Design of bioelectronic interfaces by exploiting hinge-bending motions in proteins.

We report a flexible strategy for transducing ligand-binding events into electrochemical responses for a wide variety of proteins. The method exploits ligand-mediated hinge-bending motions, intrinsic to the bacterial periplasmic binding protein superfamily, to establish allosterically controlled interactions between electrode surfaces and redox-active, Ru(II)-labeled proteins. This approach allows the development of protein-based bioelectronic interfaces that respond to a diverse set of analytes. Families of these interfaces can be generated either by exploiting natural binding diversity within the superfamily or by reengineering the specificity of individual proteins. These proteins may have numerous medical, environmental, and defense applications.

Characteristics of circulating growth hormone in women after acute heavy resistance exercise.

The effects of exercise on the molecular nature of secreted human growth hormone (GH) or its biological activity are not well understood. Plasma from women (average age 23.6 yr, n = 35), drawn before and after an acute heavy resistance exercise test, was fractionated by size exclusion chromatography into three size classes, namely, > 60 kDa (fraction A), 30-60 kDa (fraction B), and < 30 kDa (fraction C), before GH assay. Concentrations of GH in these fractions, as well as in unfractioned plasma, were measured by the Nichols immunoradiometric assay, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) polyclonal competitive RIA, Diagnostic Systems Laboratory's immunofunctional assay (measures dimerization-capable species), and the rat tibial bioassay. Significantly increased circulating GH concentrations of two- to fourfold were observed when immunoassays in unfractionated plasma samples were used, but they showed no significant change with use of the rat tibial bioassay. Significant exercise-induced increases in GH were found in fractions B and C but not in fraction A. Because chemical reduction of the samples before GH immunoassay significantly increased GH concentrations in fractions B and C (Nichols and NIDDK kits) after exercise, it is concluded that exercise may specifically increase release of disulfide-linked hormone molecules and/or fragments. Finally, because most of the GH released after exercise was able to dimerize the GH receptor in vitro, it is also concluded that these forms have the two intact binding sites required to initiate signal transduction in target cells.

Synthesis and characterization of a ruthenium(II)-based redox conjugate for reagentless biosensing.

Synthesis of a novel sulfhydryl-specific, tetraammine Ru(II)polypyridyl complex, [Ru(II)(NH(3))(4)(1,10-phenanthroline-5-maleimide)](PF(6))(2), which exhibits environment-sensitive electrochemical properties is described. When conjugated to an allosteric site in a genetically engineered mutant of maltose binding protein, the formal potential of the conjugated redox probe is shifted to higher potential upon maltose binding. The magnitude of this potential shift was used to measure maltose affinity of the protein-redox conjugate complex and to monitor maltose concentration in solution. These results are presented in context of reagentless biosensing.

Rational design of nascent metalloenzymes.

Understanding the early genesis of new enzymatic functions is one of the challenges in protein design, mechanistic enzymology, and molecular evolution. We have experimentally mimicked starting points in this process by introducing primitive iron and oxygen binding sites at various locations in thioredoxin, a small protein lacking metal centers, by using computational design. These rudimentary active sites show emerging enzymatic activities that select to varying degrees between different oxygen chemistries. Even within these nascent enzymes, mechanisms by which different reactions are controlled can be discerned. These involve both stabilizing and destabilizing interactions imposed on the metal center by the surrounding protein matrix.

The catalytic pathway of cytochrome p450cam at atomic resolution.

Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.

Construction of a novel redox protein by rational design: conversion of a disulfide bridge into a mononuclear iron-sulfur center.

A mononuclear iron-sulfur center, capable of reversible electron transfer, has been introduced into thioredoxin, a protein devoid of such sites, using an automated, structure-based design algorithm. One of the sites predicted by the Dezymer computer program to introduce a tetrahedral tetrathiolate iron center included the intrinsic Cys32-Cys35 disulfide of wild-type thioredoxin and two additional mutants, Trp28Cys and Ile75Cys, thereby converting a disulfide into a metal-based redox center. This designed protein forms a 1:1 monomeric complex with FeIII, whose electronic absorption and EPR spectra closely resemble those of the rubredoxins, as intended. CoII spectra provided further confirmation of tetrahedral tetrathiolate metal coordination. The designed protein is capable of undergoing successive cycles of oxidation and reduction. The computer-generated design only took into account the geometry of the primary coordination shell around the metal. We have therefore demonstrated that simple geometrical considerations can be sufficient to reproduce the dominant electronic structure and reactivity of a simple metal-based redox center.

Reduced oxy intermediate observed in D251N cytochrome P450cam.

Cytochrome P450s are ubiquitous heme proteins responsible for various oxidative metabolic processes. The overall rate-determining step in the catalytic cycle of native cytochrome P450cam is the reduction of the dioxygen complex, which has made detection of catalytic intermediates after this reduction impossible. However, for the site-specific mutant D251N cytochrome P450cam (which affects proton transfer near the catalytic center), the overall rate-determining step occurs after the reduction of oxy-P450. As a consequence, we have observed in the UV-visible spectrum during catalytic turnover a new intermediate that is one electron reduced from oxy-P450 with an intact dioxygen bond.

Effects of plasma on adhesion of biofilm forming Pseudomonas aeruginosa and Staphylococcus epidermidis to fibrin substrate.

Bacterial adhesion has been identified as the critical initial step in the pathogenesis of foreign body related infection. Recent investigations have shown microbial binding to implanted polymeric materials using specific adhesion of bacteria to immobilized plasma proteins, such as fibrin. These proteins are though to function as bridging molecules to facilitate bacterial colonization of the surface. The authors' results indicated a significant reduction in adhesion of biofilm forming Pseudomonas aeruginosa and coagulase negative Staphylococcus epidermidis to immobilized fibrin strands in the presence of platelet poor plasma (PPP) as compared to studies performed with phosphate buffered saline and Hank's balanced salt solution. A 10-fold decrease in the number of adherent bacteria was noted for samples exposed to PPP as compared to control samples. The effective range of PPP concentrations capable of producing the marked decrease in binding to fibrin strands was determined to be 1-100% for P. aeruginosa and 4-100% for S. epidermidis.

Magnetic field enhancement of antibiotic activity in biofilm forming Pseudomonas aeruginosa.

Device related infection initiated by biofilm bacteria are often difficult to resolve with antimicrobial therapy. Study results indicate that application of static magnetic fields may enhance the activity of gentamicin against biofilm forming Pseudomonas aeruginosa adherent to a polymer substrate. Results indicate a maximal reduction of 86.5 +/- 7.2% (n = 6) in the number of adherent viable bacteria compared with a control for samples exposed to a 5 gauss (G) magnetic field and gentamicin. The effect appears to be limited to magnetic fields between 5 and 20 G. Experiments using glass, Chronoflex (Polymedica, Golden, CO), Biomer (Ethicon, Somerville, NJ), and polystyrene substrate showed that the effect was independent of substrate surface. Autoradiograms from In111 uptake experiments showed that bacteria colonizing the substrate surface were significantly reduced in samples subjected to a magnetic field and gentamicin.

Physicians' recognition of and response to child abuse: Northern Ireland and the U.S.A.

There is a long history of professional and public concern with the problems of recognizing and reporting child abuse. The research reported here compares recognition of and response to potential abuse by physicians in Northern Ireland and the United States. An experimental study using vignettes in the USA and hospital data, showed that physicians' judgments were moderately affected by ethnic status (black-white) and socioeconomic status of the family, in addition to level of injury to the child. We conducted a comparative study to test whether the social cleavage based on religion (Protestant-Catholic in North Ireland), would affect the recognition of and response to child abuse by medical practitioners in North Ireland. While there was a slight tendency for responses to be affected by socioeconomic status and religion, the results were not statistically significant, as was true for the level of injury to the child. We also compared "diagnostic behavior," signs used to diagnose abuse and causal notions concerning abuse. Medical practitioners in the two countries differ with respect to (1) the amount and type of information desired about a case, and (2) the factors believed to "cause" abuse, but are in general agreement about the "signs" of abuse.