Quaternary structure of the neuronal protein NAP-22 in aqueous solution

• Published in: Biochimica et biophysica acta Vol. 1650; no. 1; pp. 50 - 58
• Main Authors: Epand, Richard M., Braswell, Emory H., Yip, Christopher M., Epand, Raquel F., Maekawa, Shohei
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 21.08.2003
• Subjects: Animals; Calmodulin-Binding Proteins - chemistry; Calmodulin-Binding Proteins - metabolism; Calmodulin-Binding Proteins - ultrastructure; Cholesterol - metabolism; Cytoskeletal Proteins; Liposomes; Membrane protein; Membrane raft; Microscopy, Atomic Force; NAP-22; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - metabolism; Nerve Tissue Proteins - ultrastructure; Phosphatidylcholines - metabolism; Protein myristoylation; Protein self-association; Protein Structure, Quaternary; Rats; Ultracentrifugation; Water - metabolism; Protein self-association; Membrane protein; Ultracentrifugation; NAP-22; Protein myristoylation; Membrane raft
• ISSN: 1570-9639; 0006-3002; 1878-1454; 1878-2434
• DOI: 10.1016/S1570-9639(03)00191-2

NAP-22, a myristoylated, anionic protein, is a major protein component of the detergent-insoluble fraction of neurons. After extraction from the membrane, it is readily soluble in water. NAP-22 will partition only into membranes with specific lipid compositions. The lipid specificity is not expected for a monomeric myristoylated protein. We have studied the self-association of NAP-22 in solution. Sedimentation velocity experiments indicated that the protein is largely associated. The low concentration limiting s value is ∼1.3 S, indicating a highly asymmetric monomer. In contrast, a nonmyristoylated form of the protein shows no evidence of oligomerization by velocity sedimentation and has an s value corresponding to the smallest component of NAP-22, but without the presence of higher oligomers. Sedimentation equilibrium runs indicate that there is a rapidly reversible equilibrium between monomeric and oligomeric forms of the protein followed by a slower, more irreversible association into larger aggregates. In situ atomic force microscopy of the protein deposited on mica from freshly prepared dilute solution revealed dimers on the mica surface. The values of the association constants obtained from the sedimentation equilibrium data suggest that the weight concentration of the monomer exceeds that of the dimer below a total protein concentration of 0.04 mg/ml. Since the concentration of NAP-22 in the neurons of the developing brain is ∼0.6 mg/ml, if the protein were in solution, it would be in oligomeric form and bind specifically to cholesterol-rich domains. We demonstrate, using fluorescence resonance energy transfer, that at low concentrations, NAP-22 labeled with Texas Red binds equally well to liposomes of phosphatidylcholine either with or without the addition of 40 mol% cholesterol. Thus, oligomerization of NAP-22 contributes to its lipid selectivity during membrane binding.