Changes in the redox equilibrium of cells influence a host of cell functions. Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator. Ratios of fluorescence from excitation at 400 and 490 nm indicate the extent of oxidation and thus the redox potential while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 (GFP with mutations C48S, S147C, and Q204C) and roGFP2 (the same plus S65T) with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H 2 O 2 -stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid.Cells have elaborate homeostatic mechanisms to regulate the thiol-disulfide redox status of their internal compartments. Most thiol groups within the cytoplasm are normally reduced. Very few are present as disulfides. It has been speculated that the cytoplasm is reducing because many metabolic reactions evolved before oxygen became abundant in the atmosphere (1). Modest alterations in the thiol-disulfide equilibrium could have major consequences in the cell, including defective protein folding or enzyme activity (because many enzymes have a cysteine in their active site). When excess oxidation overwhelms the reductive capabilities of the cell, death results. Despite the dangers of excessive oxidation, cells sometimes use redox adjustments as signaling events, such as in the activation of transcription factors (NF-B and AP-1), caspases, protein tyrosine phosphatases, or GTPases (Ras). Thus, changes in the redox equilibrium influence a host of cell functions, including but not limited to growth, stress responses, differentiation, metabolism, cell cycle, communication, migration, gene transcription, ion channels, and immune responses (for reviews see Refs. 2-6). Alterations in the redox equilibrium are reflected in changes of the glutathione/glutathione-disulfide ratio (GSH/ GSSG) and the reduced/oxidized thioredoxin ratio. Glutathione is found in high concentrations in cells (5-10 mM) and is considered to be the major player in maintaining intracellular redox equilibrium. Ratios of GSH to GSSG are reported to range from 100 to 300:1 (7, 8), but these measurements have been problematic because they require destruction of the tissue, during which great care must be taken not to allow further oxidation. The major source of error is the determination of GSSG concentration, because this species is at low abundance yet is ...

B lymphocytes use somatic hypermutation (SHM) to optimize immunoglobulins. Although SHM can rescue single point mutations deliberately introduced into nonimmunoglobulin genes, such experiments do not show whether SHM can efficiently evolve challenging novel phenotypes requiring multiple unforeseeable mutations in nonantibody proteins. We have now iterated SHM over 23 rounds of fluorescence-activated cell sorting to create monomeric red fluorescent proteins with increased photostability and far-red emissions (e.g., 649 nm), surpassing the best efforts of structure-based design. SHM offers a strategy to evolve nonantibody proteins with desirable properties for which a high-throughput selection or viable single-cell screen can be devised. directed evolution ͉ mPlum ͉ Ramos ͉ red fluorescent protein

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Gas-phase acidities (GA or ΔG acid) of acidic di- and tripeptides are determined for the first time. The peptides studied are composed of inert alanine (A) residues and one X residue of either aspartic acid (D) or glutamic acid (E): AX, XA, AAX, AXA, and XAA. Experimental GAs were measured by the thermokinetic method of deprotonation ion/molecule reactions in a Fourier transform ion cyclotron resonance mass spectrometer. Calculated GAs were obtained by composite correlated molecular orbital theory at the G3­(MP2) level for deprotonation of carboxylic acid groups both at the C-terminus and at the side chain. Excellent agreement was found between experimental and calculated GA values. There is a slight preference for peptides with D being more acidic than analogous peptides with E, which agrees with the GAs of the corresponding amino acids. Experiments showed that peptides are more acidic (lower numerical GA values) when the acidic residue is located at the C-terminus (i.e., AX or AAX). The lowest energy form of deprotonated AAE has a unique structure where the longer side chain of E allows the two carboxylates, which are in close proximity, to share the proton. The tripeptides are less acidic (higher GA value) by 3–7 kcal/mol when the acidic residue is in the center. The tripeptides are more acidic (by 2–10 kcal/mol) than dipeptides containing the same acidic residue at the same location.