The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFB binding sequences and monitoring tumor necrosis factor ␣-mediated NFB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFB translocation to the nucleus.Because of its easily detected green fluorescence, the green fluorescent protein (GFP) 1 from the jellyfish Aequorea victoria is a widely used reporter in studies of gene expression and protein localization (1-4). GFP fluorescence does not require any substrate or cofactor (5); hence it is possible to use it in many species for live cell detection purposes. The fluorescence of GFPs is dependent on the key sequence Ser-Tyr-Gly (amino acids 65-67). This sequence undergoes spontaneous oxidation to form a cyclized chromophore (6). Enhanced GFP (EGFP) contains mutations of Ser to Thr at amino acid 65 and Phe to Leu at position 64 and is encoded by a gene with humanoptimized codons (7-9). Crystallographic structures of wildtype GFP and the mutant S65T reveal that the GFP tertiary structure resembles a barrel (10, 11). GFP is a single chain polypeptide of 238 amino acids (12). Most of these amino acids form ␤ sheets that are compacted through an antiparallel structure to form the barrel. An ␣-helix containing the chromophore is located inside the barrel, which shields it from the external environment. The compact structure makes GFP very stable under a variety of conditions, including treatment with protease (1). The stability of GFP limits its application in some studies, including transcriptional induction studies.Cellular proteins differ widely in their stabilities. Rapid turnover in proteins is often caused by signals that induce protein degradation. In some cases, the signal is a primary sequence such as the PEST sequence, a sequence possibly correlated with protein degradation (13,14)...

Polyamine-mediated degradation of vertebrate ornithine decarboxylase (ODC) is associated with the production of antizyme, a reversible tightly binding protein inhibitor of ODC activity. The interaction of antizyme with a binding element near the N terminus of ODC is essential but not sufficient for regulation of the enzyme by polyamines (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-2562, 1992. We now show that a second element present at the C terminus is required for the degradation process. Antizyme caused a conformational change in ODC, which made the C terminus of ODC more accessible. Blocking the C terminus with antibody prevented degradation. Tethering the C terminus by creating a circularly permuted, enzymatically active form of ODC prevented antizyme-mediated degradation. These data elucidate a form of feedback regulation whereby excess polyamines induce destruction of ODC, the enzyme that initiates their biosynthesis.Proteins that control important cellular processes are commonly short lived (30). This requires a means of recognizing them and tagging them for destruction. A few protein pairs have been identified, such that the first binds to and apparently targets the second for degradation, e.g., the human papillomavirus oncoprotein E6 and the tumor suppressor p53 (5,33,39). How these interactions direct proteolysis has not been determined. We (18) and others (4,7,15,25,26) have shown that ornithine decarboxylase (ODC), a short-lived enzyme, and antizyme, a tightly binding protein inhibitor of ODC, represent such a pair: antizyme accelerates the intracellular destruction of ODC. This interaction subserves a form of feedback regulation: ODC initiates the synthesis of polyamines, excess polyamines induce the production of antizyme (7, 15), and antizyme both inhibits ODC activity and leads to its destruction (26). Evidence for this chain of events comes from correlations among cellular polyamine pool size, antizyme level, and the rate of ODC turnover (25), and, more recently, the observation that forced expression of cloned antizyme accelerates the turnover of ODC (27). Conversely, if ODC is structurally altered so as to disrupt the site of antizyme binding, the ability of antizyme to inhibit enzymatic activity in vitro is abolished and the enzyme is no longer regulated by polyamines in vivo (18). Although it has been maintained that intracellular polyamines decrease ODC by reducing the translation of its mRNA, we have considered and negated these claims (9,18,38).Mouse ODC is a homodimer of subunits that each contains 461 amino acids. Turnover can proceed along two different pathways, constitutive and polyamine dependent. The C terminus is both necessary and sufficient to make an ODC protein constitutively unstable.

We summarize the development and utilization of organic interfacial materials in solar cells, photodetectors and light-emitting diodes based on organic–inorganic halide perovskites.

The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is a widely used marker for gene expression and protein localization studies. Dissection of the structure of the protein would be expected to shed light on its potential applications to other fields such as the detection of protease activity. Using deletion analysis, we have defined the minimal domain in GFP required for fluorescence to amino acids 7-229. This domain starts at the middle of the first small ␣ helix at the N terminus of GFP and ends immediately following the last ␤ sheet. Studies of the amino acids at both termini of the minimal domain revealed that positions 6 and 7 at the N terminus are Glu-specific. (1996) Nat. Biotechnol. 14, 1246 -1251) that a tightly packed structure exists in the protein. We also generated internal deletions within the loop regions of GFP according to its crystal structure and found that all such deletions eliminated GFP fluorescence. The Green Fluorescent Protein (GFP)1 from the jellyfish Aequorea victoria has been widely used as a reporter in the determination of gene expression and protein localization (1-4). GFP cDNA can be expressed in various cells or organisms with an easily detected fluorescence in the absence of any substrate or cofactor (5). GFP is a 27-kDa, single-chain polypeptide of 238 amino acids (6). A key sequence of Ser-TyrGly at amino acids 65-67 near the N terminus functions as the GFP fluorophore (7). These three amino acids undergo spontaneous oxidation to form a cyclized chromophore responsible for the fluorescence of GFP (8). Enhanced GFP (EGFP) is a mutant of GFP with a 35-fold increase in fluorescence (9 -11). This variant has mutations of Ser to Thr at amino acid 65 and Phe to Leu at position 64 and is encoded by a gene with optimized human codons (10).Recent crystallographic studies of wild-type GFP and the mutant S65T indicate that the GFP tertiary structure resembles a barrel (12, 13). It consists of 11 antiparallel ␤ sheets and a single central ␣ helix surrounded by the ␤ sheets. The chromophore resides in the center of the barrel, completely shielded from the extral environment. There are three small ␣ helices within the protein, one of which is located at the N terminus. Most of the ␤ sheets are connected by small loops of one to four amino acids. Two larger connecting loops, beginning at positions 128 and 188, contain 15 and 9 amino acids, respectively.The compact structure of GFP presumably contributes to its unusual resistance to proteolysis and denaturation. This structure might also explain the observation that few amino acids can be removed from the termini of GFP without affecting its activity. Based on deletion analysis, Dopf and Horiagon (1996) concluded that amino acids 2-232 are the minimal domain required for the fluorescence of wild-type GFP (14). N-terminal deletions of amino acids 2-8 abolished activity. In contrast, the C terminus of GFP is slightly less critical. A deletion of amino acids 233-238 has activity, but the slightly larger deletion of amino a...

A centimeter-sized organic-inorganic hybrid lead-based perovskite CH 3 NH 3 PbI 3 (MAPbI 3 ) single crystal was obtained by using a modified fast and inverse-temperature growth method. The optical properties of this single crystal at room and low temperatures were studied in terms of optical absorption and photoluminescence measurements. The single crystal exhibited optical properties with a band-gap of 1.53 eV, which is comparable to a reported value. The temperature-dependent UV-vis spectra of this perovskite single crystal showed a unique structural phase transition as the temperatures varied. The These thermoelectric characteristics would be useful for potential thermoelectric applications if the electrical conductivity of this crystal is improved by tuning its composition.

A dithienobenzodithiophene‐based π‐conjugated polymer consisting of fluorinated benzotriazole and benzothiadiazole is successfully applied through anti‐solvent method to passivate the defects of perovskite crystals. The fluorinated polymer interacts with under coordinated Pb2+ ions in the perovskite crystals to form Pb‐F bond which effectively passivates the defects. The trap density is reduced and the charge carrier transfer between the perovskite film and Spiro‐OMeTAD is also improved after passivation with the polymer. As a result, a power conversion efficiency (PCE) of 18.03% is achieved in the champion cell. After storing in an ambient environment with 60% relative humidity for 1000 h, the device still retains 90% of the original PCE. These results demonstrate that dithienobenzodithiophene‐based π‐conjugated polymers are promising materials for passivation of perovskite films to further improve the performance and stability of perovskite solar cells.

Selective degradation by proteasomes of ornithine decarboxylase, the initial enzyme in polyamine biosynthesis, is mediated by the polyamine-inducible protein antizyme. Antizyme binds to a region near the N terminus of ornithine decarboxylase (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-3562, 1992). This interaction induces a conformational change in ornithine decarboxylase that exposes its C terminus and inactivates the enzyme (X. Li and P. Coffino, Mol. Cell. Biol. 13:1487-1492). Here we show that the C-terminal half of antizyme alone can inactivate ornithine decarboxylase and alter its conformation, but it cannot direct degradation of the enzyme, either in vitro or in vivo. A portion of the N-terminal half of antizyme must be present to promote degradation.The rapid turnover of proteins is important in regulating cell growth and metabolism. The proteasome is a proteolytic machine that selectively degrades ubiquitinated and nonubiquitinated proteins (6, 26). 20S and 26S forms of the proteasome, each with multiple subunits, have been isolated from eukaryotic cells. The mechanism whereby proteasomes distinguish appropriate substrate proteins is poorly understood (7), although some studies have shown that ubiquitination (9) or exposure of internal structural elements (such as PEST sequences [25]) can provide degradation signals.Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is among the most labile of cellular proteins. It provides a unique system with which to study regulated stability: polyamine levels within cells strongly influence the stability of ODC (18,31). By promoting the destruction of the enzyme, polyamines limit their own accumulation. This form of feedback inhibition utilizes irreversible breakdown of ODC rather than, as in allosteric regulation, reversible inhibition. The polyamine-inducible agent of destruction of ODC is a protein termed antizyme (AZ). AZ has a high affinity for ODC (11). Association of the two entrains the following series of events: ODC becomes catalytically inactive (but activity can still be restored, by dissociation of the complex); the C terminus becomes more accessible (13); in a latter step, this one irreversible and dependent on ATP, ODC is degraded by the 26S proteasome (21).A more comprehensive description of this process requires an understanding of the structural elements of both proteins that participate in their association, the effect of that association on (12). A second, at the C terminus of ODC, is required for AZ-mediated degradation (3, 4, 13). This region is made more readily reactive with C-specific antibody when AZ is present. The C terminus is not required for the enzymatic activity of ODC (16). Furthermore, truncation of the C terminus does not prevent the binding of AZ nor abolish one functional consequence of AZ binding: reversible inhibition of enzymatic activity (12). Functional studies of the mutant forms of ODC were greatly facilitated by the use of an in vitro system derived from rabbit reticulocytes in which degradatio...

No abstract