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.

Nuclear magnetic resonance studies of the N-terminal fragment of adenosine diphosphate ribosylation factor 1 in micelles and bicelles: influence of N-myristoylation.

The N-terminal fragment of adenosine diphosphate (ADP) ribosylation factor 1 (ARF1) is proposed to be involved in the guanosine triphosphate- (GTP-) dependent, reversible association of the protein with membranes through the interaction of not only the N-linked myristoyl chain but also its highly conserved N-terminal hydrophobic residues. Based on the N-terminal sequence of this protein, specifically (13)C- and (15)N-labeled peptides were synthesized with and without an N-myristoyl anchor. The behavior, including structure, dynamics, and orientation, of these peptides in a lipid environment was then studied through a combination of solution (1)H nuclear magnetic resonance (NMR) techniques in micelles and heteronuclear solid-state NMR experiments in magnetically aligned bicelles. The work presented is an extension of the previously reported characterization of the myristoylated N-terminal fragment of ARF1 [Losonczi and Prestegard (1998) Biochemistry 37, 706-716] to include a comparison to a nonmyristoylated analogue. Results indicate that both myristoylated and nonmyristoylated peptides are alpha-helical in a lipid environment and that N-myristoylation does not greatly influence the structure of the peptides. Evidence is presented suggesting association of the peptides with bilayer disks through a combination of edge and surface interactions.

Sign determination of dipolar couplings in field-oriented bicelles by variable angle sample spinning (VASS).

Residual dipolar couplings are being increasingly used as structural constraints for NMR studies of biomolecules. A problem arises when dipolar coupling contributions are larger than scalar contributions for a given spin pair, as is commonly observed in solid state NMR studies, in that signs of dipolar couplings cannot easily be determined. Here the sign ambiguities of dipolar couplings in field-oriented bicelles are resolved by variable angle sample spinning (VASS) techniques. The director behavior of field-oriented bicelles (DMPC/DHPC, DMPC/CHAPSO) in VASS is studied by 31P NMR. A stable configuration occurs when the spinning angle is smaller than the magic angle, 54.7 degrees, and the director (or bicelle normal) of the disks is mainly distributed in a plane perpendicular to the rotation axis. Since the dipolar couplings depend on how the bicelles are oriented with respect to the magnetic field, it is shown that the dipolar interaction can be scaled to the same order as the J-coupling by moving the spinning axis from 0 degree toward 54.7 degrees. Thus the relative sign of dipolar and scalar couplings can be determined.

Domain orientation and dynamics in multidomain proteins from residual dipolar couplings.

The data most commonly available for the determination of macromolecular structures in solution are NOE based distance estimates and spin-spin coupling constant based dihedral angle estimates. This information is, unfortunately, inherently short-range in nature. Thus, for many multidomain proteins, little information is available to accurately position weakly interacting domains with respect to each other. Recent studies of proteins aligned in dilute liquid crystalline solvents have shown the utility of measuring anisotropic spin interactions, such as residual dipolar couplings, to obtain unique long-range structural information. In this work, the latter approach is taken to explore the relative domain orientation in a two-domain fragment from the protein barley lectin. An approach based on singular value decomposition as opposed to simulated annealing is used to directly determine order tensors for each domain from residual (15)N-(1)H dipolar couplings, and the limitations of the two approaches are discussed. Comparison of the order tensor principal axis frames as separately determined for each domain indicates that the two domains are not oriented as in the crystal structure of wheat germ agglutinin, a highly homologous protein ( approximately 95% sequence identical). Furthermore, differences in the order tensor values suggest that the two domains are not statically positioned but are experiencing different reorientational dynamics and, to a large degree, may be considered to reorient independently. Data are also presented that suggest that a specific association occurs between one domain and the lipid bicelles comprising the liquid crystal solvent.

Order matrix analysis of residual dipolar couplings using singular value decomposition.

The measurement of anisotropic spin interactions, such as residual dipolar couplings, in partially ordered solutions can provide valuable information on biomolecular structure. While the information can be used to refine local structure, it can make a unique contribution in determining the relative orientation of remote parts of molecules, which are locally well structured, but poorly connected based on NOE data. Analysis of dipolar couplings in terms of Saupe order matrices provides a concise description of both orientation and motional properties of locally structured fragments in these cases. This paper demonstrates that by using singular value decomposition as a method for calculating the order matrices, principal frames and order parameters can be determined efficiently, even when a very limited set of experimental data is available. Analysis of 1H-15N dipolar couplings, measured in a two-domain fragment of the barley lectin protein, is used to illustrate the computational method.

Improved dilute bicelle solutions for high-resolution NMR of biological macromolecules.

Dissolving biological macromolecules in dilute bicelle solutions, which form oriented liquid crystals in the presence of a magnetic field, permits measurement of anisotropic spin interactions such as dipolar couplings [Tjandra, N. and Bax, A., Science, 278, 1111-1114]. However, the lifetimes and temperature ranges of orientation for these samples are critically dependent on sample composition and experimental conditions. This paper demonstrates that doping dilute bicelle solutions with small amounts of charged amphiphiles substantially improves the stability and degree of alignment, as well as extends the temperature range of orientation for these systems. An explanation of the dependence of bicelle aggregation on sample composition is proposed based on the DLVO theory of colloids.

Nuclear magnetic resonance characterization of the myristoylated, N-terminal fragment of ADP-ribosylation factor 1 in a magnetically oriented membrane array.

The behavior of the N-terminal fragment of human ADP-ribosylation factor 1 (ARF1) in a membranelike environment is described. This is accomplished using heteronuclear liquid crystal NMR techniques in a magnetically oriented membrane array on a selectively 13C- and 15N-labeled peptide. After full assignment of the labeled sites, residual dipolar couplings (13C-13C, 15N-1H and, 13C-15N) and chemical shift anisotropy effects (amide 13C and 15N) were measured. The experimental data were interpreted using order matrix calculations to yield orientational and dynamic information for four separate, rigid amide planes. The experimental data obtained proves that the amphipathic peptide interacts with the bilayer in a mode that is consistent with an alpha-helix having its axis parallel to the membrane surface. Possibilities of extending the employed techniques to larger and uniformly labeled systems are discussed.