NMR studies of the anti-apoptotic protein Bcl-xL in micelles.
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.
Losonczi
J A
JA
Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, IL 60064, USA.
Olejniczak
E T
ET
Betz
S F
SF
Harlan
J E
JE
Mack
J
J
Fesik
S W
SW
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology; D001665:Binding Sites; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008823:Micelles; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010742:Phospholipid Ethers / Q000737:chemistry; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000502:physiology; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012996:Solutions; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation; D014867:Water / Q000737:chemistry; D051020:bcl-X Protein
C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; C494811:BCL2L1 protein, human; D003902:Detergents; D008823:Micelles; D010742:Phospholipid Ethers; D019253:Proto-Oncogene Proteins c-bcl-2; D012996:Solutions; D051020:bcl-X Protein; D014867:Water; D010767:Phosphorylcholine; D012967:Sodium Dodecyl Sulfate; C028810:dodecylphosphocholine; D010450:Endopeptidases