Radiation damage to cryocooled protein crystals during x-ray structure determination has become an inherent part of macromolecular diffraction data collection at third-generation synchrotrons. Generally, radiation damage is an undesirable component of the experiment and can result in erroneous structural detail in the final model. The characterization of radiation damage thus has become an important area for structural biologists. The calculated dose limit of 2 ؋ 10 7 Gy for the diffracting power of cryocooled protein crystals to drop by half has been experimentally evaluated at a third-generation synchrotron source. Successive data sets were collected from four holoferritin and three apoferritin crystals. The absorbed dose for each crystal was calculated by using the program RADDOSE after measurement of the incident photon flux and determination of the elemental crystal composition by microparticle-induced x-ray emission. Degradation in diffraction quality and specific structural changes induced by synchrotron radiation then could be compared directly with absorbed dose for different dose͞dose rate regimes: a 10% lifetime decrease for a 10-fold dose rate increase was observed. Remarkable agreement both between different crystals of the same type and between apoferritin and holoferritin was observed for the dose required to reduce the diffracted intensity by half (D 1/2). From these measurements, a dose limit of D 1/2 ‫؍‬ 4.3 (؎0.3) ؋10 7 Gy was obtained. However, by considering other data quality indicators, an intensity reduction to I ln2 ‫؍‬ ln2 ؋ I0, corresponding to an absorbed dose of 3.0 ؋ 10 7 Gy, is recommended as an appropriate dose limit for typical macromolecular crystallography experiments.experimental dose limit ͉ macromolecular cryocrystallography ͉ radiation damage

Staphylococcus aureus can adhere to and invade endothelial cells by binding to the human protein fibronectin (Fn). FnBPA and FnBPB, cell wall-attached proteins from S. aureus, have multiple, intrinsically disordered, high-affinity binding repeats (FnBRs) for Fn. Here, 30 years after the first report of S. aureus/Fn interactions, we present four crystal structures that together comprise the structures of two complete FnBRs, each in complex with four of the N-terminal modules of Fn. Each Ϸ40-residue FnBR forms antiparallel strands along the triple-stranded ␤-sheets of four sequential F1 modules ( 2-5 F1) with each FnBR/ 2-5 F1 interface burying a total surface area of Ϸ4,300 Å 2 . The structures reveal the roles of residues conserved between S. aureus and Streptococcus pyogenes FnBRs and show that there are few linker residues between FnBRs. The ability to form large intermolecular interfaces with relatively few residues has been proposed to be a feature of disordered proteins, and S. aureus/Fn interactions provide an unusual illustration of this efficiency.intrinsic disorder ͉ tandem ␤-zipper ͉ host-pathogen interaction S taphylococcus aureus is a dangerous human pathogen that causes a wide range of debilitating and life-threatening infections (1). Incidence of S. aureus resistance to antibiotics (2) makes the understanding of its mechanisms of pathogenesis imperative. S. aureus/Fn interactions were first reported 30 years ago, and an S. aureus Fn-binding protein was isolated and characterized Ϸ20 years ago (3). Our recent work has dissected the 363-residue C-terminal region of FnBPA into 11 FnBRs (4) (FnBPA1-11; Fig. 1 A and B), six of which bind the NTD (N-terminal domain) of Fn (comprising modules 1-5 F1) with dissociation constants in the nanomolar range (5). The Cterminal region of FnBPB, a second S. aureus Fn-binding protein, is very similar to FnBPA but lacks one of the shorter FnBRs (5). In FnBPA, which also binds fibrinogen, the fibrinogen-and Fn-binding regions (Fig. 1 A) appear to cooperate in disease progression, with the FnBR region being particularly associated with persistence of infection (6). FnBPA/Fn interactions both mediate S. aureus invasion of (7) and activate endothelial cells, evoking both the proinflammatory and procoagulant responses typical of infective endocarditis (8). FnBPAs ability to mediate platelet activation, a key step in thrombus formation, is also likely to play a role in cardiovascular disease (9) and FnBPA has been implicated in cardiac device infections through its ability to mediate S. aureus attachment to implanted prosthetic materials (10). We previously predicted that in Fn-BPA each FnBR binds a string of three or four F1 modules in the NTD of Fn through a longer version of the tandem ␤-zipper mechanism that we discovered in Streptococcus dysgalactiae interactions with 1 F1 2 F1 (4). Results and DiscussionCrystal Structure of FnBPA-1/ 2-5 F1. Fig. 1C shows two F1 module pair/peptide structures that together comprise the structure of the most N-terminal S. aureus FnBR (...

During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins, which form ionic pores. Binding of Cry1A toxins to the cadherin receptor promotes the formation of a 250 kDa oligomer. In this work, we analyzed for the first time the structural changes presented by Cry1Ab toxin upon membrane insertion. Trp fluorescence of pure monomeric and oligomeric structures in solution and in a membrane-bound state was analyzed. Cry1Ab has nine Trp residues, seven of them in pore-forming domain I. Trp quenching analysis with iodide indicated that oligomerization caused a 27% reduction in the level of Trp exposed to the solvent. Most of the oligomeric structure (96%) inserts into the membrane as a function of the lipid:protein ratio, in contrast to the monomer (10%). Additionally, the membrane-associated oligomer presented a blue shift of 5 nm in lambda(max) of the emission spectrum, indicating a more hydrophobic environment for some Trp residues. In agreement with this, iodide was unable to quench the Trp of the membrane-bound oligomer, suggesting that a significant part of the protein may be buried in the membrane. Quenching analysis using brominated and spin-labeled phospholipids in the vesicles indicates that most of the Trp residues are located close to the membrane-water interface. Finally, ionic currents in black lipid bilayers revealed that the oligomeric structure has kinetics different from those of the monomer, producing stable channels with a high probability of being open in contrast to the monomer that exhibited unstable opening patterns. These data show that the oligomer, in contrast to the monomer, is able to interact efficiently with phospholipid membranes forming stable pores.

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