Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(II) and manganese(II) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use Fe[superscript II] as a Lewis acid under normal growth conditions but which switch to Mn[superscript II] under oxidative stress; (2) extradiol dioxygenases, which have been found to use both Fe[superscript II] and Mn[superscript II], the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, “discrimination” between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.National Institutes of Health (U.S.) (Grant GM81393

From Waste to Wares: Upcycling Plastic Bags for Relief, Aid, and Development

University of Minnesota master's thesis. Summer 2012. Degree: Master of Liberal Studies. Advisor: Tim Smith. 1 digital file (pdf)Upcycling plastic bags may be a viable method to provide basic necessities for people living in poverty. I created knitted and fused designs from plastic bags including; ropes, shoes, tarps, rainwater catchers, toilet seats, neonatal warming bags, fishing nets, and construction webbing. Plastic bags are ubiquitous in every part of the world, but particularly in the developing world where more than 40% of the population lives on less than $2.50 per day. Plastic bags are an environmental and health hazard, but they are also a useful raw material. Using simple knitting and fusing methods, it is possible to turn plastic shopping bags into useful items for relief, aid, and development needs

Biochemical and biophysical characterization of the manganese transport regulator (MntR) from Bacillus subtilis

Metal ions are employed in biology for several reasons including their ability to participate in redox chemistry, catalysis, and structural stabilization of proteins. However, the properties that make metal ions so widely utilized in biology can be potentially hazardous, particularly if abnormal quantities of these ions are accumulated. This necessitates a mechanism by which the balance between uptake of essential metal ions and efflux of excess essential or toxic metal ions, otherwise referred to as metal homeostasis, can be maintained. Bacteria employ a unique set of metal responsive transcription factors (metalloregulators) to manage this delicate balance. The biochemical and biophysical characterization of MntR, a manganese responsive regulator from the DtxR family is the focus of this thesis. Fluorescence anisotropy was used to probe the DNA-binding of wild type MntR, MntR D8M, and MntR E99C mutants to the cognate DNA recognition sequences mntH and mntA in the presence of various divalent metal ions. Our studies demonstrate the extent to which these metal ions are able to activate MntR to bind DNA. In addition, these studies shed light on the origin of metal specificity between MntR and DtxR and are in agreement with in vivo data reported in the literature. In addition to investigating the DNA- binding abilities of MntR, we also examined the metal binding affinities of this protein in order explain how it fits into the DtxR family and the general field of metalloregulatory proteins. The results demonstrate that MntR metal-binding affinities loosely follow the Irving- Williams series. Interestingly, the protein exhibits the weakest affinity for one of its cognate metal ions. Finally, the metal-mediated mechanism of DNA binding by MntR was studied. Initial investigations using circular dichroism and an environmentally-sensitive dye ANS showed that metal binding stabilizes either tertiary or quaternary structure of MntR. Subsequent studies focused on localizing these structural changes using deuterium exchange mass spectrometry (DXMS) and demonstrated that metal-binding serves to rigidify the pre-organized structure of MntR. Moreover, contrary to typical observation of transcription factors, cofactor (metal) binding does not appear to alter the structure of helix- turn-helix DNA-binding moti

Antibody detection using a FRET-based protein conformational switch

No wash, just go: Classical antibody-detection methods rely on heterogeneous detection schemes that involve multiple, time-consuming binding and washing steps. Here we present a new concept to translate the antigen–antibody interaction directly into a readily detectable fluorescent signal by using a single-chain sensor protein and taking advantage of the unique Y-shaped structure common to all antibodie

Rational design of FRET sensor proteins based on mutually exclusive domain interactions

Proteins that switch between distinct conformational states are ideal to monitor and control molecular processes within the complexity of biological systems. Inspired by the modular architecture of natural signalling proteins, our group explores generic design strategies for the construction of FRET-based sensor proteins and other protein switches. In the present article, I show that designing FRET sensors based on mutually exclusive domain interactions provides a robust method to engineer sensors with predictable properties and an inherently large change in emission ratio. The modularity of this approach should make it easily transferable to other applications of protein switches in fields ranging from synthetic biology, optogenetics and molecular diagnostics

Engineering protein switches : sensors, regulators, and spare parts for biology and biotechnology

Switch protein switch! Proteins that switch between distinct conformational states are ideal for monitoring and controlling molecular processes in biological systems. We discuss new engineering concepts for the construction of protein switches that have the potential to be generally applicable and discuss them according to their mechanism of action

Rational design of FRET sensor proteins based on mutually exclusive domain interactions

Abstract Proteins that switch between distinct conformational states are ideal to monitor and control molecular processes within the complexity of biological systems. Inspired by the modular architecture of natural signalling proteins, our group explores generic design strategies for the construction of FRET-based sensor proteins and other protein switches. In the present article, I show that designing FRET sensors based on mutually exclusive domain interactions provides a robust method to engineer sensors with predictable properties and an inherently large change in emission ratio. The modularity of this approach should make it easily transferable to other applications of protein switches in fields ranging from synthetic biology, optogenetics and molecular diagnostics