Is the structure of the N-domain of phosphoglycerate kinase affected by isolation from the intact molecule?

The structural integrity of the isolated N-domain (residues 1-174) of Bacillus stearothermophilus 3-phosphoglycerate kinase (PGK) has been investigated using heteronuclear NMR spectroscopy. Analysis of 13C chemical shifts, amide protection, and comparison of observed and expected sequential NOE intensities calculated from the crystal structure of the domain in the intact protein indicate that the secondary structure of the isolated domain is unchanged from that found in the intact molecule. Markedly shifted 1H resonances, amide protection, and long-range NOEs indicate that the tertiary structure is similarly unaffected. These results are confirmed by an excellent agreement (standard deviation 0.28 ppm) between observed H alpha chemical shifts and those calculated from the high-resolution (1.6 A) crystal structure of intact PGK [Davies et al. (1994) Acta Crystallogr. D50, 202-209]. The only region perturbed by loss of interactions with the C-domain is a small portion of the substrate-binding site (residues 148-152) whose amide protons are poorly protected from solvent. These results provide a structural basis for the analysis of the folding of the domains of PGK as isolated units and within the intact molecule [Parker et al. (1996) Biochemistry (in press)] and contrast with the notion that the native tertiary fold of the N-domain of PGK requires the whole polypeptide chain, including the entire C-domain [Mas et al. (1995) Biochemistry 34, 7931-7940]. Assignments of backbone 13C, 15N, HN, and H alpha resonances are provided.