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The cystine knot promotes folding and not thermodynamic stability in vascular endothelial growth factor

Item Type:Article
Title:The cystine knot promotes folding and not thermodynamic stability in vascular endothelial growth factor
Creators Name:Muller, Y.A. and Heiring, C. and Misselwitz, R. and Welfle, K. and Welfle, H.
Abstract:Cystine knots consist of three intertwined disulfide bridges and are considered major determinants of protein stability in proteins in which they occur. We questioned this function and observed that removal of individual disulfide bridges in human vascular endothelial growth factor (VEGF) does not reduce its thermodynamic stability but reduces its unexpected high thermal stability of 108 °C by up to 40 °C. In wild-type VEGF ({delta}Gu,250 = 5.1 kcal·mol-1), the knot is responsible for a large entropic stabilization of T{delta}Su,250 = -39.3 kcal mol-1, which is compensated for by a {delta}Hu,250 of -34.2 kcal mol-1. In the disulfide-deficient mutants, this entropic stabilization disappears, but instead of a decrease, we observe an increase in the thermodynamic stability by about 2 kcal·mol-1. A detailed crystallographic analysis of the mutant structures suggests a role of the cystine knot motif in protein folding rather than in the stabilization of the folded state. When assuming that the sequential order of the disulfide bridge formation is conserved between VEGF and glycoprotein {alpha}-subunit, the crystal structure of the mutant C61A-C104A, which deviates by a root mean square deviation of more than 2.2 A from wild-type VEGF, identifies a true folding intermediate of VEGF.
Keywords:Amino Acid Sequence, Amino Acid Substitution, Calorimetry, Circular Dichroism, Cystine, Drug Stability, Endothelial Growth Factors, Guanidine, Intercellular Signaling Peptides and Proteins, Lymphokines, Mutagenesis, Site-Directed Mutagenesis, Protein Conformation, Protein Denaturation, Recombinant Proteins, Secondary Protein Structure, Sequence Deletion, Thermodynamics, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors, X-Ray Crystallography
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:277
Number:45
Page Range:43410-43416
Date:8 November 2002
Official Publication:https://doi.org/10.1074/jbc.M206438200
PubMed:View item in PubMed

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