ARTICLE pubs.acs.org/crystal
Polymer-Induced Heteronucleation for Protein Single Crystal Growth: Structural Elucidation of Bovine Liver Catalase and Concanavalin A Forms Leila M. Foroughi,† You-Na Kang,‡ and Adam J. Matzger*,†,§ †
Department of Chemistry, ‡Life Sciences Institute, and §Macromolecular Science and Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States
bS Supporting Information ABSTRACT: Obtaining single crystals for X-ray diffraction remains a major bottleneck in structural biology; when existing crystal growth methods fail to yield suitable crystals, often the target rather than the crystallization approach is reconsidered. Here we demonstrate that polymer-induced heteronucleation, a powerful technique that has been used for small molecule crystallization form discovery, can be applied to protein crystallization by optimizing the heteronucleant composition and crystallization formats for crystallizing a wide range of protein targets. Applying these advances to two benchmark proteins resulted in dramatically increased crystal size, enabling structure determination, for a half century old form of bovine liver catalase (BLC) that had previously only been characterized by electron microscopy, and the discovery of two new forms of concanavalin A (conA) from the Jack bean and accompanying structural elucidation of one of these forms.
’ INTRODUCTION Despite significant effort expended in designing efficient protein crystallization techniques, the most widely practiced method for protein crystal growth remains the use of a battery of conditions applied to vapor diffusion formats.1 Failure of traditional screening methods to produce diffraction quality crystals is more likely to lead to reconsideration of the protein construct (e.g., truncation, mutagensis, and chemical modification of the protein) rather than the crystal growth strategy. This is unfortunate, both from an efficiency standpoint and because biological relevance becomes increasingly compromised as the native protein sequence is altered. This recognition has led to a healthy effort in the field of macromolecular crystallography to seek better crystal growth methods.2 Of particular relevance to the present work is the use of heteronucleants, including crystallization on minerals,3 silicon microarrays,4 and mesoporous gel glass,5 as well as more recent examples, such as zeolites,6 carbon nanotubes,7 and silicate powders.8 Although these heteronucleants demonstrated that a broad cross-section of material types can successfully be employed in this application, none of them have shown success as a “universal heteronucleant” that could promote crystallization for most biological systems. The ideal heteronucleant(s) need to address the problems that have hampered deployment of existing systems, including a lack of diversity in functionality, limitations in the crystallization format that can be used, and the need for protein-heteronucleant specificity. Herein, we report a heteronucleant approach that is able to overcome the limitations of other systems by utilizing the functional diversity and flexibility of r 2011 American Chemical Society
crystallization formats that is afforded by using polymers as heteronucleant surfaces. In the realm of small molecule crystallization, heteronucleation approaches to single crystal production and new form selection have met with considerable success.9 Most successful among these is the polymer-induced heteronucleation (PIHn) approach.10 By applying a functionally diverse library of heteronucleants, PIHn enables solid form selection and discovery from aqueous and organic solutions for a broad range of pharmaceuticals,10-12 as well as for other compound classes.13 To determine if this approach might be equally useful for biomolecular crystallization, PIHn was applied to the benchmark protein hen egg white lysozyme (HEWL) where crystallization in the presence of the polymers allowed nucleation rate control, as well as new form access and selective form production from several buffer conditions indicating a good measure of kinetic control.14 These results indicate that PIHn approaches for small molecules can be successfully deployed on proteins; however, the heteronucleant composition and crystallization formats as implemented were unsuitable for the vast majority of protein targets. These issues are solved by the methods presented here. In its most recent incarnation, PIHn applied to small molecule crystallization employs three polymer libraries deployed in 96-well plates categorized according to their predominant functional groups: acidic, polar nitrogen, and nonpolar aromatic.10 Each library Received: November 15, 2010 Revised: January 12, 2011 Published: February 15, 2011 1294
dx.doi.org/10.1021/cg101518f | Cryst. Growth Des. 2011, 11, 1294–1298
Crystal Growth & Design
ARTICLE
Figure 1. A schematic representation of protein crystallization utilizing PIHn. (A) Each polymer library (nonpolar aromatic, polar nitrogen, and acidic; see Materials and Methods) consists of 96 unique polymers arrayed in a 96-well plate. The highlighted wells represent two of the possible heteronucleants in the polar nitrogen library. (B) A representation of the surface of the cross-linked polymers methacrylonitrile-co-N,Ndimethylmethacrylamide (red) and 4-vinylpyridine-co-2-methyl-2-nitropropymethacrylate (blue). (C) The heteronucleation of BLC (protein solution: 40 mg/mL protein in 0.05 M sodium phosphate buffer at pH 6.8, well solution: 12% PEG 4000 and 0.05 M sodium phosphate buffer at pH 6.8)19 on the red and blue surfaces results in different packing motifs, due to the interactions between the polymers and proteins.
(Figure 1A) consists of 96 unique polymer heteronucleants that are cross-linked with 1,4-divinylbenzene (DVB) to ensure insolubility. Here, we describe changes to both the polymer composition and crystallization format that optimize PIHn for protein crystallization. To demonstrate the power and utility of this system we report: the dramatically increased crystal size of a half century old form of bovine liver catalase (BLC) that had previously only been characterized by electron microscopy,15-18 and the discovery of two new forms of concanavalin A (conA) from the Jack bean.
’ EXPERIMENTAL SECTION ConA and BLC were purchased from Sigma-Aldrich (C7275 and C40, respectively) and HEWL was purchased from Thermo Fisher Scientific (19503). They were all used without further purification. Corning glass coverslips (12-519A) and 96-well polypropylene plates were purchased from Fisher Scientific. Crystal Clear Sealing Tape (HR4511), 24-well VDXM hanging drop diffusion plates (HR3-108), and 24well Cryschem sitting drop plates (HR3-159) were purchased from Hampton Research. All monomers, salts, and buffers were purchased from Sigma-Aldrich, Fisher Scientific, or Scientific Polymer Products. Polymer Heteronucleants. 96-Well Plates For Sitting Drop Vapor Diffusion Method. Three polymer libraries (A, B, and C) were prepared similarly to the previously reported method.10 For each library the monomers, diluted 50% by volume with ethanol, were dispensed in a pairwise combination (six ratios: 100:0, 86:14, 71:29, 57:43, 43:57, 29:71, 14:86, 0:100) to a 96-well polypropylene storage plate. To these monomer solutions, 1:1 cross-linker/ethanol solutions containing 2 mol % azobisisobutyronitrile (AIBN) were added, resulting in an overall monomer to cross-linker ratio of 2:1. Using a 96 needle Hydra (Robbins Instruments), the wells were distributed into three additional plates, thus
producing four 96-well plates with a well volume in each plate of 40 μL. These plates were then flushed with nitrogen and irradiated under four 15 W UVA lamps for 2 h and were subsequently placed in a vacuum oven at 85 °C (
