Chemical modification of a variant of human MIP-1alpha; implications for dimer structure

• Published in: Protein science Vol. 9; no. 10; pp. 2047 - 2053
• Main Authors: Ashfield, J T, Meyers, T, Lowne, D, Varley, P G, Arnold, J R, Tan, P, Yang, J C, Czaplewski, L G, Dudgeon, T, Fisher, J
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01-10-2000
• Subjects: Alanine; Amino Acid Substitution; Aspartic Acid; Binding Sites; Chemokine CCL3; Chemokine CCL4; Chemokine CCL5 - chemistry; Dimerization; Genetic Variation; Humans; Macrophage Inflammatory Proteins - chemistry; Macrophage Inflammatory Proteins - genetics; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments - chemistry; Protein Conformation; Receptors, Chemokine - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin; Ultracentrifugation
• ISSN: 0961-8368; 1469-896X

A sequence variant of human MIP-1alpha, in which Asp26 has been replaced by Al alpha, has been chemically modified by the addition of 13C-labeled methyl groups at each of the lysine residues and the N-terminus. The sites of methylation have been verified by a combination of MALDI-TOF mass spectrometric experiments and tryptic digestion followed by N-terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra-centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP-alpha D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP-3, rather than the elongated or cylindrical structure determined for dimers of huMIP-1beta and RANTES.