Effects of C-terminal deletions on the conformational state and denaturation of phosphoglycerate kinase.
Phosphoglycerate kinase (PGK) contains two domains of approximately equal size, both of the alpha/beta type. An alpha-helix consisting of the middle section of the 415-amino acid polypeptide chain, and the N- and C-termini reside in the interdomain hinge region [Watson, H. C., et al. (1982) EMBO J. 1, 1635-1640]. The C-terminal end is an integral part of the N-terminal domain. The consequences of the deletion of fifteen and three C-terminal amino acids on the conformational state and on the guanidine hydrochloride-induced and thermal unfolding of PGK were investigated by using near- and far-UV CD, tryptophan fluorescence, 1-anilinonaphthalene-8-sulfonic acid binding, accessibility to chemical modification, and differential scanning calorimetry. The results of these studies indicate that the conformations of both domains and of the interdomain region were altered by these deletions. In the absence of the 15-amino acid C-terminal peptide [delta(401-415)], the N-terminal domain exhibits several characteristics of a molten globule state, whereas the C-terminal domain retains native-like, although distinctly different, tertiary structure. Deletion of three C-terminal amino acids [delta(413-415)] also globally affects PGK conformation, although to a much lesser extent. Both C-terminal deletions resulted in a significant decrease in protein stability, as demonstrated by their increased susceptibility to guanidine-induced and thermal denaturation. These results suggest that the formation of a native tertiary fold of PGK requires the presence of a complete polypeptide chain.
Mas
M T
MT
Physical Biochemistry Section, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Chen
H H
HH
Aisaka
K
K
Lin
L N
LN
Brandts
J F
JF
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006358:Hot Temperature; D008958:Models, Molecular; D016296:Mutagenesis; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013431:Sulfates / Q000494:pharmacology
D006146:Guanidines; D013431:Sulfates; C012036:sodium sulfate; D004228:Dithionitrobenzoic Acid; D010735:Phosphoglycerate Kinase; D019791:Guanidine