title: From Constructs to Crystals - Towards Structure Determination of β-barrel Outer Membrane Proteins.
abstract: Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, α-helical and β-barrel. While α-helical membrane proteins can be found in nearly all cellular membranes, β-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite β-barrel outer membrane protein (OMP), general protocols are presented for the production of target β-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.
authors.affiliation: Department of Biological Sciences, Markey Center for Structural Biology, Purdue University; nnoinaj@purdue.edu.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Noinaj
authors.affiliation: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health.
authors.forename: Stephen
authors.initials: S
authors.lastname: Mayclin
authors.affiliation: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health; National Institute of General Medical Sciences (NIGMS), National Institutes of Health.
authors.forename: Ann M
authors.initials: AM
authors.lastname: Stanley
authors.affiliation: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health; National Institute of General Medical Sciences (NIGMS), National Institutes of Health.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Jao
authors.affiliation: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health.
authors.forename: Susan K
authors.initials: SK
authors.lastname: Buchanan
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002462:Cell Membrane; D003460:Crystallization; D006090:Gram-Negative Bacteria; D008928:Mitochondria; D017433:Protein Structure, Secondary
chemical_list: D001425:Bacterial Outer Membrane Proteins