©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alignment of the Apolipophorin-III -Helices in Complex with Dimyristoylphosphatidylcholine
A UNIQUE SPATIAL ORIENTATION (*)

Vincent Raussens (1)(§), Vasanthy Narayanaswami (2), Erik Goormaghtigh (1)(¶), Robert O. Ryan (2)(**), Jean-Marie Ruysschaert (1)

From the (1) Laboratoire de Chimie Physique des Macromolécules aux Interfaces, CP206/2, Université Libre de Bruxelles, Campus Plaine, B-1050 Brussels, Belgium and the (2) Lipid and Lipoprotein Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Apolipophorin-III (apoLp-III) from Manduca sexta can exist in two alternate states: as a globular, lipid-free helix bundle or a lipid surface-associated apolipoprotein. Previous papers (Ryan R. O., Oikawa K., and Kay C. M.(1993) J. Biol. Chem. 268, 1525-1530; Wientzek M., Kay C. M., Oikawa K., and Ryan R. O.(1994) J. Biol. Chem. 269, 4605-4612) have investigated the structures and properties of apolipophorin-III from M. sexta in the lipid-free state and associated to lipids. Association of apoLp-III with dimyristoylphosphatidylcholine vesicles leads to the formation of uniform lipid discs with an average diameter and thickness of 18.5 ± 2.0 and 4.8 ± 0.8 nm, respectively. These discs contain six molecules of apoLp-III. Geometrical calculations based on these data, together with x-ray crystallographic data from the homologous L. migratoria apoLp-III (Breiter D. R., Kanost M. R., Benning M. M., Wesenberg G., Law J. H., Wells M. A., Rayment I., and Holden H. M.(1991) Biochemistry 30, 603-608), have allowed the presentation of a model of lipid-protein interaction, in which the -helices of the apoLp-III orient perpendicular to the phospholipid chains and surround the lipid disc. Here, using polarized Fourier transform-attenuated total reflection infrared spectroscopy, we provide the first experimental evidence of a unique perpendicular orientation of the -helices with respect to the fatty acyl chains of the phospholipids in the disc.


INTRODUCTION

Apolipophorin-III (apoLp-III)() is an abundant hemolymph protein in the sphynx moth, Manduca sexta, that normally exists in a lipid-free state (Shapiro and Law, 1983). ApoLp-III is a single chain polypeptide of 166 amino acids (M = 18,300), which exists as a monomer in solution and is rich in -helix structure (Cole et al., 1987; Kawooya et al., 1986). The three-dimensional structure of lipid-free apoLp-III from Locusta migratoria has been elucidated by x-ray crystallography (Holden et al., 1988; Breiter et al., 1991). L. migratoria apoLp-III contains five long amphipathic -helices in a bundle connected by short loops. There is an approximate 50% homology between M. sexta and L. migratoria apoLp-III amino acid sequences, and these apoLp-IIIs have been found to be functionally indistinguishable (Van der Horst et al., 1988). The two apoLp-IIIs likely possess similar amphipathic helix folding motives (Wientzek et al., 1994). When presented with a lipid surface, it was suggested that apoLp-III undergoes a conformational change that exposes the hydrophobic faces of the helices, thereby permitting interaction with lipid surfaces. Association of apoLp-III with DMPC vesicles results in the formation of uniform discs with an average diameter and width of 18.5 ± 2 nm and 4.8 ± 0.8 nm, respectively. Calculations based on the experimental characterization of these complexes together with the x-ray crystallographic data from L. migratoria apoLp-III allowed the presentation of a model of lipid-protein interaction (Wientzek et al., 1994). In this model, apolipoprotein helices are oriented perpendicular to the lipid acyl chains and surround the phospholipid disc.

To provide experimental evidence of the validity of this model, Fourier transform infrared-attenuated total reflection (ATR) spectroscopy was used here to gain information about the secondary structure and orientation of apoLp-III with regard to the lipid bilayer. This technique has been applied successfully to deal with membrane structural problems in our laboratory (Goormaghtigh et al., 1990, 1991a, 1991b, 1994a, 1994b; Challou et al., 1994; Sonveaux et al., 1994, Vandenbussche et al., 1992). The results on the secondary structure of the protein in the apoLp-IIIDMPC complexes are in good agreement with the previous CD measurements (Ryan et al., 1993). About the orientation, we provide the first experimental evidence of a unique orientation of the apoLp-III helical domains perpendicular to the lipid acyl chains. This topology is quite opposite to that described for the apoA1DMPC (Brasseur et al., 1990) and apoA1dipalmitoylphosphatidylcholine complexes (Hefele Wald et al., 1990), where the amphipathic helices were oriented parallel to the acyl chains.


EXPERIMENTAL PROCEDURES

Materials

Dimyristoylphosphatidylcholine was purchased from Sigma. Phospholipid (choline) enzymatic colorimetric analysis kit was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Deuterium oxide was from Janssen Chimica (Geel, Belgium).

Purification of Apolipophorin III

apoLp-III was isolated and purified from hemolymph of adult M. sexta with some modifications of the method by Wells et al.(1985). The purity of the apoLp-III was evaluated by SDS-polyacrylamide gel electrophoresis, and the pure apoLp-III was stored lyophilized at -20 °C.

Preparation of DMPC Vesicles

DMPC vesicles were prepared by modification of the method of Swaney(1980). DMPC was dissolved in chloroform:methanol mixture (3:1), evaporated to dryness as a thin film under an inert atmosphere, and dried further for 2 h, followed by the addition of 10 mM Tris-HCl, pH 7.5. The mixture was warmed to 50 °C, vortexed, and then sonicated in a bath sonicator for about 20 min.

Preparation and Isolation of DMPCapoLp-III Complexes

The preparation and isolation of DMPCapoLp-III complexes were performed as described by Swaney(1980) and Rifici et al.(1985) with some modifications. ApoLp-III was dissolved in 10 mM Tris-HCl, pH 7.5, and added to the DMPC vesicles at a lipid:protein ratio of 2.5:1 (w/w) (molar ratio of about 70:1). After a brief mixing in a vortexer, the mixture was incubated at 24 °C for 18 h. The density of the sample was adjusted to 1.21 g/ml with KBr in a final volume of 2.5 ml and placed in a 5-ml Quick-Seal tube, layered with 0.9% saline, and centrifuged at 65,000 rpm for 3 h at 4 °C. At the end of the centrifugation, fractions of 0.5 ml were removed from the top of the tube and analyzed for protein and phospholipid content. The DMPCapoLp-III complexes were localized by the coenrichment of protein and phospholipid, pooled, and dialyzed extensively against 10 mM Tris-HCl, pH 7.5. The protein content of the final DMPCapoLp-III complexes was evaluated by amino acid analysis (Beckman System 6300 Amino Acid Analyzer, System Gold Version 6/01) and the phospholipid content by colorimetric kit for choline. The complex had a lipid:protein weight ratio of 7.1:1 (molar ratio of 190:1). Control DMPC alone and apoLp-IIIDMPC complexes were adsorbed to carbon-coated grids, stained with 2% sodium phosphotungstate, and viewed under a Philips FM 420 electron microscope operated at 100 kV (Wientzek et al., 1994). In addition to apoLp-III complexes, DMPC vesicles without added apoLp-III were employed in parallel control experiments.

IR Spectroscopy

Spectra were recorded on a Perkin-Elmer infrared spectrophotometer 1720X equipped with a Perkin-Elmer microspecular reflectance accessory and a polarizer mount assembly with a gold wire grid element. The internal reflection element was a germanium ATR plate (50 20 2 mm, Harrick EJ2121) with an aperture angle of 45° yielding 25 internal reflections. 128 accumulations were performed to improve the signal/noise ratio. The spectrophotometer was continuously purged with air dried on a silicagel column (5 130 cm) at a flow rate of 7 liters/min. Spectra were recorded at a nominal resolution of 2 cm. At the end of the scan, they were transferred from the memory of the spectrophotometer to a computer for subsequent treatments. All the measurements were made at 20 °C.

Samples Preparation

Oriented multilayers were formed by slow evaporation of 100 µl of the sample on one side of the ATR plate. The ATR plate was then sealed in a universal sample holder (Perkin-Elmer 186-0354) and hydrated by flushing DO-saturated N (room temperature) for 3 h.

Secondary Structure Determination

Fourier self-deconvolution was applied to increase the resolution of the spectra in the amide I` region, which is the most sensitive to the secondary structure of proteins. The self-deconvolution was carried out using a Lorentzian line shape for the deconvolution and a Gaussian line shape for the apodization. To quantify the area of the different components of amide I` revealed by the self-deconvolution, a least square iterative curve fitting was performed to fit Lorentzian line shapes to the spectrum between 1700 and 1600 cm. Prior to curve fitting, a straight base line passing through the ordinates at 1700 and 1600 cm was subtracted. The spectrum arising from the lipid part of the system was found to be completely flat between 1700 and 1600 cm and was therefore not subtracted. To avoid introducing artifacts due to the self-deconvolution procedure, the fitting was performed on the non-deconvolved spectrum. The proportion of a particular structure is computed to be the sum of the area of all the fitted Lorentzian bands having their maximum in the frequency region where that structure occurs divided by the area of all the Lorentzian bands having their maximum between 1689 and 1615 cm (Goormaghtigh et al., 1990).

Orientation of the Secondary Structures

For the amount of material used throughout this work, film thickness remains small when compared to the IR wavelength. This allows the ``thin film'' approximation to be used for the establishment of the equations describing the dichroic ratio as a function of the orientational order parameter (Goormaghtigh and Ruysschaert, 1990). The potential energy distribution of amide I consists in about 80% (C=O), 10% (C-N), and 10% (N-H). In an -helix, the main transition dipole moment lies approximately parallel to the helical axis, while in an anti-parallel -sheet, the polarization is predominantly perpendicular to the fiber axis. It is therefore possible to determine the mean orientation of the -helix and -sheet structures from the orientation of the peptide bond C=O group (Goormaghtigh and Ruysschaert, 1990). When this information was desired, additional spectra were recorded with parallel (0°) and perpendicular (90°) polarized incident light with respect to a normal to the ATR plate. Polarization was expressed as the dichroic ratio R = A/A. The mean angle between the helix axes and a normal to the ATR plate surface was then calculated from R. In these calculations, a 27° angle between the long axis of the -helix and the C=O dipole moment was considered (Goormaghtigh and Ruysschaert, 1990; Goormaghtigh et al., 1994a). The (CH) transition at 1200 cm, whose dipole lies parallel to the all-trans hydrocarbon chains, was used to characterize the lipid acyl chain orientation (Fringeli and Günthard, 1981).

CD Spectroscopy

Spectra were recorded between 260 and 190 nm with a resolution of 0.5 nm on a Jasco J-710 spectropolarimeter controlled by Jasco software. The spectropolarimeter was continuously purged with N. The cell used was a cylindral quartz cell with a pathlength of 1 mm. The spectra were the result of four accumulations at a scan speed of 50 nm/min with a response time of 0.125 s. The concentration of the samples was 0.15 mg of protein/ml.


RESULTS AND DISCUSSION

Fig. 1 , A and B, and 2, A and B, show the IR spectra of a pure DMPC preparation and of the apoLp-IIIDMPC complex (190:1 lipid:protein molar ratio), respectively. The amide I region of the spectrum (1700-1600 cm) (Fig. 2, A and B) reveals a symmetric peak with a maximum at 1653 cm characteristic of a mainly helical structure in good agreement with the CD (Ryan et al., 1993) and x-ray (Breiter et al., 1991) data. Since the CD spectra of the apoLp-IIIDMPC complexes provided evidence that apoLp-III becomes essentially completely helical after interaction with DMPC (Ryan et al., 1993), we assigned the 1653 cm band to the -helix structure. After exposure of the sample to DO-saturated N for 10 h, the amide I` region shifted from 1653 to 1646 cm (Fig. 3, A and B). Compilation of literature data on the behavior of the -helix amide I component (Goormaghtigh et al., 1994a) reveals that a 1646-cm frequency is well within the range of frequencies characteristic of deuterated helices. To make sure that the shift of the amide I maximum from 1653 to 1646 cm we observed after deuteration was not due to a change in the protein secondary structure upon incubation in the presence of DO, we exchanged the aqueous buffer of a similar sample by either DO or HO by dialysis and recorded CD spectra. It turned out that this CD spectrum of the protein in DO was exactly superimposable to the CD spectrum of the sample dialyzed against HO (data not shown). We can therefore assign unambiguously the shift of the amide I to the deuteration of the -helices of the apoLp-III.


Figure 1: IR spectra of a DMPC film. SpectrumA was obtained with 90° polarized light and spectrumB with 0° polarized light. TraceC represents the difference between spectrumA and spectrumB. A positive deviation in spectrumC from a horizontal base line indicates a higher absorbance at 90° than at 0° and reflects a preferential perpendicular orientation of the dipole transition, relative to the germanium surface. A negative deviation in spectrumC indicates that the dipole is preferentially oriented parallel to the germanium surface. The optical density amplitudes of spectraA-C are 0.255, 0.211, and 0.128, respectively. The sample has been deuterated as described under ``Experimental Procedures.''




Figure 2: IR spectra of the apoLp-IIIDMPC complex. SpectrumA was obtained with 90° polarized light and spectrumB with 0° polarized light. TraceC represents the difference between spectrumA and spectrumB. The optical density amplitudes of spectraA-C are 0.239, 0.141, and 0.30, respectively.




Figure 3: IR spectra of the deuterated apoLp-IIIDMPC complex. SpectrumA was obtained with 90° polarized light and spectrumB with 0° polarized light. TraceC represents the difference between spectrumA and spectrumB. The optical density amplitudes of spectraA-C are 0.255, 0.152, and 0.30, respectively. The sample has been deuterated as described under ``Experimental Procedures.''



Components of the amide I` revealed by Fourier self-deconvolution were quantified by a least square iterative curve fitting as described (Goormaghtigh et al., 1990). The calculated percentage of helical secondary structure (70-80%) (data not shown) is in good agreement with the Provencher-Glöckner analysis of the CD spectra, revealing a completely -helical structure for the apoLp-III upon complex formation (Wientzek et al., 1994).

The amide I region of the spectra (1700-1600 cm) was used to determine the orientation of the -helices in apoLp-III relative to the DMPC hydrocarbon chains of the bilayers in the apoLp-IIIDMPC complex. A dichroic spectrum, obtained by subtracting the spectrum recorded with 0° polarized light from the spectrum recorded with 90° polarized light, has a positive deviation when the dipole moment was oriented perpendicular to the germanium crystal plane and a negative deviation when the dipole moment was oriented parallel to the germanium plane (Goormaghtigh and Ruysschaert, 1990). Here, for both the undeuterated (Fig. 2C) and deuterated (Fig. 3C) samples, the amide I band is strongly 0° polarized, as shown by a negative deviation located in the helix region of the amide I band, indicating an orientation of the -helices parallel to the germanium plate, i.e. parallel to the lipid bilayer and perpendicular to the acyl chains. This orientation of the helices is confirmed by the positive deviation in the amide II band at 1549 cm corresponding to the -helix amide II band (Goormaghtigh et al., 1994a). Indeed, the amide I and amide II polarizations are perpendicular (Krimm and Bandekar, 1986; Tsuboi, 1962). A more quantitative estimation of the helix orientation can be obtained after iterative least square curve fitting applied to the deconvolved polarized spectra, which allows the computation of the dichroic ratio for the -helix components in the apoLp-IIIDMPC complex. While the isotropic dichroic ratio (i.e. the dichroic ratio measured for a dipole with an isotrope distribution) was 1.51, it was 1.21 for the amide I component, indicating that the helix axis makes a maximum tilt with the germanium surface of 20°. This value is a maximum estimate since taking into account additional sources of disorder, e.g. a mosaic spread (Rothschild and Clark, 1979), would further reduce this tilt.

The orientation of the lipid acyl chains was also assessed. In DMPC, the hydrocarbon chain in the -position in the gel state is all-trans from the ester group to the methyl group. This conformation allows a resonance to occur between the ester group and the CH groups of the chain, giving rise to the so-called (CH) progression between 1200 and 1350 cm (peaks at 1201, 1230, 1257, 1280, 1304, and 1328 cm) (Fig. 1-3). The peak at 1200 cm was chosen to characterize the lipid acyl chain orientation (Fringeli and Günthard, 1981). The strong 90° polarization of this absorption peak (Fig. 1-3) indicates that the all-trans hydrocarbon chains of DMPC are oriented nearly normal to the germanium surface. The measured dichroic ratio is 3.4-4.0. Accordingly, the maximum tilt between acyl chain and a normal to the germanium surface is equal to 20°. Additional source of disorder would reduce this tilt. These data indicate that the lipid hydrocarbon chains and the -helices of the protein are oriented almost perpendicular to each other in the apoLp-IIIDMPC complex.

These results experimentally confirm the previously proposed model of discoidal DMPC particles containing -helical segments of apoLp-III organized perpendicular to the phospholipid acyl chains on the periphery of the discoidal structure. It is to the best of our knowledge the first experimental evidence of a protein interacting with the lipid core and orienting normal with respect to the acyl chains.

A significant shift of the amide I peak from 1653 to 1646 cm was associated to the deuteration of the apoLp-III helical domains. On the opposite, such a shift has not been described after deuteration of the apoA1dipalmitoylphosphatidylcholine discoidal complex (Hefele Wald et al., 1990) and is not observed for membrane and soluble -helix-rich proteins tested so far in our laboratory. This raises the question of the helix structure (, angles, hydrogen bonding). However, the frequency of the undeuterated form of the protein is in good agreement with a large set of data collected in the literature as reported by Goormaghtigh et al. (1994a). This demonstrates that the geometry and hydrogen bonding in the helices is close to this of a standard -helix since the amide I frequency is very sensitive to these parameters. CD data confirm on the other hand the large amount of helix structure. We suggest that the peculiar distribution of the hydrophilic and hydrophobic amino acids on the two sides of the apoLp-III helices and the interaction with the lipid bilayer is responsible for the amplitude of the shift. The high extent of apoLp-III helices deuteration could be related to its interaction with the lipid bilayer. The apoLp-III helices are large and normal with respect to the acyl chains. The length of apoLp-III helices and the contours of the lipid discs could give to the helices a curved shape, which would increase the accessibility of their amide hydrogen, involved in hydrogen bonds, to the solvent and favor the hydrogen/deuterium exchange. Hydrogen/deuterium exchange has been shown to be much more rapid in amphipathic helices distorted to a curved structure than in non-distorted helices (Zhou et al., 1992).


FOOTNOTES

*
This work was performed with the financial support of the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
An Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture fellow.

A research associate of the National Fund for Scientific Research (Belgium).

**
A Medical Research Council of Canada Scientist and Senior Scholar of the Alberta Heritage Foundation for Medical Research.

The abbreviations used are: apoLp-III, apolipophorin III; ATR, attenuated total reflection; DMPC, dimyristoylphosphatidylcholine; IR, infrared.


ACKNOWLEDGEMENTS

We thank R. Bradley and D. G. Scabra for assistance with electron microscopy and H. de Jongh for assistance with CD spectrometry.


REFERENCES
  1. Brasseur, R., De Meutter, J., Vanloo, B., Goormaghtigh, E., Ruysschaert, J.-M., and Rosseneu, M.(1990) Biochim. Biophys. Acta 1043, 245-252 [Medline] [Order article via Infotrieve]
  2. Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M.(1991) Biochemistry 30, 603-608 [Medline] [Order article via Infotrieve]
  3. Challou, N., Goormaghtigh, E., Cabiaux, V., Conrath, K., and Ruysschaert, J.-M.(1994) Biochemistry 33, 6902-6910 [Medline] [Order article via Infotrieve]
  4. Cole, K. D., Fernando-Warnakulasuriya, G. J. P., Boguski, M. S., Freeman, M., Gordon, J. I., Clark, W. A., Law, J. H., and Wells, M. A. (1987) J. Biol. Chem. 262, 11794-11800 [Abstract/Free Full Text]
  5. Fringeli, U. P., and Günthard, H. H.(1981) in Membrane Spectroscopy (Grell, E., ed) pp 270-332, Springer-Verlag, Berlin
  6. Goormaghtigh, E., and Ruysschaert, J.-M.(1990) in Molecular Description of Biological Membrane Components by Computer Aided Conformational Analysis (Brasseur, R., ed) pp. 285-329, CRC Press, Inc., Boca Raton, FL
  7. Goormaghtigh, E., Cabiaux, V., and Ruysschaert, J.-M.(1990) Eur. J. Biochem. 193, 409-420 [Abstract]
  8. Goormaghtigh, E., De Meutter, J., Szoka, F., Cabiaux, V., Parente, R. A., and Ruysschaert, J.-M. (1991a) Eur. J. Biochem. 195, 421-429 [Abstract]
  9. Goormaghtigh, E., Vigneron, L., Knibiehler, M., Lazdunski, C., and Ruysschaert, J.-M. (1991b) Eur. J. Biochem. 202, 1299-1305 [Abstract]
  10. Goormaghtigh, E., Cabiaux, V., and Ruysschaert, J.-M. (1994a) Subcell. Biochem. 23, 329-450 [Medline] [Order article via Infotrieve]
  11. Goormaghtigh, E., Vigneron, L., Scarborough, G. A., and Ruysschaert, J.-M. (1994b) J. Biol. Chem. 269, 27409-27413 [Abstract/Free Full Text]
  12. Holden, H. M., Kanost, M. R., Law, J. H., Rayment, I., and Wells, M. A. (1988) J. Biol. Chem. 263, 3960-3962 [Abstract/Free Full Text]
  13. Kawooya, J. K., Meredith, S. C., Wells, M. A., Kézdy, F. J., and Law, J. H.(1986) J. Biol. Chem. 261, 13588-13591 [Abstract/Free Full Text]
  14. Krimm, S., and Bandekar, J.(1986) Adv. Protein Chem. 38, 181-364 [Medline] [Order article via Infotrieve]
  15. Rifici, V. A., Eder, H. A., and Swaney, J. B.(1985) Biochim. Biophys. Acta 834, 205-214 [Medline] [Order article via Infotrieve]
  16. Rothschild, K. J., and Clark, N. A.(1979) Biophys. J. 5, 473-488
  17. Ryan, R. O., Oikawa, K., and Kay, C. M.(1993) J. Biol. Chem. 268, 1525-1530 [Abstract/Free Full Text]
  18. Shapiro, J. P., and Law, J. H.(1983) Biochem. Biophys. Res. Commun. 115, 924-931 [Medline] [Order article via Infotrieve]
  19. Sonveaux, N., Conrath, K., Capiau, C., Brasseur, R., Goormaghtigh, E., and Ruysschaert, J.-M.(1994) J. Biol. Chem. 269, 25637-25645 [Abstract/Free Full Text]
  20. Swaney, J. B.(1980) J. Biol. Chem. 255, 877-881 [Free Full Text]
  21. Tsuboi, M.(1962) J. Polym. Sci. 58, 139-153
  22. Van der Horst, D. J., Ryan, R. O., Van Heusden, M. C., Schulz, T. K. F., Van Doorn, J., Law, J. H., and Beenakkers, A. M. T.(1988) J. Biol. Chem. 263, 2027-2033 [Abstract/Free Full Text]
  23. Vandenbussche, G., Clercx, A., Clercx, M., Curstedt, T., Johansson, J., Jörnvall, H., and Ruysschaert, J.-M.(1992) Biochemistry 31, 9169-9176 [Medline] [Order article via Infotrieve]
  24. Wald, J. H., Goormaghtigh, E., De Meutter, J., Ruysschaert, J.-M., and Jonas, A.(1990) J. Bio. Chem. 265, 20044-20050 [Abstract/Free Full Text]
  25. Wells, M. A., Ryan, R. O., Prasad, S. V., and Law, J. H.(1985) Insect Biochem. 15, 565-571
  26. Wientzek, M., Kay, C. M., Oikawa, K., and Ryan, R. O.(1994) J. Biol. Chem. 269, 4605-4612 [Abstract/Free Full Text]
  27. Zhou, N. E., Zhu, B.-Y., Sykes, B. D., and Hodges, R. S.(1992) J. Am. Chem. Soc. 114, 4320-4326

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.