Robert D. Litwiller

Bio: Robert D. Litwiller is an academic researcher from University of Rochester. The author has contributed to research in topics: Protein subunit & Calcineurin. The author has an hindex of 5, co-authored 7 publications receiving 175 citations.

Calcineurin subunit interactions: Mapping the calcineurin B binding domain on calcineurin A

Abstract: Recombinant forms of the A and B subunits of the protein phosphatase calcineurin were produced in Escherichia coli, reconstituted into a heterodimer and purified to homogeneity. The reconstituted heterodimer exhibited properties like that of bovine brain calcineurin. This included calmodulin-stimulated activity and a subunit stoichiometry and Stokes radius consistent with native-like structure. In order to map the region on the A subunit where calcineurin B binds, a series of overlapping 20-residue peptides corresponding to this putative domain were synthesized. Using isolated calcineurin A and B subunits, an assay that relied upon peptide inhibition of calcineurin B stimulation of calcineurin A activity was developed. All five peptides, but not a control peptide, inhibited calcineurin B-dependent stimulation of calcineurin A although with different potencies. The three most effective inhibitory peptides spanned calcineurin A residues 338-377. These three peptides also altered the electrophoretic mobility of the isolated calcineurin B subunit during native polyacrylamide gel electrophoresis indicating a direct interaction between these peptides and calcineurin B. The peptide corresponding to residues 348-367 was also able to block binding of calcineurin B to the catalytic subunit.

Calcineurin : form and function

Abstract: Calcineurin is a eukaryotic Ca2+- and calmodulin-dependent serine/threonine protein phosphatase. It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, which contains an act...

Studying noncovalent protein complexes by electrospray ionization mass spectrometry

Abstract: Electrospray ionization mass spectrometry has been used to study protein interactions driven by noncovalent forces The gentleness of the electrospray ionization process allows intact protein complexes to be directly detected by mass spectrometry Evidence from the growing body of literature suggests that the ESI-MS observations for these weakly bound systems reflect, to some extent, the nature of the interaction found in the condensed phase Stoichiometry of the complex can be easily obtained from the resulting mass spectrum because the molecular weight of the complex is directly measured For the study of protein interactions, ESI-MS is complementary to other biophysical methods, such as NMR and analytical ultracentrifugation However, mass spectrometry offers advantages in speed and sensitivity The experimental variables that play a role in the outcome of ESI-MS studies of noncovalently bound complexes are reviewed Several applications of ESI-MS are discussed, including protein interactions with metal ions and nucleic acids and subunit protein structures (quaternary structure)

Superoxide dismutases and superoxide reductases

Abstract: Superoxide, O2•–, is formed in all living organisms that come in contact with air, and, depending upon its biological context, it may act as a signaling agent, a toxic species, or a harmless intermediate that decomposes spontaneously Its levels are limited in vivo by two different types of enzymes, superoxide reductase (SOR) and superoxide dismutase (SOD) Although superoxide has long been an important factor in evolution, it was not so when life first emerged on Earth at least 35 billion years ago At that time, the early biosphere was highly reducing and lacking in any significant concentrations of dioxygen (O2), very different from what it is today Consequently, there was little or no O2•– and therefore no reason for SOR or SOD enzymes to evolve Instead, the history of biological O2•– probably commences somewhere around 24 billion years ago, when the biosphere started to experience what has been termed the “Great Oxidation Event”, a transformation driven by the increase in O2 levels, formed by cyanobacteria as a product of oxygenic photosynthesis1 The rise of O2 on Earth caused a reshaping of existing metabolic pathways, and it triggered the development of new ones2 Its appearance led to the formation of the so-called “reactive oxygen species” (ROS), for example, superoxide, hydrogen peroxide, and hydroxyl radical, and to a need for antioxidant enzymes and other antioxidant systems to protect against the growing levels of oxidative damage to living systems Dioxygen is a powerful four-electron oxidizing agent, and the product of this reduction is water 1 When O2 is reduced in four sequential one-electron steps, the intermediates formed are the three major ROS, that is, O2•–, H2O2, and HO• 2 3 4 5 Each of these intermediates is a potent oxidizing agent The consequences of their presence to early life must have been an enormous evolutionary challenge In the case of superoxide, we find the SOD and SOR enzymes to be widely distributed throughout current living organisms, both aerobic and anaerobic, suggesting that, from the start of the rise of O2 on Earth, the chemistry of superoxide has been an important factor during evolution The SORs and three very different types of SOD enzymes are redox-active metalloenzymes that have evolved entirely independently from one another for the purpose of lowering superoxide concentrations SORs catalyze the one-electron reduction of O2•– to give H2O2, a reaction requiring two protons per superoxide reacted as well as an external reductant to provide the electron (eq 6) SODs catalyze the disproportionation of superoxide to give O2 and H2O2, a reaction requiring one proton per superoxide reacted, but no external reductant (eq 7) 6 7 All of the SOR enzymes contain only iron, while the three types of SODs are the nickel-containing SODs (NiSOD), the iron- or manganese-containing SODs (FeSOD and MnSOD), and the copper- and zinc-containing SODs (CuZnSOD) Although the structures and other properties of these four types of metalloenzymes are quite different, they all share several characteristics, including the ability to react rapidly and selectively with the small anionic substrate O2•– Consequently, there are some striking similarities between these otherwise dissimilar enzymes, many of which can be explained by considering the nature of the chemical reactivity of O2•– (see below) Numerous valuable reviews describing the SOD and SOR enzymes have appeared over the years, but few have covered and compared all four classes of these enzymes, as we attempt to do here Thus, the purpose of this Review is to describe, compare, and contrast the properties of the SOR and the four SOD enzymes; to summarize what is known about their evolutionary pathways; and to analyze the properties of these enzymes in light of what is known of the inherent chemical reactivity of superoxide