Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway.

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.

A photoswitchable orange-to-far-red fluorescent protein, PSmOrange.

We report a photoswitchable monomeric Orange (PSmOrange) protein that is initially orange (excitation, 548 nm; emission, 565 nm) but becomes far-red (excitation, 636 nm; emission, 662 nm) after irradiation with blue-green light. Compared to its parental orange proteins, PSmOrange has greater brightness, faster maturation, higher photoconversion contrast and better photostability. The red-shifted spectra of both forms of PSmOrange enable its simultaneous use with cyan-to-green photoswitchable proteins to study four intracellular populations. Photoconverted PSmOrange has, to our knowledge, the most far-red excitation peak of all GFP-like fluorescent proteins, provides diffraction-limited and super-resolution imaging in the far-red light range, is optimally excited with common red lasers, and can be photoconverted subcutaneously in a mouse. PSmOrange photoswitching occurs via a two-step photo-oxidation process, which causes cleavage of the polypeptide backbone. The far-red fluorescence of photoconverted PSmOrange results from a new chromophore containing N-acylimine with a co-planar carbon-oxygen double bond.

Monomeric red fluorescent proteins with a large Stokes shift.

Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463/624 and 460/605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 mum the breast cancer cells show significant polarization towards vessels in living mice.

Impact of benzo[a]pyrene-2'-deoxyguanosine lesions on methylation of DNA by SssI and HhaI DNA methyltransferases.

DNA damage caused by the binding of the tumorigen 7R,8S-diol 9S,10R-epoxide (B[a]PDE), a metabolite of bezo[a]pyrene, to guanine in CpG dinucleotide sequences could affect DNA methylation and, thus, represent a potential epigenetic mechanism of chemical carcinogenesis. In this work, we investigated the impact of stereoisomeric (+)- and (-)-trans-anti-B[a]P-N(2)-dG adducts (B(+) and B(-)) on DNA methylation by prokaryotic DNA methyltransferases M.SssI and M.HhaI. These two methyltransferases recognize CpG and GCGC sequences, respectively, and transfer a methyl group to the C5 atom of cytosine (C). A series of 18-mer unmethylated or hemimethylated oligodeoxynucleotide duplexes containing trans-anti-B[a]P-N(2)-dG adducts was generated. The B(+) or B(-) residues were introduced either 5' or 3' adjacent or opposite to the target 2'-deoxycytidines. The B[a]PDE lesions practically produced no effect on M.SssI binding to DNA but reduced M.HhaI binding by 1-2 orders of magnitude. In most cases, the benzo[a]pyrenyl residues decreased the methylation efficiency of hemimethylated and unmethylated DNA by M.SssI and M.HhaI. An absence of the methylation of hemimethylated duplexes was observed when either the (+)- or the (-)-trans-anti-B[a]P-N(2)-dG adduct was positioned 5' to the target dC. The effects observed may be related to the minor groove conformation of the bulky benzo[a]pyrenyl residue and to a perturbation of the normal contacts of the methyltransferase catalytic loop with the B[a]PDE-modified DNA. Our results indicate that a trans-anti-B[a]P-N(2)-dG lesion flanking a target dC in the CpG dinucleotide sequence on its 5'-side has a greater adverse impact on methylation than the same lesion when it is 3' adjacent or opposite to the target dC.

2-Pyrimidinone as a probe for studying the EcoRII DNA methyltransferase-substrate interaction.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the 5' em leader CC*T/AGG em leader 3' DNA sequence and catalyzes the transfer of the methyl group from S-adenosyl-l-methionine to the C5 position of the inner cytosine residue (C*). Here, we study the mechanism of inhibition of M.EcoRII by DNA containing 2-pyrimidinone, a cytosine analogue lacking an NH(2) group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2-Pyrimidinone was incorporated into the 5' em leader CCT/AGG em leader 3' sequence replacing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal denaturation of 2-pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2-pyrimidinone and to a greater destabilizing influence of 2-pyrimidinone substitution for thymine than that for cytosine. Binding of M.EcoRII to 2-pyrimidinone containing DNA and methylation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the DNA-M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.EcoRII. In the absence of the cofactor S-adenosyl-l-methionine, proton transfer to the C5 position of 2-pyrimidinone occurs and in the presence of S-adenosyl-l-methionine, methyl transfer to the C5 position of 2-pyrimidinone occurs.

DNA duplexes containing photoactive derivatives of 2'-deoxyuridine as photocrosslinking probes for EcoRII DNA methyltransferase-substrate interaction.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.