of 310 -helical and α-helical hydrogen bonds. High S2 values in relatively disordered parts of a protein may be due to fluctuating networks of hydrogen bonds between the backbone and the side chains, which restrict the motion of N-H bond vectors.