title: Crystallization of proteins from crude bovine rod outer segments.
abstract: Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques.
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA; Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA. Electronic address: byb@case.edu.
authors.forename: Bo Y
authors.initials: BY
authors.lastname: Baker
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA; Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Sahil
authors.initials: S
authors.lastname: Gulati
authors.affiliation: Center for Proteomics and Bioinformatics, Center for Synchrotron Biosciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Wuxian
authors.initials: W
authors.lastname: Shi
authors.affiliation: Center for Proteomics and Bioinformatics, Center for Synchrotron Biosciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Benlian
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA; Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Phoebe L
authors.initials: PL
authors.lastname: Stewart
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA; Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA. Electronic address: kxp65@case.edu.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
mesh_terms: D000818:Animals; D002417:Cattle; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods*; D005136:Eye Proteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation; D012374:Rod Cell Outer Segment / Q000737:chemistry*
chemical_list: D005136:Eye Proteins; D008565:Membrane Proteins
keywords: Creatine kinase-B type; Glyceraldehyde 3-phosphate dehydrogenase; Opsin; Phosphodiesterase 6; Photoreceptor; Protein crystallization; Rod outer segment proteins; Rods; Transducin; l-Lactate dehydrogenase