NMR studies of the anti-apoptotic protein Bcl-xL in micelles.

Losonczi JA, Olejniczak ET, Betz SF, Harlan JE, Mack J, Fesik SW
Biochemistry. 2000 39 (36): 11024-33

PMID: 10998239 · DOI:10.1021/bi000919v

The Bcl-2 family of proteins play a pivotal role in the regulation of programmed cell death. One of the postulated mechanisms for the function of these proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporated into small detergent micelles. This form of Bcl-x(L) lacks the loop (residues 49-88) between helix 1 and helix 2 and the putative C-terminal transmembrane helix (residues 214-237). Below the critical micelle concentration (CMC), Bcl-x(L) binds detergents in the hydrophobic groove that binds to pro-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary structure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous solution, the structure of the protein when dissolved in dodecylphosphocholine (DPC) micelles consists of several alpha-helices separated by loops. However, the length and position of the individual helices of Bcl-x(L) in micelles differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and limited proteolysis. In addition, the mobility of the micelle-bound form of Bcl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and location of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dynamic structure in micelles, with helices 1 and 6 and possibly helix 5 partially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.

MeSH Terms (23)

Amino Acid Sequence Apoptosis bcl-X Protein Binding Sites Circular Dichroism Detergents Endopeptidases Humans Hydrolysis Micelles Molecular Sequence Data Nuclear Magnetic Resonance, Biomolecular Phospholipid Ethers Phosphorylcholine Protein Conformation Protein Structure, Secondary Protein Structure, Tertiary Proto-Oncogene Proteins c-bcl-2 Sodium Dodecyl Sulfate Solutions Structure-Activity Relationship Ultracentrifugation Water

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