Assessing Micellar Interaction and Growth in Detergent Solutions Used to Crystallize Integral Membrane Proteins Patrick J. Loll, † Carl Hitscherich, Jr., ‡ Vladimir Aseyev, ‡,§ Margaret Allaman, ‡ and John Wiencek* ,‡ Department of Biochemistry, Drexel University College of Medicine, 245 North 15th Street, Philadelphia, Pennsylvania 19102-1192, and Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa 52242 Received August 1, 2002; Revised Manuscript Received September 16, 2002 ABSTRACT: Integral membrane proteins are solubilized in aqueous solutions by the addition of detergent, creating protein-detergent complexes (PDCs), which can then be crystallized. Interactions between the detergent moieties of PDCs contribute significantly to their crystallization behavior. Interaction forces can be quantified using the second osmotic virial coefficient (B 22 ). The B 22 behavior of protein-free detergent micelles is a good predictor of the behavior of the corresponding PDCs under similar conditions, suggesting that detergent B 22 measurements can be used as a screening tool when crystallizing PDCs. However, if the micelle size varies, B 22 measurements will not accurately reflect micelle-micelle forces. We therefore examined micelle size in a model detergent system, using small-angle X-ray scattering and static and dynamic light scattering, assessing the effects of temperature, detergent concentration, and precipitant on B 22 and micelle size. In the absence of poly(ethylene glycol) (PEG), decreases in B 22 principally reflect increases in micelle-micelle attractive forces and do not reflect significant changes in micelle size. In the presence of PEG, the apparent hydrodynamic radius of detergent micelles shows a similar dependence upon micelle concentration as in the absence of PEG, suggesting that PEG does not effect significant changes in micelle size but rather acts by enhancing interaction forces between micelles. 1. Introduction Roughly one-quarter to one-third of all proteins are thought to be integral membrane proteins. 1 These molecules are critically important to the functioning of living cells and are under intensive investigation as potential drug targets. However, relatively little is known about membrane protein structure: of the tens of thousands of proteins of known structure, fewer than 1% are membrane proteins. This is largely due to the difficulties associated with obtaining crystals suitable for X-ray diffraction analysis. 2,3 Insights into the basic mechanisms controlling membrane protein crystal growth are required so that rational strategies may be devised to improve the success rate for crystallization. Membrane proteins have evolved to exist in the anisotropic, amphipathic environment of biological mem- branes. Hence, these proteins typically contain both regions that are embedded in the lipid bilayer and regions that are exposed to aqueous solution and are soluble in neither aqueous solutions nor organic sol- vents. However, the addition of detergents can mask the hydrophobic section of the protein, creating a water soluble protein-detergent complex (PDC). Crystals of membrane proteins for X-ray diffraction analysis are most frequently obtained by direct crystal- lization of PDCs. Because PDCs can contain as much as 50% detergent by weight, it is not surprising that PDC crystallization is exquisitely sensitive to the prop- erties of the detergent(s) solutions used. For example, crystallization of PDCs appears to be particularly favor- able under conditions that lie near the solution’s cloud point. 4-7 The cloud point is a phase transition that occurs in solutions containing detergent micelles. At the cloud point, micelles coalesce and separate from the aqueous phase, causing microscopic droplets of detergent- rich phase to be dispersed throughout the solution and giving rise to a characteristic turbidity (hence the term “cloud point”). The attractive forces between detergent micelles that mediate micelle aggregation and subse- quent phase separation are also expected to mediate attractions between the detergent moieties of PDCs. Thus, it seems reasonable that when solution conditions approach the cloud point, the interactions between PDCs can be sufficiently attractive to bring PDCs into close contact, allowing crystal lattice contacts to be formed. 8 This hypothesis has been examined using the bacter- ial outer membrane protein OmpF porin, one of many proteins that crystallizes near the solution cloud point. 9 Measurements of the second osmotic virial coefficient (B 22 ) were used to quantify interparticle attractive forces in solutions containing micelles and OmpF PDCs. 10 B 22 values for PDCs were found to become more negative as the crystallization conditions are approached, corre- sponding to increasingly attractive forces between PDCs. Earlier work has prompted the suggestion that there exists a “crystallization slot” for soluble proteins, an optimum range of B 22 values in which crystals are most likely to form. 11-14 In an analogous fashion, the B 22 values for pure detergent micelles become more negative as the solution cloud point is approached. In fact, B 22 * To whom correspondence should be addressed. Tel: (319)353-2377. Fax: (319)335-1415. E-mail: john-wiencek@uiowa.edu. † Drexel University College of Medicine. ‡ University of Iowa. § Permanent address: Institute of Macromolecular Compounds, Russian Academy of Science, Bolshoi Prospect 31, 199004, St. Peters- burg, Russia. CRYSTAL GROWTH & DESIGN 2002 VOL. 2, NO. 6 533 - 539 10.1021/cg025563w CCC: $22.00 © 2002 American Chemical Society Published on Web 10/09/2002