Strategy for membrane protein crystallization exemplified with OmpA and OmpX.
ABSTRACT The bacterial outer membrane proteins OmpA and OmpX were modified in such a manner that they yielded bulky crystals diffracting X-rays isotropically beyond 2 A resolution and permitting detailed structural analyses. The procedure involved semi-directed mutagenesis, mass production into inclusion bodies, and (re)naturation therefrom; it should be applicable for a broader range of membrane proteins.
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ABSTRACT: Strategies for growing protein crystals have for many years been essentially empirical, the protein, once purified to a certain homogeneity, being mixed with a selection of crystallization agents selected in a more or less trial-and-error fashion. Screening for the correct conditions has been made easier through automation and by the introduction of commercially available crystallization kits. Many parameters can be changed in these experiments, such as temperature, pH, and ionic strength, but perhaps the most important variable has been ignored, namely the protein. The crystallization properties of a protein vary greatly: some crystallize readily, whereas others have proven extremely difficult or even impossible to obtain in a crystalline state. The possibility of altering the intrinsic characteristics of a protein for crystallization has become a feasible strategy. Some historical perspectives and advances in this area will be reviewed.Journal of Structural Biology 05/2003; 142(1):88-97. DOI:10.1016/S1047-8477(03)00041-8 · 3.37 Impact Factor
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ABSTRACT: Type IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenic Escherichia coli (EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, from Pseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer. Secretins are multimeric proteins that allow the passage of secreted toxins and surface structures through the outer membranes (OMs) of Gram-negative bacteria. To date, there have been no atomic structures of the C-terminal region of a secretin, although electron microscopy (EM) structures of the complex are available. This work provides a detailed topology prediction of the membrane-spanning domain of a type IV pilus (T4P) secretin. Our study used innovative techniques to provide new and comprehensive information on secretin topology, highlighting similarities and differences among secretin subfamilies. Additionally, the techniques used in this study may prove useful for the study of other OM proteins. Copyright © 2015 Lieberman et al.mBio 03/2015; 6(2). DOI:10.1128/mBio.00322-15 · 6.88 Impact Factor
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ABSTRACT: A generic protocol that utilizes a dual hexahistidine-maltose-binding protein (His6-MBP) affinity tag has been developed for the production of recombinant proteins in Escherichia coli. The MBP moiety improves the yield and enhances the solubility of the passenger protein while the His-tag facilitates its purification. The fusion protein (His6-MBP-passenger) is purified by immobilized metal affinity chromatography (IMAC) on nickel-nitrilotriacetic acid (Ni-NTA) resin and then cleaved in vitro with His6-tobacco etch virus protease to separate the His6-MBP from the passenger protein. In the final step, the unwanted byproducts of the digest are absorbed by a second round of IMAC, leaving nothing but the pure passenger protein in the flow-through fraction. Endogenous proteins that bind to the Ni-NTA resin during the first IMAC step also do so during the second round of IMAC. Hence, the application of two successive IMAC steps, rather than just one, is the key to obtaining crystallization-grade protein with a single affinity technique.Methods in Molecular Biology 02/2007; 363:1-19. DOI:10.1007/978-1-59745-209-0_1 · 1.29 Impact Factor