Chemical and functional aspects of posttranslational modification of proteins.

This paper reviews the chemical and functional aspects of the posttranslational modifications of proteins, which are achieved by the addition of various groups to the side chain of the amino acid residue backbone of proteins. It describes the main prosthetic groups and the interaction of these groups and the apoenzyme in the process of catalysis, using pyridoxal catalysis as an example. Much attention is paid to the role of posttranslational modification of proteins in the regulation of biochemical processes in live organisms, and especially to the role of protein kinases and their respective phosphotases. Methylation and acetylation reactions and their role in the "histone code", which regulates genome expression on the transcription level, are also reviewed. This paper also describes the modification of proteins by large hydrophobic residues and their role in the function of membrane-associated proteins. Much attention is paid to the glycosylation of proteins, which leads to the formation of glycoproteins. We also describe the main non-enzymatic protein modifications such as glycation, homocysteination, and desamida-tion of amide residues in dibasic acids.

[Photoactivated analogues of the initiating substrates of RNA polymerase II based on arylazide derivatives of NTP gamma-amidophosphate: synthesis and chemical and photochemical reactions of functional groups].

Photoactivatable derivatives Ar-NH-(CH2)n-NHpppB (where Ar = p-azidophenyl (A1), 5-azido-2-nitrobenzoyl (A2), or 4-azido-2,3,5,6-tetrafluorobenzoyl (A3) group; B = Ado or Guo; n = 2, 3, or 4) were synthesized. The phosphoroamidate bond stability was found to depend on the structure of both the heterocyclic and the photoactivatable groups. The derivative with A3, Ado, and n=3 is hydrolyzed with regeneration of aryl azide and ATP, whereas the other derivatives are stable in aqueous solutions. The photoanalogues with A1 and A2, B = Ado, and n = 2 or 4 were found to behave as initiating substrates toward the RNA polymerase II from Saccharomyces cerevisiae under the conditions of specific transcription initiation and control of the adenovirus late promoter. The photolysis of N-(4-azidophenyl)-1,4-diaminobutane and N-(5-azido-2-nitrobenzoyl)-1,3-diaminopropane, two functional fragments of the photoaffinity reagents, in aqueous solutions was established to result in the formation of p-benzoquinone diimine and p-nitro-N-arylhydroxylamine derivatives, respectively. The arylhydroxylamine derivatives undergo a number of transformations in aqueous solution leading to nitroso derivatives. We concluded that it is these nitroso derivatives (products of nitrene transformation, rather than the nitrene itself) that may modify proteins with reagents containing p-nitrophenylazide fragment.

Photoanalogues of the initiation substrates of the RNA polymerase II, 5-azido-2-nitrobenzoyl derivatives of the ATP gamma-amidophosphate: the possible photoinduced degradation of the functional group to an N-arylhydroxylamine.

Photoanlogues of the initiation substrates of the RNA polymerase II, N3ArNH(CH2)(n)NHpppA where N3Ar is 5-azido-2-nitrobenzoyl group (n = 2 or 4) were synthesized, allowing the preparation of photoreactive oligonucleotides in situ by RNA polymerase II for application as photolabels. Photolysis of p-nitro-substituted aromatic azide in aqueous medium was investigated. Using the azoxy-coupling reaction it was possible to determine whether a nitrene or p-nitrophenyl hydroxylamine azoxy compound is the trappable intermediate that is generated at ambient temperature in aqueous solution.

p-Azidophenyl phosphate is a photoactivatable phosphorylating reagent and p-benzoquinone monoimine precursor.

p-Azidophenyl phosphate (I) has been exposed to ultraviolet light (lambda=313 nm) in aqueous solution with or without Lys. Analysis of the photoproducts by means of UV-VIS, IR, (1)H, (13)C and (31)P NMR spectroscopy has revealed that under irradiation of I inorganic phosphate (P(i)) is released, and p-benzoquinone monoimine (II) and p-benzoquinone (III) have appeared. The electrophilic nature of the intermediate results in a high tendency to react with lysine molecules, whereas the reaction with water is less favourable when I is irradiated in the presence of Lys. The product formed in this case is a phosphoramidate whose structure has been tentatively supported by (31)P NMR spectroscopy. These results imply that a p-azidophenyl phosphate is a highly potent aryl nitrene-precursor, which can be transformed easily into p-benzoquinone monoimine and is able to phosphorylate nucleophilic groups of amino acids. This finding is of great importance for the discussions about the chemical nature of protein photomodification products when p-azidophenyl phosphate derivatives are used as modifying reagents.

Long-lived reactive intermediate photogenerated from N-(5-azido-2-nitrobenzoyl)-N'-(D-biotinyl)-1,2-diaminoethane as an affinity reagent to streptavidin.

Irradiation of a complex between N-(5-azido-2-nitrobenzoyl)-N'-(D-biotinyl)-1,2-diaminoethane (I) and streptavidin with light of 313 nm led to the covalent attachment of the photobiotin analogue I to the protein. Streptavidin could also be labelled in the dark with prephotolyzed I. These results indicate that a long-lived reactive intermediate was formed upon irradiation. Moreover, after cleavage of labelled streptavidin with proteinase K this intermediate appears to be covalently attached to the same peptide as the one obtained by direct photoaffinity labelling. An iminosulfurane II derived from the reaction of biotin sulfur atom with aryl nitrene is responsible for the dark-labelling reaction. The photoproduct II converts in an aqueous solution almost completely into N-(5-amino-2-nitrobenzoyl)-N'-(D-(S-oxo)biotinyl)-1,2-diaminoethane (the half-life of II is 10 days).

Affinity labeling of RNA-polymerase II in the transcriptionally active complex by a phosphorylating analog of the initiation substrate.

Affinity modification of RNA-polymerase II by a phosphorylating analog of the initiation substrate carrying a zwitterionic 5;-terminal phosphate group with a 4-N,N-dimethylaminopyridine residue (DMAP-pA) was studied during specific transcription initiation controlled by the late adenoviral promotor. Super-selective affinity labeling and standard conditions of affinity modification resulted in labeling a polypeptide with molecular weight corresponding to that of the third subunit of the enzyme, RPB3 (45 kD). The initiation substrate (ATP) protects RNA-polymerase II from modification. The third subunit may be involved in the formation of the substrate-binding site of the enzyme.

Study of the chemical structures of the photo-cross-linking products between Tyr and the 5-azido-2-nitrobenzoyl residue.

Irradiation of N-(tyrosyl)-N'-(5-azido-2-nitrobenzoyl)-1,2-diaminoethane (I) initiates chemical reactions that lead to different products depending on the experimental conditions. All of these products are attributed to the reactions of triplet 4-nitrobenzoyl nitrene (4NBN). The reactions of triplet 4NBN with the tyrosyl residue result in the formation of two distinct products: compound II, which is unstable in aqueous solution, and the stable compound cyclo-[1-(4'-nitro-3'-benzoyl)-2-(aminotyrosyl)-N,N'-ethylenediami ne] (III). The formation of II is detected only in aerobic conditions. The unstable photoproduct II converts almost completely into compound III when its solution is concentrated. The photoproducts II and III have absorption spectra that are close to those of the photolabelled peptides. This finding is important for speculating about the chemical nature of the photomodification products of protein tyrosyl residues by the arylazide group.

Synthesis of new photocross-linking 5-C-base-substituted UTP analogs and their application in highly selective affinity labelling of the tick-borne encephalitis virus RNA replicase proteins.

A new photocross-linking 5-C-base-substituted UTP analogs, carrying 4-azidoperfluorobenzoyl and 4-azidoaniline residues were synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation (lambda > 300 nm) and subsequent [alpha-32P]NTP incorporation.

5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2'-deoxy- uridine-5'-triphosphate substitutes for thymidine-5'-triphosphate in the polymerase chain reaction.

The DNA targets may be labeled and simultaneously amplified in the polymerase chain reaction (PCR) using a pair of respective primers after elongation with nucleoside-5'-triphosphates carrying photoreactive groups. The amplified DNA may be subsequently photoactivated by irradiation above 300 nm, resulting in photo-cross-linking of the strands. For this goal 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-, 5-{N-[N'-(4-azido-2,3,5, 6-tetrafluorobenzoyl)-3-aminopropionyl]aminomethyl}-, and 5-{N-[N'-(2-nitro-5-azidobenzoyl)-3-aminopropionyl]aminomethyl}-2'-de oxyuridine-5'-triphosphate (VII, VIa, and VIb) derivatives have been synthesized. It was found that VII is capable of efficiently elongating DNA primers with both Klenow fragment DNA polymerase I and Thermus aquaticus DNA polymerase. Thereto, it turned out to provide quantitative incorporation in DNA as revealed by the formation of the full-length amplificate by PCR in the presence of this photoreactive analogue without any dilution with natural dTTP. On the contrary, it was found, that incorporation of VIa and VIb do not permit further DNA replication.

[New approach to the study of interaction of amino acid side groups with aryl azides]. / Novyi podkhod k izucheniiu vzaimodeistviia aminokislot po ikh bokovym radikalam s arilazidami.

A new approach to the study of the interaction of amino acid side chains with photoreactive aryl azides was proposed. This approach was based on the drawing together of the reacting groups by the attachment of the reacting compounds to complementary oligonucleotides. Cystamine, histamine, and 1,6-hexamethylenediamine mimicking the cystine, histidine, and lysine residues, respectively, were attached to the 3'-terminal phosphate of the oligonucleotide GGTATCp through a phosphamide bond and used as the targets for photomodification. Derivatives of the oligonucleotide pGATACCAA with the fragment N3C6H4NH- attached directly to its 5'-end by a phosphamide bond or through the spacer -(CH2)nNH- (where n is 2, 4, and 6) were used as photoreagents. Their derivatives containing the same spacer and the N3C6F4CO-NH(CH2)3NH- or 2-N3,5-NO2-C6H3CO-NH(CH2)3NH- residues were also used. The duplexes were photomodified by irradiation with 300-350 nm wavelength light. The maximal yields of the photo-cross-linking were from 22 to 68%. The reagents containing p-azidoaniline residue were found to be the most effective toward the targets. The maximum yields of the photomodification products modeling the side chains of cysteine and lysine were found to vary from 40 to 67% and to depend on the length and the structure of the spacers used. The duplex with the target bearing the imidazole residue (the histidine model) manifested a yield decreased to 25%. This fact was in a good agreement with the data of computer modeling that indicated an unfavorable mutual displacement of the imidazole residue and the photoreactive group.

[Use of photo-anchoring of DNA probes for fluorescent in situ hybridization]. / Ispol'zovanie fotoprishivaemykh DNK-zondov dlia fluorestsentnoi gibridizatsii in situ.

A possibility was investigated to use photo-crosslinking DNA probes for fluorescent in situ hybridization (FISH). DNA probes were modified by incorporating photonucleotides in these, containing a photoreactive group (tetrafluorobenzazid) and capable of making covalent bonds with the examined DNA, when irradiated in 300-330 nm region. The photonucleotide was incorporated into the probe either by nick-translation, or upon elongation of the hybridized probe by the Kljonow fragment. It has been shown that the DNA probe, cross-linking to a chromosome as a result of covalent bonds, is not removed from the place of hybridization under consequent denaturating washing, which makes it possible to carry out the following DNA hybridization with selective conservation of signals obtained due to previous hybridization. This peculiarity of photo-linking DNA probes makes it possible to use them for the two-step DNA hybridization. To demonstrate this, preparations of human chromosomes were investigated. On the first step, chromosomal DNA was hybridized by means of DNA probe having nucleotide sequences of centromeric regions of chromosomes 13 and 21, the probe being linked to chromosomal DNA by the photonucleotide. Following the denaturation treatment of the preparation, and after the second chromosomal DNA hybridization with cosmid DNA, containing chromosome 13 DNA nucleotide sequence, the signal in chromosome 13 centromeric region was retained to serve a marker of this chromosome, thus fascilitating its easier identification following the hybridization of its DNA with cosmic DNA. The denaturation stability of photo-crosslinking probes opens some new possibilities in technology of DNA in situ hybridization.

Photoaffinity labeling as an approach to study supramolecular nucleoprotein complexes.

The modern approaches for studying the detailed structure of nucleoprotein complexes involved in replication and transcription, based on the use of nucleic acids with photoreactive groups incorporated into definite positions of polynucleotide chain, are considered. Methods of preparation of photoreactive nucleic acids of this type are presented. Their use for positioning of RNA polymerase III and transcription factors as well as of the main participants of the replication machinery at the respective templates is described. A survey of the data concerning the amino acid residues modified in the course of photoaffinity labeling of proteins is also presented and some complications are discussed.

Synthesis of azidoaniline derivatives of oligonucleotides and investigation of their photochemical behavior.

A series of aryl azides, p-N3C6H4NH(CH2)nNH2 with n = 2-6, have been synthesized and used to prepare oligonucleotide derivatives carrying photoreactive the p-azidoaniline residue. Reactive moieties have been coupled to the 5'-terminal phosphate of d(pGATACCAA) [compounds IV(b), IV(c), and IV(e) with n = 3, 4, and 6, respectively] and of d(pGCC) [compound V(b) with n = 3] via a phosphoamide bond. Irradiation at wavelengths over > 300 nm of IV(b) and V(b) (n = 3) resulted in cleavage of the P-N bond. However, under the same reaction conditions, the P-N bond remained intact for compounds containing longer spacers [IV(c) and IV(e)]. Intraduplex reaction of the latter derivatives with d(GGTATCp)NH(CH2)6NH2 resulted in cross-linking dependent on the presence of an aliphatic amino group. The results obtained have demonstrated that the azidoaniline derivatives of oligonucleotides capable of the affinity modification of a specific target can be prepared. However, the sufficiently long aliphatic spacer group is necessary to prevent P-N bond cleavage within the photoreactive oligonucleotide.

Catalytic site-specific cleavage of a DNA-target by an oligonucleotide carrying bleomycin A5.

Oligonucleotide reagents have been created which are capable of catalytic site-specific cleavage of DNA-targets. The oligonucleotide reagent Blm-R-pd(CCAAACA) bearing the bleomycin A5 (Blm-RH) residue was used to degrade the DNA-target pd(TGTTTGGCGAAGGA). It has been shown that at equimolar reagent: target concentration the bleomycin oligonucleotide derivative can repeatedly cleave the target at G9, G7, T5, T4 and T3 in site-specific manner. This paper demonstrates that with a 10-fold excess of the DNA-target relative to the reagent 30% degradation of the target was observed primarily at a single position G7. The paper also shows that one reagent molecule containing bleomycin A5 residue was capable to degrade three molecules of the DNA-target. The catalytic activity of Blm-R-pd(CCAAACA) was the highest in the temperature range close to the melting temperature of the reagent-target complex, that is under conditions where the oligonucleotide reagent can form a complementary complex and easily dissociate to interact with the next molecule of the target. The number of target molecules degraded by the bleomycin reagent is limited by the degradation of the antibiotic residue itself.

[Photoaffinity modification of amino acid derivatives of oligonucleotides in a complementary complex]. / Fotoaffinnaia modifikatsiia aminokislotnykh proizvodnykh oligonukleotidov v komplementarnom komplekse.

Intracomplex photochemical interaction of photoactive derivatives R-CONH(CH2)3NH-pGATACCAA, where R= p-azidotetrafluorophenyl (I) or 2-nitro-5-azidophenyl (II), and 5'-phospho-p-azidoanilide pGATACCAA (III) with a target - oligonucleotide *pGGTATCp (IV) and its derivatives *pGGTATCp-NH(CH2)3NHX, where X = H (V), Phe (VI), or Lys (VII), was studied. According to electrophoretic data, photoreagent (I) gives rise to a covalent photoadduct with compound (IV) as well as with derivatives (VI) and (VII). In the case of reagent (II), only targets (V) - (VII) including aliphatic amino groups participate in the photocoupling. Upon irradiation of the duplexes comprising photoreagent (III) and targets (V)-(VII), the process is accompanied by the cleavage of the reagent's oligonucleotide moiety off the photomodification product. A plausible mechanism of the cleavage is discussed.

[Catalytic cleavage of target DNA in a complementary complex by a bleomycin oligonucleotide derivative]. / Kataliticheskoe rasshcheplenie DNK-misheni bleomitsinovym proizvodmym oligonukleotida v sostave komplementarnogo kompleksa.

For the first time, reagents that are capable of catalytic site-specific cleavage of DNA were synthesized based on oligonucleotides. One molecule of reagent (Blm-R)pd(CCAAACA) containing bleomycin A5 residue (Blm-RH) could degrade three molecules of target DNA pd(TGTTTGGCGAAGGA). The reagent displayed maximum catalytic activity in a temperature range that was close to the melting temperature of the reagent-target DNA complex. The number of the target DNA molecules that could be degraded by the reagent was limited by the degradation of the antibiotic residue itself during the oxidative degradation of the target DNA. The reagent catalyzed the subsequent degradation of residues G7, T5, T4, and T3 of the target DNA at an equimolar reagent-target DNA ratio. When the tenfold excess of the target DNA was used, the reagent degraded primarily the single G7 residue of the target DNA by 30%.