Kinetic stability of designed glycosylation mutants of Coprinus cinereus peroxidase.
The effect of glycans and surface mutations on protein unfolding induced by heat or urea has been studied. Removal of the only native high mannose type glycan in the N142P, N142T, and N142D CIP mutants reduced the lifetime to half of that of wtCIP at irreversible conditions of unfolding. The effect was moderate at reversible conditions. Five glycomutants designed to have 0, 1, 2, 4 and 6N glycans showed a correlation between increased carbohydrate mass and increased stability toward irreversible unfolding. The results are in agreement with a dampening effect of glycans on backbone fluctuation in both the native and the unfolded states. However, experiments in reversible conditions were less clear because of additional effects of an increasing number of amino acid substitutions and aggregation. Examples of strong effects from minor surface changes were also observed.
Tams
J W
JW
Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark. jwtams@biobase.dk
Welinder
K G
KG
D001665:Binding Sites; D003302:Coprinus / Q000201:enzymology*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006418:Heme / Q000032:analysis; D007700:Kinetics; D009154:Mutation; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011134:Polysaccharides / Q000502:physiology; D017510:Protein Folding; D013696:Temperature
D005656:Fungal Proteins; D006023:Glycoproteins; D011134:Polysaccharides; D006418:Heme; D010544:Peroxidases