Comparison of femtosecond laser and continuous wave UV sources for protein-nucleic acid crosslinking

Crosslinking proteins to the nucleic acids they bind affords stable access to otherwise transient regulatory interactions. Photochemical crosslinking provides an attractive alternative to formaldehyde-based protocols, but irradiation with conventional UV sources typically yields inadequate product amounts. Crosslinking with pulsed UV lasers has been heralded as a revolutionary technique to increase photochemical yield, but this method had only been tested on a few protein-nucleic acid complexes. To test the generality of the yield enhancement, we have investigated the benefits of using approximately 150 fs UV pulses to crosslink TATA-binding protein, glucocorticoid receptor and heat shock factor to oligonucleotides in vitro. For these proteins, we find that the quantum yields (and saturating yields) for forming crosslinks using the high-peak intensity femtosecond laser do not improve on those obtained with low-intensity continuous wave (CW) UV sources. The photodamage to the oligonucleotides and proteins also has comparable quantum yields. Measurements of the photochemical reaction yields of several small molecules selected to model the crosslinking reactions also exhibit nearly linear dependences on UV intensity instead of the previously predicted quadratic dependence. Unfortunately, these results disprove earlier assertions that femtosecond pulsed laser sources provide significant advantages over CW radiation for protein-nucleic acid crosslinking.     

UV-Induced Nucleic Acid–Protein Cross-Linking: Manual on Planning of Irradiation Experiments and Calculation of Absorbed Dose and Quantum Yield

We have developed methods of photochemical quantitation of photobiological studies on UV-induced nucleic acid–protein cross-linking. Cases relating to incoherent low-intensity UV sources, laser UV sources and high-intensity laser UV sources are considered. In the case of low-intensity UV radiation the most important point is the correct determination of absorbed dose. The laser UV pulse energy is easily measured and the short-pulse irradiation also has the advantage of "freezing" the conformation of complexes under study. However, the use of high-intensity laser UV irradiation leads to realizations of two-quantum processes both in nucleic acid chromophores–bases and in solvent–water, which complicates singificantly the processing of results. In this paper methods for calculating the absorbed dose and the quantum yield of cross-linking for all above-mentioned cases are given as well as practical advice.     

PHOTOLYSIS OF THE SINGLE TRYPTOPHAN RESIDUE OF EEL TROPONIN C

Abstract— The tryptophan (TRP) residue of eel troponin C was selectively degraded by direct UV irradiation, at 302 nm, in Ar-saturated solution. Depending on the absence or presence of calcium ions, this TRP residue is exposed to aqueous medium or buried in a hydrophobic environment. Tryptophan loss was determined by both absorption and fluorescence spectroscopy and by amino acid analysis. The photodegradation yield was significantly higher for the exposed TRP residue than for the buried ones. These results give more detail on previous observations on several other proteins and corroborate the predominant influence of the polarity on the photosensitivity of a TRP residue in polypeptidic structures.     

Investigation of methods suitable for the matrix-assisted laser desorption/ionization mass spectrometric analysis of proteins from ribonucleoprotein complexes

A variety of protein isolation and purification techniques for ribonucleoprotein (RNP) complexes were investigated for their compatibility with downstream analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ribosomal proteins from Escherichia coli 70S ribosomes were obtained using methods such as phenol extraction and precipitation by organic solvents or acids. Under optimal conditions, more than 90% of the expected ribosomal proteins were detected in a single MALDI-MS experiment. The most effective approach combined ribosome denaturation by buffer exchange with acid precipitation of the ribosomal ribonucleic acids. An improved acid precipitation approach, involving the sequential additions of acetic and trifluoroacetic acid, yielded more complete protein coverage while minimizing loss of ion signal from lower molecular weight proteins. With phenol extraction, substantial gains in ion abundance of higher molecular weight proteins are noted, although some of the lower molecular weight proteins were not efficiently extracted. These results illustrate several effective approaches for protein isolation from protein complexes such as RNPs that are MALDI-MS compatible, and these approaches should extend the use of MALDI-MS for proteomics-based analyses of other protein-nucleic acid complexes.     

FORMATION OF PROTEIN-BOUND 3,4-DIHYDROXYPHENYLALANINE IN COLLAGEN TYPES I AND IV EXPOSED TO ULTRAVIOLET LIGHT

Abstract— Collagen was exposed to an ultraviolet (UV) lamp that emitted predominantly in the UVB range. The cross-linking of collagen type I and type IV by UV irradiation was observed. Amino acid analyses revealed that Tyr residues in both collagen types I and IV were decreased by irradiation. In collagen type IV, losses of His and Met residues were also observed. These losses of collagen type IV may be due to the degradation of Trp, which exists in collagen type IV and decreased drastically during UV irradiation. To clarify the mechanism of Tyr modification in both types of collagen, the degradation products of Tyr were analyzed. Dityrosine, which is a dimer of the Tyr residue, could not be detected in the acid hydrolysates of UV-irradiated collagen. However, 3,4-dihydroxyphenylalanine, DOPA, was detected in the hydrolysates using HPLC with an electrochemical detector. The amounts of DOPA in the acid hydrolysates of collagen exposed to UV light for 24 h were approximately 350 pmol/mg protein (collagen type IV) and 80 pmol/mg protein (collagen type I). The DOPA formed may partially contribute to photoaging of the skin.     

Mapping protein interfaces by a trifunctional cross-linker combined with MALDI-TOF and ESI-FTICR mass spectrometry

Chemical cross-linking of protein complexes has gained renewed interest in combination with mass spectrometric analysis of the reaction products as it allows a rapid mapping of protein interfaces, which is crucial for understanding protein/protein interactions. The identification of cross-linking products from the complex mixtures created after the cross-linking reaction, however, remains a daunting task. To facilitate the identification of cross-linking products, we explore the use of the commercially available biotinylated cross-linking reagent sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3'-dithiopropionate). This trifunctional cross-linker possesses one amine-reactive and one photo-reactive site and, additionally, allows an affinity-based enrichment of cross-linker containing species. As a model system, we chose the Ca(2+)-dependent complex between calmodulin and its target peptide M13, which represents a part of the C-terminal sequence of the skeletal muscle myosin light chain kinase. After the cross-linking reaction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and one-dimensional gel electrophoresis were employed to check for the extent of cross-linking product formation. The cross-linking reaction mixtures were subjected to tryptic in-solution digestion. Biotinylated peptides, e.g., peptides that had been modified by the cross-linker as well as cross-linked peptides, were enriched on monomeric avidin beads after several washing steps had been performed. Peptide mixtures were analyzed by MALDI-TOFMS, nano-high-performance liquid chromatography (HPLC)/nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS), and tandem MS. We demonstrate that an enrichment of cross-linker containing species allows a more efficient identification of interacting amino acid sequences in protein complexes. This strategy is expected to be especially beneficial for investigating large protein assemblies.     

Induction of Lambda Prophage by 213 nm Laser Radiation: A Quantitative Comparison with 193 nm Excimer Radiation Using Image Analysis

Abstract— We compared the DNA damage produced by radiation from two UV laser wavelengths, 213 nm and 193 nm, with that produced by noncoherent 254 nm radiation. Following irradiation of Escherichia coli BR339, a bacteriophage lambda lysogen containing the lacZ gene, prophage induction was measured by assaying for β-galactosidase. Because of the limited penetration by UV laser wavelengths an agar overlay of the lysogen was used as the irradiation target. Irradiation of 254 nm was performed in buffer suspension followed by transfer of 5 μL spots onto assay plants. Computer image analysis was used to monitor the rate of product formation, observed as an increase in optical density of the irradiated zones on assay plates. We found that the rate of product formation was a more reproducible unit of comparison than the optical density present at the end of the reaction. Although the rate of product formation was not linearly related to enzyme concentration, the data could be fit to a simple logarithmic function. Using this method, we concluded that the DNA damaging ability of 213 nm radiation was 10 times more efficient than 193 nm radiation and about 100 times less efficient than 254 nm noncoherent radiation.     

Covalently modified silicon and diamond surfaces: resistance to nonspecific protein adsorption and optimization for biosensing

We report the direct covalent functionalization of silicon and diamond surfaces with short ethylene glycol (EG) oligomers via photochemical reaction of the hydrogen-terminated surfaces with terminal vinyl groups of the oligomers, and the use of these monolayers to control protein binding at surfaces. Photochemical modification of Si(111) and polycrystalline diamond surfaces produces EG monolayers linked via Si-C bond formation (silicon) or C-C bond formation (diamond). X-ray photoelectron spectroscopy was used to characterize the monolayer composition. Measurements using fluorescently labeled proteins show that the EG-functionalized surfaces effectively resist nonspecific adsorption of proteins. Additionally, we demonstrate the use of mixed monolayers on silicon and diamond and apply these surfaces to control specific versus nonspecific binding to optimize a model protein sensing assay.     

A Preparative Method for Crosslinking Proteins to DNA in Nuclei by Single-pulse UV Laser Irradiation

Abstract— Photocrosslinking of proteins to DNA by single-pulse UV laser has been used only in analytical experiments, carried out with reconstituted complexes of a single DNA binding protein and a labeled target sequence. Here we propose a large-scale technique for irradiation of nuclei, generating preparative quantities of covalently linked protein-DNA complexes for further analysis of the partner molecules. The use of a flow cuvette allows a milligram of DNA in either nuclei or chromatin to be irradiated by a single pulse within few minutes. The efficiency of crosslinking varies from 6 to 12% of the total nuclear proteins. The presence of histones and other chromosomal proteins in the crosslinked protein-DNA complexes was demonstrated by using specific antibodies. The irradiation procedure can be fully automated using a microcomputer.     

菁染料和份菁染料溶液的光降解机理的研究

利用UV-Vis吸收光谱仪和光化学反应器,研究了菁染料和份菁染料溶液的光降解动力学,认为染料在乙腈溶液中的光褪色反应服从假一级或零级动力学.利用GC/MS光谱仪检测了染料的光降解产物.与相应的份菁染料相比,携带正电荷的菁染料具有相对较好的光稳定性.研究结果表明,菁染料光降解反应的中间体可能是染料的半氧化态Dye⁺,并利用纳秒级闪光光解技术研究了Dye⁺的瞬态吸收光谱.     

Probing the Binding Region of the Single-Stranded DNA-Binding Domain of Rat DNA Polymerase β Using Nanosecond-Pulse Laser-Induced Cross-Linking and Mass Spectrometry

In recent years, there has been a significant number of studies in which UV light has been used as a reagent to induce cross-links in nucleic acid-protein complexes. An area of considerable interest among those interested in structural biology is the garnering of information about the sites of cross-linking within the protein and nucleic acid members of photolinked conjugates, under the assumption that such knowledge should lead to identification of contact regions or sites within the native complexes. In this paper, we present our results from a photocross-linking study of the complex of the single-stranded DNA-binding domain of rat DNA polymerase β (pol β-ss) with the oligonucleotide d(ATATATA). In this study, we have used single nanosecond laser pulses as the cross-linking reagent and matrix-assisted laser desorp-tion/ionization-time of flight mass spectrometry as an analytical tool to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Six cross-linked peptides have been identified in tryptic digests of the protein-oligonucleotide conjugates that result from irradiation of the pol β-ss-d(ATATATA) complex with a single laser pulse. Comparisons with NMR data in the literature for the same complex show that each of the cross-linked peptides contains amino acids that are in contact with the nucleic acid component of the complex.     

聚甲基丙烯酸环氧丙酯的改性及其光敏性能的研究

<正> 以远紫外线作曝光光源的光刻技术具有较高的分辨率及设备简单等优点,已在国际上引起了广泛的重视。聚甲基丙烯酸环氧丙酯(PGMA)作为负性电子束抗蚀剂有较高灵敏度(4×10~(-7)库仑/平方厘米)和良好的分辨率(0.5微米)。但以远紫外线曝光时它成为光降解型且敏度低,然而在此体系中引进对光敏感的肉桂酰基团后,能使它从原     

COMPARATIVE POTENCY OF DIFFERENT UV SOURCES IN REDUCING THE DENSITY AND ANTIGEN-PRESENTING CAPACITY OF LANGERHANS CELLS IN C3H MICE

Abstract Although broadband UV-B irradiation has been shown to induce selective immunosuppression in a variety of experimental systems, the wavelength dependence of the immunomodulation and the initial events in the skin remain unclear. In the present study three UV lamps were used at suberythermal doses on C3H mice: a conventional broadband UV-B source (270–350 nm), a narrowband UV-B source (311–312 nm) and a UV-A source (320400 nm). Their effects on the photoisomerization of the naturally occumng trans- isomer of urocanic acid (UCA) to cis- UCA, on the density of Langerhans cells and on the ability of epidermal cells to stimulate allogeneic lymphocytes in the mixed skin lymphocyte reaction (MSLR) were ascertained. Broadband UV-B irradiation was more efficient than narrowband UV-B at reducing the density and function of Langerhans cells, while UV-A irradiation was least effective. These changes were most pronounced immediately following irradiation, were dose dependent and were only detected in UV-exposed areas of skin. There was a close correlation between the UV-induced reduction in Langerhans cell density and the formation of cis -UCA in the epidermis. This correlation was not detected between the reduction in the MSLR response following UV irradiation in vivo and cis-UCA formation.     

Photobinding of ketoprofen in vitro and ex vivo

Ketoprofen (KP), a non-steroidal anti-inflammatory drug of the 2-aryl propionic class, has been shown to produce photoallergic side effects as well as cutaneous photosensitizing properties that induce other phototoxic effects. In the present study we investigated photobinding of ketoprofen to both human serum albumin (HSA), a model protein, and to ex vivo pig skin and its photodegradation. Results demonstrate that photoadduct formation and photodegradation progressively increased with irradiation time where they reach a maximum. Maximum photobinding to the viable layer of the epidermis was about 7-8% of the initial radiolabelled KP added, in the region of 15-30 min UV irradiation. These results were comparable to in vitro results that were seen with photobinding of KP to HSA; in this case, the quantity of covalently bound material was approximately 10% of the initial, after a maximum of 18 min irradiation. It was found by HPLC analysis that the KP decrease is accompanied by an increase of the corresponding photoproduct, decarboxylated ketoprofen (DKP). The yield of DKP reaches a maximum at around 15 min. DKP appears to play an important role in vitro and ex vivo, being the major photoproduct and responsible for the photobinding process. Using micro-autoradiographical techniques we investigated the penetration and distribution of ketoprofen in ex vivo pig skin in greater detail. It was apparent that percutaneous absorption was taking place and that most of the ketoprofen was predominately localised in fibroblasts in the papillary dermis. No other specific localisation within the skin architecture was identified. Although there were differences in the quantities of bound ketoprofen within the different layers of the skin, these levels did not appear to correlate with irradiation time.     

Synthesis and photochemistry of 1-iodocyclohexene: influence of ultrasound on ionic vs. radical behaviour

Simultaneous application of UV light and ultrasonic irradiation to a reaction mixture containing 1-iodocyclohexene is reported. The irradiation of 1-iodocyclohexene in methanol was carried out with or without addition of zinc. The effect of ultrasound or mechanical stirring on this solid-liquid system was also compared. The irradiation of 1-iodocyclohexene in methanol in the presence of zinc increases the yield of the nucleophilic trapping product, compared with the yield after irradiation in the absence of zinc. The photodegradation of 1-iodocyclohexene was slightly accelerated after addition of zinc. A rapid formation of radical product was accompanied by substantial decrease of 1-iodocyclohexene after application of ultrasound and irradiation without the zinc. The ultrasound significantly affects the photobehaviour of this reaction, predominantly its radical route. The joint application of ultrasound and zinc contributes positively to the production of radical and ionic products. The sonochemical stirring is more effective than mechanical stirring.     

Photoregulation of PRMT-1 Using a Photolabile Non-Canonical Amino Acid

Protein methyltransferases are vital to the epigenetic modification of gene expression. Thus, obtaining a better understanding of and control over the regulation of these crucial proteins has significant implications for the study and treatment of numerous diseases. One ideal mechanism of protein regulation is the specific installation of a photolabile-protecting group through the use of photocaged non-canonical amino acids. Consequently, PRMT1 was caged at a key tyrosine residue with a nitrobenzyl-protected Schultz amino acid to modulate protein function. Subsequent irradiation with UV light removes the caging group and restores normal methyltransferase activity, facilitating the spatial and temporal control of PRMT1 activity. Ultimately, this caged PRMT1 affords the ability to better understand the protein’s mechanism of action and potentially regulate the epigenetic impacts of this vital protein.     

A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters

Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest as targets for chemical intervention. Herein we describe a systematic evaluation of split-proteins, specifically the green fluorescent protein, beta-lactamase, and several luciferases, for their ability to function as reporters in completely cell-free systems to allow for the extremely rapid and sensitive determination of a wide range of biomolecular interactions without the requirement for laborious transfection, cell culture, or protein purification (12-48 h). We demonstrate that the cell-free split-luciferase system in particular is amenable for directly interrogating protein-protein, protein-DNA, and protein-RNA interactions in homogeneous assays with very high sensitivity (22-1800 fold) starting from the corresponding mRNA or DNA. Importantly, we show that the cell-free system allows for the rapid (2 h) identification of target-site specificity for protein-nucleic acid interactions and in evaluating antagonists of protein-protein and protein-peptide complexes circumventing protein purification bottlenecks. Moreover, we show that the cell-free split-protein system is adaptable for analysis of both protein-protein and protein-nucleic acid interactions in artificial cell systems comprising water-in-oil emulsions. Thus, this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.     

INTERACTION OF DISULFIDE GROUPS WITH NUCLEIC ACID BASES

Abstract— Low temperature luminescence studies have provided evidence for interactions between the disulfide group of cystamine and adenine bases in cystamine-poly(A) complexes and in cystamine-adenosine aggregates. A quenching of adenine phosphorescence by cystamine was observed. At room temperature complex formation between cystamine and poly(A) was followed by proton magnetic resonance, absorption spectrophotometry and fluorescence using competition with a fluorescent peptide to monitor the binding process. These base-disulfide interactions could be involved in the photochemistry of protein-nucleic acid complexes.