Unmasking of Proteins by in Vitro Methylation of Rat Liver Rna During Azo Dye Carcinogenesis

In liver parenchyma of rats fed the azo dye 4-dimethylaminoazobenzene cytoplasmic ribonucleic acid (RNA) stains intensely with toluidine blue in sites of neoplastic transformation and hepatomas. With the mercury-bromphenol blue method, the staining intensity of proteins was found to be much weaker in these areas than in surrounding tissue. A 4 hr treatment at 60 C in a mixture of methanol and HCI completely abolished the increased staining of RNA with toluidine blue, but restored protein stainability with bromphenol blue in preneoplastic foci and tumors; the staining intensity of proteins was then comparable to that in surrounding liver. These results suggest that proteins were masked by the RNA responsible for hyperbasophilia, and that in vitro addition of methyl groups to such RNA is equal in effect to RNase extraction which was previously found to unmask cytoplasmic proteins.     

Detection in milk of a 60,000 Mr mouse and a 68,000 Mr human phosphorylated glycoprotein by histochemical analysis on polyacrylamide gels

Proteins in colostrum and skimmed milk from humans and mice were separated by electrophoresis on polyacrylamide gels and stained with Coomassie blue (CB), Ethyl-Stains-all (ESA), and periodic acid-Schiff (PAS) to investigate changes that may occur in milks throughout lactation. In mouse colostrum but not in mature mouse milk, a PAS-positive protein of apparent molecular weight of 60,000 stained prominently blue with ESA. A protein in human milk with a molecular weight of 68,000 stained similarly but was present throughout lactation. The intensity of blue staining of these minor proteins in milk approached that obtained with casein phosphoproteins. The metachromatic dye ESA stains phosphoproteins and sialic acid-rich glycoproteins blue to blue-green. Removal of phosphorus from the former and sialic acid from the latter results in those proteins staining red with ESA. The intensity of blue staining of the 60,000 and 68,000 Mr proteins was diminished but not lost following treatment with phosphatase. It was eliminated following neuraminidase digestion of the mouse protein and mild acid hydrolysis of the human protein. Coomassie blue staining of the proteins was not affected by these procedures. Following electrophoresis of milk and milk fractions in a non-sodium dodecyl sulfate-containing system, the proteins were identified by their characteristic staining properties with ESA and isolated.     

Advantages and disadvantages of silver staining of proteins in polyacrylamide gel

Sensitivity of protein staining with Serva blue G-250 (Coomassie brilliant blue G-250 analogue) in polyacrylamide gel was determined. It has been shown that protein staining with 0.1% Serva blue G-250 results in the recovery of 80 to 35 ng of single protein, that is almost 10 times higher than reported previously for Coomassie brill. blue G-250 (or R-250) staining. The comparison of the sensitivity of Serva blue G-250 protein staining in PAAG and AgNO3 has shown that AgNO3 staining was approximately 18-30 (but not 100 times, as it had been thought before) times more effective for the majority of proteins under study. Silver staining of some proteins, for instance ribonuclease and a number of retrovirus-specific structural proteins, was of lower efficacy. Thus, to obtain reliable results protein electrophoresis in PAAG should be followed by both staining procedures.     

Blue copper proteins: a comparative analysis of their molecular interaction properties

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (I) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.     

Direct visualization of collagens and procollagens in polyacrylamide gels.

This report describes a procedure that results in the rapid visual identification of collagens and procollagens in polyacrylamide gels. The technique results in a pink stain for collagenous proteins and a blue stain for all other proteins. The color difference has been evaluated spectrophotometrically. The absorbance maxima for collagen-Coomassie blue R250 complexes in gels is 520–535 nm, and the maxima for all other protein-Coomassie blue R250 complexes that we tested is 550–560 nm. This technique will facilitate the identification of collagenous proteins in complex mixtures of proteins derived from cell membranes, whole cell extracts, conditioned media, and extracellular matrices. We use the technique to detect procollagens in human diploid fibroblast conditioned media. The technique is simple, relatively rapid, has utility for proteins extracted by a variety of methods, and is applicable to all polyacrylamide gel systems in general use.     

Improved Blue,Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae

Fluorescent protein fusions are a powerful tool to monitor the localization and trafficking of proteins. Such studies are particularly easy to carry out in the budding yeast Saccharomyces cerevisiae due to the ease with which tags can be introduced into the genome by homologous recombination. However, the available yeast tagging plasmids have not kept pace with the development of new and improved fluorescent proteins. Here, we have constructed yeast optimized versions of 19 different fluorescent proteins and tested them for use as fusion tags in yeast. These include two blue, seven green, and seven red fluorescent proteins, which we have assessed for brightness, photostability and perturbation of tagged proteins. We find that EGFP remains the best performing green fluorescent protein, that TagRFP-T and mRuby2 outperform mCherry as red fluorescent proteins, and that mTagBFP2 can be used as a blue fluorescent protein tag. Together, the new tagging vectors we have constructed provide improved blue and red fluorescent proteins for yeast tagging and three color imaging.     

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins regulate gibberellin signaling and photomorphogenesis in Arabidopsis

Cryptochromes are blue light photoreceptors that mediate various light responses in plants and mammals. In Arabidopsis (Arabidopsis thaliana), cryptochrome 1 (CRY1) mediates blue light-induced photomorphogenesis, which is characterized by reduced hypocotyl elongation and enhanced anthocyanin production, whereas gibberellin (GA) signaling mediated by the GA receptor GA-INSENSITIVE DWARF1 (GID1) and DELLA proteins promotes hypocotyl elongation and inhibits anthocyanin accumulation. Whether CRY1 control of photomorphogenesis involves regulation of GA signaling is largely unknown. Here, we show that CRY1 signaling involves the inhibition of GA signaling through repression of GA-induced degradation of DELLA proteins. CRY1 physically interacts with DELLA proteins in a blue light-dependent manner, leading to their dissociation from SLEEPY1 (SLY1) and the inhibition of their ubiquitination. Moreover, CRY1 interacts directly with GID1 in a blue light-dependent but GA-independent manner, leading to the inhibition of the interaction between GID1 with DELLA proteins. These findings suggest that CRY1 controls photomorphogenesis through inhibition of GA-induced degradation of DELLA proteins and GA signaling, which is mediated by CRY1 inhibition of the interactions of DELLA proteins with GID1 and SCF^SLY1, respectively.

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins inhibit gibberellin (GA)-induced degradation of DELLA proteins to regulate GA signaling and photomorphogenesis.     

Blue, red and blue plus red light control of chloroplast pigment and pigment-proteins in corn mesophyll cells: Irradiance level-quality interaction

Corn ( Zea mays L. cv. OP Golden Bantum) was grown under low irradiance blue, red or blue plus red light. Red was more effective than blue light for synthesis of Chl a, b and light-harvesting proteins (LHC-2) associated with photosystem 2(PS2). Blue light was slightly more effective for synthesis of light-harvesting proteins (LHC-1) associated with photosystem 1 (PS1), but below a fluence rate of 1 μmol m⁻² s⁻¹ the response to blue vs that to red depended on irradiance level. Blue light containing a small amount of red light was as effective as red light for Chl a and b synthesis, but no more effective than blue light for LHC-2 synthesis. Adding small amounts of blue light to red repressed the effect of red light on LHC-2 synthesis and produced irradiance response curves similar to those produced by blue alone for LHC-2 synthesis. This repression by blue light depended on the ratio of red to blue and the level of the blue light.     

PAS/LOV proteins: A proposed new class of plant blue light receptor

The light, oxygen or voltage (LOV) domain belongs to the Per-ARNT-Sim (PAS) superfamily of domains, and functions with the flavin chromophore as a module for sensing blue light in plants and fungi. The Arabidopsis thaliana PAS/LOV proteins (PLPs), of unknown function, possess an N-terminal PAS domain and a C-terminal LOV domain. Our recent analysis using yeast two-hybrid and Escherichia coli protein production systems reveals that the interactions of Arabidopsis PLPs with several proteins diminish under blue light illumination and that the PLP LOV domain may bind to a flavin chromophore. These results suggest that PLP functions as a blue light receptor. Homologs of PLP exist in rice, tomato and moss. The LOV domains of these PLP homologs form a distinct group in phylogenetic analysis. These facts suggest that PLP belongs to a new class of plant blue light receptor.Key words: PAS, LOV, blue light, protein-protein interaction, photoreceptor     

The influence of axial ligands on the reduction potential of blue copper proteins

 The reduction potentials of blue copper sites vary between 180 and about 1000 mV. It has been suggested that the reason for this variation is that the proteins constrain the distance between the copper ion and its axial ligands to different values. We have tested this suggestion by performing density functional B3LYP calculations on realistic models of the blue copper proteins, including solvent effects by the polarizable continuum method. Constraining the Cu-S_Met bond length to values between 245 and 310 pm (the range encountered in crystal structures) change the reduction potential by less than 70 mV. Similarly, we have studied five typical blue copper proteins spanning the whole range of reduction potentials: stellacyanin, plastocyanin, azurin, rusticyanin, and ceruloplasmin. These studies included the methionine (or glutamine) ligand as well as the back-bone carbonyl oxygen group that is a ligand in azurin and is found at larger distances in the other proteins. The active-site models of these proteins show a variation in the reduction potential of about 140 mV, i.e., only a minor part of the range observed experimentally (800 mV). Consequently, we can conclude that the axial ligands have a small influence on the reduction potentials of the blue copper proteins. Instead, the large variation in the reduction potentials seems to arise mainly from variations in the solvent accessibility of the copper site and in the orientation of protein dipoles around the copper site. Received: 7 April 1999 / Accepted: 26 July 1999     

Dimerization and blue light regulation of PIF1 interacting bHLH proteins in Arabidopsis

Phytochrome Interacting Factor 1 (PIF1), a basic helix-loop-helix (bHLH) protein, functions as a negative regulator of various facets of photomorphogenesis. To indentify PIF1-interacting proteins, we performed yeast two-hybrid screening using PIF1 as a bait and identified a group of proteins including PIF1 itself, PIF3 and long hypocotyl in far-red 1 (HFR1), an atypical HLH protein. Directed yeast two-hybrid interaction assays showed that PIF1 can form heterodimers with all other PIFs as well as with HFR1. PIF1 and PIF3 interacted with each other in both in vitro and in vivo co-immunoprecipitation assays. PIF1-PIF3 heterodimer also bound to a G-box DNA sequence element in vitro. To understand the biological significance of these interactions, a pif1pif3 double mutant was obtained and characterized. Analyses of the single and double mutants showed that PIF3 plays a prominent role in repressing photomorphogenesis under continuous blue light conditions. pif1 and pif3 showed additive phenotypes more prominently under discontinuous blue light conditions. Similar to PIF1, PIF3 was also rapidly phosphorylated, poly-ubiquitylated and degraded in response to blue light. PIF3 also interacted with phytochromes in response to blue light. A PIF3 mutant defective in interaction with both phyA and phyB displayed reduced degradation under blue light, suggesting that phy-interaction was necessary for the blue light-induced degradation of PIF3. Taken together, these data suggest a combinatorial control of photomorphogenesis by bHLH proteins in response to light in Arabidopsis.     

Alcian blue as a protein stain for sodium alkyl sulfate-polyacrylamide gels: Application to analysis of polypeptide components of the human platelet

The cationic dye, Alcian blue, previously used as a glycoprotein-specific stain on cellulose acetate and polyacrylamide gels, was found to be capable of staining a variety of purified proteins and each of the components of the human platelet presently identifiable with Coomassie blue R or periodic acid-Schiff (PAS) reagent in sodium alkyl sulfate-polyacrylamide gel electrophoretic preparations. Evidence was obtained to indicate that staining of detergent-protein complexes by Alcian blue occurs by virtue of the affinity of the stain for accessible sulfate groups of detergent molecules, especially sodium tetradecyl sulfate, hydrophobically associated with polypeptide chains. Thus, Alcian blue fails to stain nonglycosylated proteins when pure sodium dodecyl sulfate (C₁₂) is used as the detergent, but does so readily when small quantities of sodium tetradecyl sulfate are also present. The advantages of using Alcian blue to determine platelet protein composition and to make quantitative comparisons between bands in sodium alkyl sulfate gels are discussed.     

A faithful double stain of proteins in the polyacrylamide gels with Coomassie blue and silver

The conditions for prior fixing of proteins in a gel in order to attain a greater degree of faithful silver staining and sensitivity were examined. Fixing with formaldehyde enhanced the retention of proteins in a gel, particularly basic proteins such as histones and ribosomal proteins. The gel, one stained with Coomassie blue and following the removal of the free dyes, is capable of undergoing silver staining, and, moreover, the prestain considerably enhanced the staining intensity of various proteins differing in basicity in subsequent silver staining. Coupling the formaldehyde fixation with Coomassie brilliant blue prestain afforded a reproducible and pronounced stainability of various proteins.     

The effect of staining on the immunoreactivity of nitrocellulose bound proteins

It was found that Coomassie blue staining of proteins electroblotted onto nitrocellulose resulted in a significant decrease in subsequent immunoreactivity, relative to unstained proteins. This was not uniform, and resulted in distortion of the pattern detected by immunoreaction as well as an overall decrease in sensitivity. Staining with amido black resulted in no such alteration. Additionally, it was observed that amido black is completely leached from the nitrocellulose strip during immunoreaction, whereas no leaching is apparent with Coomassie blue. This may contribute to the observed effect of staining on immunoreactivity.     

Studies and critique of Amido Black 10B, Coomassie Blue R, and Fast Green FCF as stains for proteins after polyacrylamide gel electrophoresis.

The properties of amido black 10B (C.I. 20470), Coomassie blue R (C.I. 42660), and fast green FCF (C.I. 42053) as protein stains, along with a few comments on Coomassie blue G (C.I. 42655), are presented and dye impurities and their effects on protein-dye binding within gels are discussed. All three dyes produced metachromatic effects with some proteins. Problems encountered with long-term stability and fixation of certain maize seed proteins are reported along with procedures for overcoming them. The low solubility of Coomassie blue R in trichloroacetic acid prevented maximum staining and destaining within a reasonable time, whereas other solvents allowed diffusion of some proteins during staining. Coomassie blue R binds to proteins in much higher amounts than do amido black and fast green, which accounts for its sensitivity in detection of protein bands in gels. Procedures for obtaining maximum contrast with photographs are also outlined.     

Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins

The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. Herein we report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein "toolkit".     

Practical three color live cell imaging by widefield microscopy

Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope.