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Biochimica et Biophysica Acta 1808 (2011) 287–297

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Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m e m

The prediction and characterization of YshA, an unknown outer-membrane protein from Salmonella typhimurium
Thomas C. Freeman Jr., Samuel J. Landry, William C. Wimley ⁎Department of Biochemistry, Tulane University Health Sciences Center, 1430 Tulane Ave SL-43, New Orleans, LA 70112, USA

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We have developed an effective pathway for the prediction and characterization of novel transmembrane βbarrel proteins. The Freeman–Wimley algorithm, which is a highly accurate prediction method based on the physicochemical properties ofexperimentally characterized transmembrane β barrel (TMBB) structures, was used to predict TMBBs in the genome of Salmonella typhimurium LT2. The previously uncharacterized product of gene yshA was tested as a model for validating the algorithm. YshA is a highly conserved 230-residue protein that is predicted to have 10 transmembrane β-strands and an N-terminal signal sequence. All of thephysicochemical and spectroscopic properties exhibited by YshA are consistent with the prediction that it is a TMBB. Specifically, recombinant YshA localizes to the outer membrane when expressed in Escherichia coli; YshA has a β-sheet-rich secondary structure with stable tertiary contacts in the presence of detergent micelles or when reconstituted into a lipid bilayer. When in a lipid bilayer, YshA forms amembrane-spanning pore with an effective radius of ~ 0.7 nm. Taken together, these data substantiate the predictions made by the Freeman–Wimley algorithm by showing that YshA is a TMBB protein. © 2010 Elsevier B.V. All rights reserved.

Article history: Received 12 June 2010 Received in revised form 13 September 2010 Accepted 15 September 2010 Available online 20 September 2010 Keywords:Salmonella Beta barrel Outer-membrane protein Structure prediction

1. Introduction The proteins that span the outer membrane of Gram-negative bacteria fold into the structural motif known as the transmembrane β-barrel (TMBB), examples of which are also found in the outer membranes of mitochondria and chloroplasts [1]. The only other membrane-spanning structure is the α-helical bundle, which can haveas few as one or more than 20 transmembrane helices, and is found in the cytoplasmic membranes of all forms of life [2]. Although 2–3% of the genes in Gram-negative bacteria encode TMBBs, they represent less than 1% of all solved protein structures from Gram-negative organisms. Because TMBBs, like most membrane proteins, are difficult to crystallize or study otherwise in vitro, many groups haveturned to computational structure prediction methods. Membrane-spanning helices can be predicted using a relatively straightforward prediction algorithm that can identify the 19–25 contiguous hydrophobic residues which constitute a single membrane-spanning helix [3–5]. However, developing an accurate algorithm for TMBB prediction has been more troublesome due to the cryptic nature of the TMBBstructure [6]. The structures of TMBB proteins consist of a contiguous sequence of 8–24 anti-parallel β-strands which meander in and out of the membrane in a cylindrical geometry. This arrangement gives the

⁎ Corresponding author. E-mail address: wwimley@tulane.edu (W.C. Wimley). 0005-2736/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2010.09.008

overallstructure a barrel-like appearance with the lumen of the barrel transverse to the surface of the lipid bilayer. The meandering TM βstrands are connected by short turns of about five residues at one opening of the barrel, and loops of varying lengths at the other opening. Each strand is laterally connected to the next by anti-parallel, inter-strand hydrogen bonds, including the first N-terminal strand and...
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