© Copyright 1997 by the American Chemical Society
VOLUME 101, NUMBER 11, MARCH 13, 1997
LETTERS Crystallization of a Polar Protein and Small Molecules from the Aqueous Compartment of Lipidic Cubic Phases† Ehud M. Landau,*,‡ Gabriele Rummel,‡ Sandra W. Cowan-Jacob,§ and Jurg P. Rosenbusch‡ Biozentrum, UniVersity of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland, and Core Drug DiscoVery Technologies, K-681.5.47 Ciba Geigy AG, CH-4002 Basel, Switzerland ReceiVed: October 28, 1996; In Final Form: January 30, 1997X
Lipidic cubic phases allow crystallization of both water-soluble compounds (salts and hydrophilic proteins) and hydrophobic molecules. Unlike the crystallization of the membrane protein bacteriorhodopsin, the nucleation and growth of sodium chloride, glycine and lysozyme crystals are independent of the type of cubic phase and the packing arrangements of the lipidic compartments. The crystals obtained diffracted X-rays to better than 1.5 Å (small molecules) and 2.0 Å (lysozyme). Crystallization in lipidic cubic phases is assisted by the resistance of these matrices toward doping with a wide variety of detergents, precipitants, and additives.
Lipidic cubic phases, first described by Luzzati et al.,1 are complex materials composed of two distinct, structurally welldefined, immiscible, hydrophilic, and hydrophobic compartments. These highly viscous, transparent materials have been described to exist in two types: closed micellar and bicontinu† Abbreviations: CHAPSO, (3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate); C8E6-11, a heterodisperse distillation fraction of an octyl moiety linked to 6-11 oxyethylene units; di-CnPC, dialkanoyl phosphatidylcholine, where the subscript n denotes the number of CH2 groups in the alkyl chains; DDAO, dimethyldodecyl aminoxide; DMSO, dimethyl sulfoxide; HD, hexanediol; HT, heptanetriol; MO, 1-monooleoylrac-glycerol, C18:1c9; MP, 1-monopalmitoleoyl-rac-glycerol, C16:1c9; MPD, 2,2,4-methylpentanediol; octyl-HESO, octyl hydroxyethanesulfoxide; octylPOE, octyl poly(oxyethylene); β-OG, octyl-β-glucopyranoside; PEG, poly(ethylene glycol); PLPC, palmitoyl lysophosphatidylcholine; ZW, Zwittergent 3-10. * Author to whom correspondance should be addressed at: Department of Microbiology, Biozentrum, University of Basel, Klingelbergstr. 70, CH4056 Basel, Switzerland. FAX: +41-61-267-2118. E-mail: landau@ ubaclu.unibas.ch. ‡ University of Basel. § Ciba Geigy, Basel. X Abstract published in AdVance ACS Abstracts, March 1, 1997.
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ous. The main difference between them is the supramolecular assembly of the constituent lipid molecules; in the former, these are packed in discrete, discontinuous micelles, while in the latter system, they form curved, continuous bilayers.1,2 In either array, intercommunicating water channels pervade the entire phase. Due to their unique structural and dynamic properties, these stable and versatile matrices are of interest in several fields, ranging from material sciences,3 in which they may be used as reaction vessels or templates for the synthesis of structures with novel properties, to cell biology, where compartmentation plays a significant role in metabolic regulation in ViVo.4 We have initiated an investigation exploring the potentials of lipidic cubic phases as crystallization matrices for a variety of compounds, ranging from soluble compounds to membrane proteins. We have advanced the hypothesis that, upon nucleation, bicontinuous phases should allow crystallization of membrane proteins by virtue of their lateral diffusion in the curved bilayer, whereas in discrete micelles, this type of crystal growth is predicted not to occur. Our recent results with the © 1997 American Chemical Society
1936 J. Phys. Chem. B, Vol. 101, No. 11, 1997
Letters
TABLE 1: Stability of Two Cubic Phases upon Incorporation of Additivesa
this postulate, we have now investigated the crystallization of polar solutes in micellar and bicontinuous lipidic cubic phases. Prerequisite to such a study is the knowledge of the susceptibility of cubic phases toward the incorporation of additives that are used routinely in the crystallization of proteins, i.e. precipitants, salts, and also detergents. Different cubic phases were doped with wide ranges of concentrations of these additives. Their tolerance was monitored as a function of time in a range commensurate with protein crystallization (arbitrarily set to >30 days). Stability was assessed by observing the quasisolid, highly viscous texture and the transparency of the matrices. For our purpose, these properties may be regarded as sufficient criteria to establish the integrity of the cubic phases. It is noteworthy that deliberate perturbations, caused by adding organic solvents or by inducing phase transitions, resulted in the matrices being liquefied and often turning opaque. The results of these experiments, presented in Table 1, show the maximal amounts of additives at which the closed micellar cubic phases (palmitoyl lysophosphatidylcholine/water), and the bicontinuous phase (1-monooleyl-rac-glycerol/water) retained their texture and transparency. Poly(ethylene glycol) (PEG2000) was tolerated over a very narrow concentration range in the bicontinuous cubic system only. Yet, for the purpose of crystallization, this limitation can often be offset by the addition of salt or precipitants such as MPD, to which cubic phases are resistant. Additions of organic solvents, such as chloroform, diethyl ether, or dimethyl sulfoxide, however, promptly dissolved the lipidic matrices (Table 1). On the basis of these results, we set out to test the crystallization of soluble compounds from aqueous compartments of lipidic cubic phases. Three substances were tested: NaCl, glycine, and the polar protein lysozyme (14.4 kDa). They yielded excellent crystals independent of the cubic phases used. In all cases, crystals grew to their mature sizes within the host matrices, without affecting either transparency or solid-like texture thereof. The crystals of small molecules diffracted X-rays to the resolution limit of the detector used (
