shifts in the B1 domain of protein G

We report chemical shifts for H N , N, and C 0 nuclei in the His-tagged B1 domain of pro- tein G (GB1) over a range of pH values from pH 2.0 to 9.0, which fit well to stand- ard pH-dependent equations. We also report a 1.2 Aresolution crystal structure of GB1 at pH 3.0. Comparison of this crys- tal structure with published crystal struc- tures at higher pHs provides details of the structural changes in GB1 associated with protonation of the carboxylate groups, in particular a conformational change in the C-terminus of the protein at low pH. An additional change described recently is not seen in the crystal structure because of crystal contacts. We show that the pH-dependent changes in chemical shifts can be almost entirely understood based on structural changes, thereby providing insight into the relationship between struc- ture and chemical shift. In particular, we describe through-bond effects extending up to five bonds, affecting N and C 0 but not H N ; through-space effects of carboxylates, which fit well to a simple electric field model; and effects due to conformational change, which have a similar magnitude to many of the direct effects. Finally, we dis- cuss cooperative effects, demonstrating a lack of cooperative unfolding in the helix, and the existence of a b-sheet ''iceberg'' extending over three of the four strands. This study therefore extends the application of chemical shifts to understanding protein structure.