In This Issue

460 Unique interactions between the chromophore and glutamate 16 lead to far-red emission in a red fluorescent protein

Xiaokun Shu, Lei Wang, Leslie Colip, Karen Kallio, and S. James Remington

Shortly after the Nobel prize-winning discovery that GFP could be used to label proteins within living cells, the search for red emitting variants was initiated. Recent work by Roger Tsien and others has led to the discovery of monomeric far-red fluorescent proteins, which are useful as noninvasive labels in animals. In this report, structural and spectroscopic studies help to clarify the mechanisms leading to the ∼650 nm emission maximum of mPlum. Crystal structures of mPlum and two blue shifted mutants reveal that a unique hydrogen bond between glutamate 16 and the chromophore is required for the far-red emission. The ultimate goal of a fluorescent protein emitting in the so-called “near-IR window” (650-900 nm) is now in sight.

372 Genetic selection system for improving recombinant membrane protein expression in E. coli

Elizabeth Massey-Gendel, Anni Zhao, Gabriella Boulting, Hye-Yeon Kim, Michael A. Balamotis, Len M. Seligman, Robert K. Nakamoto, and James U. Bowie

Many proteins, particularly membrane proteins, cannot be produced in sufficient quantities for biochemical or structural characterization. In this report, Massey-Gendel et al. describe a selection system to isolate mutant strains of E. coli that improve the expression of a target protein. Strains are selected by virtue of a drug resistance marker fused to the C-terminus of the protein target. E. coli strains generated using their system improved expression of some membrane proteins over 75-fold.

467 Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

George T. Lountos, Brian P. Austin, Sreedevi Nallamsetty, and David S. Waugh

Crystal structures of cleaved (cyan) and uncleaved (red) forms of the YscU cytoplasmic domain, an essential component of the type III secretion system in Yersinia pestis, have been solved and refined up to atomic resolution. Autocleavage of the cytoplasmic domain triggers the recognition and export of translocators at the proper time during assembly of the type III secretion apparatus. These crystallographic studies reveal conformational changes induced upon autocleavage of YscU. In particular, there is a substantial movement of the the N-terminal α-helical region that links the cytosolic domain of YscU to the membrane, which may correspond to the molecular switch that influences substrate specificity.

348 An approach to characterizing single-subunit mutations in multimeric prepores and pores of anthrax protective antigen

Blythe E. Janowiak, Alan Finkelstein, and R. John Collier

Anthrax toxin has recently emerged as a tractable system for studying how catalytic subunits of toxins cross a membrane. The protective antigen (PA) moiety of this toxin forms an heptameric pore in the endosomal membrane of mammalian cells and translocates the two enzymic moieties across the membrane. Janowiak et al. report a protocol for isolating heptameric precursor of the pore (prepore) containing an inhibitory mutation in only one of the seven subunits. An Ala replacement mutation for either of two key residues inhibited the transport function of the heptamer almost completely, but by different mechanisms, depending on the specific residue mutated.