Interpretation of Seemingly Contradictory Data: Low NMR S 2 Order Parameters Observed in Helices and High NMR S 2 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 S 2 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 S 2 order parameter restraining MD simulations, this paradox has been resolved. Low S 2 values in helices are due to the presence of a mixture of 3 10 ‐helical and α‐helical hydrogen bonds. High S 2 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.