Orthorhombic Crystal Form of Bacteriorhodopsin Nucleated on Benzamidine Diffracting to 3·6 Å Resolution

doi:10.1006/jmbi.1993.1570

Journal of Molecular Biology

Volume 234, Issue 1, 5 November 1993, Pages 156-164

https://doi.org/10.1006/jmbi.1993.1570 Get rights and content

Abstract

Freshly formed benzamidine crystals were found to provide a suitable nucleation surface for crystallisation of bacteriorhodopsin. At 20°C and 1% octylglucoside pseudohexagonal needles of bacteriorhodopsin nucleated on the benzamidine surface. At 4°C and reduced detergent concentration (0·5%) a new, better-ordered orthorhombic crystal form of bacteriorhodopsin was formed by heterogeneous nucleation on benzamidine. Polarised absorption spectroscopy and flash photolysis experiments were used to show that the crystalline bacteriorhodopsin is photoactive in both forms. The M-intermediate absorbs maximally at 405nm and formation of M does not disturb the crystal lattice. The plate-shaped crystals diffract to a resolution of 3·6 Å along the a and b directions and to 4·2 Å in the c direction. The most likely space group of the crystals in C₂₂₂ (a = 107·5 Å, b = 117·0 Å, c = 69·5 Å). The crystals are built from layers of bacteriorhodopsin molecules that are tilted from the c axis by about 45°. In addition, as a background to the discovery of the new crystal form, the influence of different detergents, additives and precipitants on the formation of pseudohexagonal needles is presented.

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While detergents with small micelles such as octyl-β-d-maltoside (8M) or nonyl-β-d-maltoside (9M) hardly cover the hydrophobic surface of the protein leading to protein aggregation, detergents with large micelles such as tridecyl-β-d-maltoside (13 M) usually tend to engulf the entire protein [53]. The addition of small amphiphiles such as heptane-1,2,3-triol and benzamidine is frequently able to improve crystallography because they reduce detergent micelles thereby enhancing crystal contacts [54–56]. Alternative strategies to induce crystallization are the use of additives such as small micelle detergents, heavy metals, salts and organic solvents [57].
The field of Membrane Protein Structural Biology has grown significantly since its first landmark in 1985 with the first three-dimensional atomic resolution structure of a membrane protein. Nearly twenty-six years later, the crystal structure of the beta2 adrenergic receptor in complex with G protein has contributed to another landmark in the field leading to the 2012 Nobel Prize in Chemistry. At present, more than 350 unique membrane protein structures solved by X-ray crystallography (http://blanco.biomol.uci.edu/mpstruc/exp/list, Stephen White Lab at UC Irvine) are available in the Protein Data Bank. The advent of genomics and proteomics initiatives combined with high-throughput technologies, such as automation, miniaturization, integration and third-generation synchrotrons, has enhanced membrane protein structure determination rate. X-ray crystallography is still the only method capable of providing detailed information on how ligands, cofactors, and ions interact with proteins, and is therefore a powerful tool in biochemistry and drug discovery. Yet the growth of membrane protein crystals suitable for X-ray diffraction studies amazingly remains a fine art and a major bottleneck in the field. It is often necessary to apply as many innovative approaches as possible. In this review we draw attention to the latest methods and strategies for the production of suitable crystals for membrane protein structure determination. In addition we also highlight the impact that third-generation synchrotron radiation has made in the field, summarizing the latest strategies used at synchrotron beamlines for screening and data collection from such demanding crystals. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.
A transporter converted into a sensor, a phototaxis signaling mutant of bacteriorhodopsin at 3.0 Å
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Bicelle-grown crystals of BR_A215T diffract to 3.0 Å resolution and belong to space group C2 (Table 1). This is the same space group, albeit with different unit cell dimensions, that was reported for wild-type BR crystals obtained by vapor diffusion,13 for which a 2.9-Å structure was published subsequently (PDB code: 1BRR).14 However, only two of the unit cell dimensions are similar, whereas the shortest axis has a length of 80.2 Å for 1BRR type II crystals and a length of 61.5 Å for BR_A215T type I crystals.
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The stability of a protein can be improved by finding the appropriate conditions, which in turn helps crystallization. In several cases, the use of additives [46–50], detergent mixtures [51,52], or lipids [53–55] has been important for the successful structure determination of membrane proteins. The key is to find a compound that stabilizes the protein in solution.
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High stability is a prominent characteristic of integral membrane proteins of known atomic structure. But rather than being an intrinsic property, it may be due to a selection exerted by biochemical procedures prior to structure determination, since solubilization results in the transient exposure of membrane proteins to solution conditions. This may cause structural perturbations that interfere with 3D crystallization and hence with X-ray analysis. This problem also affects the preparation of samples for electron crystallography and NMR studies and may account for the fact that high-resolution structures of representatives of whole groups, such as transport proteins and signal transducers, have not been elucidated so far by any method. A knowledge of the proportion of labile proteins among membrane proteins, and of the kinetics of their denaturation, is therefore necessary. Establishing stability profiles, developing methods to maintain lateral pressure, or preventing contact with water (or both) should prove significant in establishing the structures of conformationally flexible proteins.
Approaches to determining membrane protein structures to high resolution: Do selections of subpopulations occur?
2001, Micron
Citation Excerpt :
Electron crystallography of the trimeric porin, which contains water channels in β-barrels, yielded resolutions, in projection, of 3.5–4 Å for two different porins (Sass et al., 1989; Jap et al., 1990), while a 3D tomographic reconstruction resulted in a map at 6 Å (Jap, 1986). The structure of the proton-pumping bacteriorhodopsin with its hydrophobic α-helical bundle initially yielded 3D-crystals diffracting to 8 Å with crystallization from detergent solution (Michel and Oesterhelt, 1980) and, more recently, to 2.9 Å (Schertler et al., 1993; Essen et al., 1998), while crystals obtained from lipidic cubic phases yielded resolution of <2 Å (Belrhali et al., 1999; Luecke et al., 1999a,b). Lactose permease, a well-studied proton–solute symporter and a paradigm for a conformationally flexible protein (Kaback and Wu, 1999) has, despite great efforts, failed to crystallize in three dimension so far, and stained 2D lattices have yielded a resolution of 20 Å (Zhuang et al., 1999).
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Journal of Molecular Biology

Regular ArticleOrthorhombic Crystal Form of Bacteriorhodopsin Nucleated on Benzamidine Diffracting to 3·6 Å Resolution

Abstract

Regular Article
Orthorhombic Crystal Form of Bacteriorhodopsin Nucleated on Benzamidine Diffracting to 3·6 Å Resolution