Is the Structure of the N-Domain of Phosphoglycerate Kinase Affected by Isolation from the Intact Molecule?
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- 作者：Laszlo L. P. Hosszu ; C. Jeremy Craven ; James Spencer ; Martin J. Parker ; Anthony R. Clarke ; Mark Kelly ; and Jonathan P. Waltho
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：January 14, 1997
- 年：1997
- 卷：36
- 期：2
- 页码：333 - 340
- 全文大小：269K
- 年卷期：v.36,no.2(January 14, 1997)
- ISSN：1520-4995
文摘
The structural integrity of the isolated N-domain (residues1-174) of Bacillus stearothermophilus 3-phosphoglycerate kinase (PGK) has been investigated usingheteronuclear NMR spectroscopy.Analysis of 13C chemical shifts, amide protection, andcomparison of observed and expected sequentialNOE intensities calculated from the crystal structure of the domain inthe intact protein indicate that thesecondary structure of the isolated domain is unchanged from that foundin the intact molecule. Markedlyshifted 1H resonances, amide protection, and long-rangeNOEs indicate that the tertiary structure is similarlyunaffected. These results are confirmed by an excellent agreement(standard deviation 0.28 ppm) betweenobserved H
chemical shifts and those calculated from thehigh-resolution (1.6 Å) crystal structure ofintact PGK [Davies et al. (1994) ActaCrystallogr. D50, 202-209]. The only regionperturbed by lossof interactions with the C-domain is a small portion of thesubstrate-binding site (residues 148-152)whose amide protons are poorly protected from solvent. Theseresults provide a structural basis for theanalysis of the folding of the domains of PGK as isolated units andwithin the intact molecule [Parker etal. (1996) Biochemistry (in press)] and contrast withthe notion that the native tertiary fold of the N-domainof PGK requires the whole polypeptide chain, including the entireC-domain [Mas et al. (1995)Biochemistry34, 7931-7940]. Assignments of backbone13C, 15N, HN, and Hresonances are provided.
chemical shifts and those calculated from thehigh-resolution (1.6 Å) crystal structure ofintact PGK [Davies et al. (1994) ActaCrystallogr. D50, 202-209]. The only regionperturbed by lossof interactions with the C-domain is a small portion of thesubstrate-binding site (residues 148-152)whose amide protons are poorly protected from solvent. Theseresults provide a structural basis for theanalysis of the folding of the domains of PGK as isolated units andwithin the intact molecule [Parker etal. (1996) Biochemistry (in press)] and contrast withthe notion that the native tertiary fold of the N-domainof PGK requires the whole polypeptide chain, including the entireC-domain [Mas et al. (1995)Biochemistry34, 7931-7940]. Assignments of backbone13C, 15N, HN, and Hresonances are provided.