Glycine to alanine substitutions in helices of glyceraldehyde-3-phosphate dehydrogenase: effects on stability.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken was expressed in and purified from Escherichia coli. To investigate the physical basis of possible protein stabilization strategies, the effect of substitutions of glycine residues by alanine in helical regions was determined. One Gly to Ala substitution (G316A) located in the central core of the subunit was found to strongly stabilize the protein, while the other mutations are neutral or destabilize the protein. The effect seen for the stabilizing mutant in irreversible heat denaturation correlates with the first transition in folding equilibrium experiments that is observable by fluorescence, but not with the one detected by circular dichroism measurements or in dilution-induced dissociation experiments. The stabilizing effect of a Gly to Ala substitution therefore does not seem to be caused by an entropic effect on the unfolded state. Rather, an internal cavity is filled by the substitution G316A, probably stabilizing the native state. In large oligomeric proteins, imperfect packing may be a frequent cause of limited stability.
Ganter
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C
Genzentrum der Universität München, Max-Planck-Institut für Biochemie, Martinsried, FRG.
Plückthun
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A
D000409:Alanine / Q000737:chemistry; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000409:Alanine; D005998:Glycine