NMR in Micelles

Solution NMR methods rely on rapid molecular reorientation for line narrowing, and can be successfully applied to membrane proteins in micelles (Henry and Sykes 1994; Williams et al. 1996; Almeida and Opella 1997; Gesell et al. 1997; MacKenzie et al. 1997; Arora et al. 2001; Fernandez et al. 2001; Hwang et al. 2002; Ma et al. 2002; Mascioni et al. 2002; Oxenoid et al. 2002; Sorgen et al. 2002; Crowell et al. 2003; Krueger-Koplin et al. 2004; Howell et al. 2005). The size limitation is substantially more severe than for globular proteins, because the many lipid molecules associated with each polypeptide slow its overall reorientation rate. Micelles afford rapid and effectively isotropic reorientation of the protein, and their amphipathic nature simulates that of membranes, offering a realistic alternative to organic solvents for studying membrane proteins. Moreover, for the proteins examined by both solution and solid-state NMR, similar structural features have been found in micelle and bilayer samples (Lee et al. 2003; Mesleh et al. 2003).

The first step in solution NMR studies of proteins is the preparation of folded, homogeneous, and well-behaved samples, and several lipids are available for membrane protein solubilization (Krueger-Koplin et al. 2004). For membrane-bound proteins, small micelles containing approximately 60 lipids and one protein provide a generally effective model membrane environment, without the damaging effects of organic solvents. The primary goal in micelle preparation is to reduce the effective rotational correlation time of the protein so that resonances will have the narrowest possible linewidths. Careful handling of the protein throughout the purification is essential, since subtle changes in the protocol can have a significant impact on the quality of the resulting spectra. It is essential to optimize the protein concentration, lipid nature and concentration, counter ions, pH and temperature, in order to obtain well-resolved NMR spectra, with narrow *H and 1SN resonance linewidths.

Although high-quality solution NMR spectra can be obtained even for some large helical membrane proteins in micelles (Krueger-Koplin et al. 2004; Oxenoid et al. 2004; Howell et al. 2005), there are only very few cases where it has been possible to measure and assign sufficiently long-range NOEs for structure determination. This limitation can be overcome by preparing weakly aligned micelle samples for the measurement of RDCs (Bax et al. 2001; Prestegard and Kishore 2001) from the backbone amide sites, and the analysis of these orientation restraints in terms of dipolar waves (Mesleh et al. 2002; Lee et al. 2003; Mesleh et al. 2003; Mesleh and Opella 2003). Stressed polyacrylamide gels provide an ideal orientable medium for membrane proteins in micelles, because they do not suffer from the drawbacks of bicelles, which bind tightly to membrane proteins, or phage particles, which are destroyed by micelles (Sass et al. 2000; Tycko et al. 2000; Chou et al. 2001; Meier et al. 2002; Howell et al. 2005). Another useful approach to compensate for insufficient NOEs involves the combination of site-directed spin labeling and NMR (Battiste and Wagner 2000), where distances derived from paramagnetic broadening of NMR resonances are used to determine global fold. In addition, spin label probes and metal ions can be incorporated within the micelles in order to probe protein insertion (Papavoine et al. 1994; Van Den Hooven et al. 1996; Jarvet et al. 1997; Damberg et al. 2001; Sorgen et al. 2002; Kutateladze et al. 2004).

Determining the Structures of Proteins in Micelles

The measurements of as many homonuclear 1H/1H NOEs as possible among the assigned resonances provide the short-range and long-range distance restraints required for structure determination (Clore and Gronenborn 1989, Wuthrich 1989, Ferentz and Wagner 2000). These are supplemented by other structural restraints, such as spin-spin coupling constants, chemical shift correlations, deuterium exchange data, and RDCs in order to assign resonances and to characterize the secondary structure of the protein. The HSQC (heteronuclear single quantum coherence) spectra of samples in D20 solutions identify the most stable helical residues, and can provide useful information on the topology of membrane proteins in micelles (Czerski et al. 2000). In addition, hydrogen-deuterium fractionation experiments extend the range of exchange rates that can be monitored to identify more subtle structural features (Veglia et al. 2002).

The two-dimensional HSQC spectra also serve as the basis for the measurement of the *H and 1SN relaxation parameters of protein backbone amide sites, which are useful for describing protein dynamics. The heteronuclear 1H-1SN NOEs of the backbone amide sites provide remarkably direct and sensitive information on local protein dynamics (Gust et al. 1975; Boguski et al. 1987; Bogusky et al. 1988). They can be measured with and without *H irradiation to saturate the *H magnetization (Farrow et al. 1994).

RDCs are extremely useful both for structure refinement, and for the de novo determination of protein folds (Tolman et al. 1995; Clore and Gronenborn 1998; Delaglio et al. 2000; Fowler et al. 2000; Hus et al. 2000; Mueller et al. 2000). During refinement, these measurements supplement an already large number of chemical shifts, approximate distance measurements, and dihedral angle restraints. Among the principal advantages of anisotropic spectral parameters in solution NMR spectroscopy is that they can report on the global orientations of separate domains of a protein and of individual bonds relative to a reference frame, which reflects the preferred alignment of the molecule in the magnetic field. This does not preclude their utility in characterizing the local backbone structure of a protein molecule.

The RDCs and RCSAs measured in solution NMR experiments provide direct angular restraints with respect to a molecule-fixed reference frame (Bax et al. 2001; Prestegard and Kishore 2001; Lee et al. 2003). They are analogous to the non-averaged dipolar couplings and chemical shift anisotropics measured in solid-state NMR experiments (Marassi and Opella 2000; Wang et al. 2000; Marassi 2001). These orientation restraints are the principal mechanism for overcoming the limitations resulting from having few reliable long-range NOEs available as distance restraints, often encountered with samples of membrane-bound proteins in micelles.

Dipolar waves are very effective at identifying the helical residues in membrane-bound proteins and the relative orientations of the helical segments, and also serve as indices of the helix regularity in proteins (Mesleh et al. 2002). The magnitudes of the RDCs are plotted as a function of residue number and fitted to a sine wave with a period of 3.6 residues (Mesleh et al. 2002; Mesleh et al. 2003; Mesleh and Opella 2003). The quality of fit is monitored by a scoring function in a four-residue sliding window and the phase of the fit. Dipolar waves from solution NMR data give relative orientations of helices in a common molecular frame. On the other hand, Dipolar waves from solid-state NMR data give absolute measurements of helix orientations because the polypeptides are immobile and the samples have a known alignment in the magnetic field.

tBid in Micelles

The cleavage of Bid by caspase-8 results in a C-terminal product, tBid, which targets mitochondria and induces apoptosis with strikingly enhanced activity. To characterize the conformation of tBid in lipid environments, we obtained its CD (circular dichroism) and solution NMR 1H/1SN HSQC spectra in the absence or in the presence of lipid micelles (Fig. 2.3) (Gong et al. 2004). The HSQC spectra of proteins are the starting point for additional multidimensional NMR experiments that lead to structure determination. In these spectra, each 15N-labeled protein site gives rise to a single peak, characterized by *H and 1SN chemical shift frequencies that reflect the local environment. In addition, the peak linewidths and line-shapes, and their dispersion in the *H and 1SN frequency dimensions, are sensitive indicators of protein conformational stability and aggregation state.

In the absence of lipids, the CD spectrum of tBid displays minima at 202 nm and222 nm, characteristic of predominantly helical proteins (Fig. 2.3a, solid line). However, while tBid retains its helical conformation even when it is separated from the 60-residue N-terminal segment, many of the resonances in its HSQC spectrum cannot be detected (Fig. 2.3b), suggesting that the protein aggregates in solution, adopts multiple conformations, or undergoes dynamic conformational exchange on the NMR time-scales. This is consistent with the dramatic changes in the physical properties of the protein that result from caspase-8 cleavage.

When tBid is dissolved in lipid micelles its HSQC spectrum changes dramatically, and single, well-defined 1H/1SN resonances are observed for each 15N-la-beled NH site, indicating that it adopts a unique conformation in this environment (Figs. 2.3c, d). Several lipids are available for protein solubilization, and we tested both SDS and LPPG for their ability to yield high-quality HSQC spectra of tBid for structure determination. Both gave excellent spectra where most of the 130 amide resonances of tBid could be resolved; for example the resonances from the five Gly amide sites are resolved in SDS (Fig. 2.3c), and four out of five are resolved in LPPG (Fig. 2.3d). Both SDS and LPPG are negatively charged but they differ in the lengths of their hydrocarbon chains (C12 for SDS; C16 for LPPG), and their polar headgroups (sulfate for SDS; phosphatidylglycerol for LPPG), thus the differences in the *H and 1SN chemical shifts between the two HSQC spectra most

Fig.2.3. tBid adopt well-defined helical folds in lipid micelles. The CD spectra in (a) were obtained at 25 °C for tBid in aqueous solution (solid line), SDS micelles (broken line), or LPPG micelles (dottedline). The 1H/15N HSQC NMR spectra in (b,c,d) were obtained at 40°Cfor uniformly 15N-labeled tBid in (a) aqueous solution, (b) SDS micelles, or (c) LPPG micelles. Aqueous samples were in 20mM sodium phosphate, pH 5; SDS micelle samples were in 20mM sodium phosphate, pH 7, 500 mM SDS; and LPPG micelle samples were in 20 mM sodium phosphate, pH 7,100 mM LPPG

Fig.2.3. tBid adopt well-defined helical folds in lipid micelles. The CD spectra in (a) were obtained at 25 °C for tBid in aqueous solution (solid line), SDS micelles (broken line), or LPPG micelles (dottedline). The 1H/15N HSQC NMR spectra in (b,c,d) were obtained at 40°Cfor uniformly 15N-labeled tBid in (a) aqueous solution, (b) SDS micelles, or (c) LPPG micelles. Aqueous samples were in 20mM sodium phosphate, pH 5; SDS micelle samples were in 20mM sodium phosphate, pH 7, 500 mM SDS; and LPPG micelle samples were in 20 mM sodium phosphate, pH 7,100 mM LPPG

likely reflect the different lipid environments. The spectrum in LPPG has excep-tionallywell-dispersed resonances with homogeneous intensities and linewidths. LPPG was recently identified as a superior lipid for NMR studies of several membrane proteins (Krueger-Koplin et al. 2004), and is particularly interesting for this study because it is a close analog of cardiolipin and monolysocardiolipin, the major components of mitochondrial membranes that bind tBid. The limited chemical shift dispersion in the two spectra is typical of helical proteins in micelles, and this is confirmed by the corresponding CD spectra, which are dominated by minima at 202 nm and 222 nm, and thus show that tBid retains a predominantly helical fold in both SDS and LPPG (Fig. 2.3a, broken and dashed lines).

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