X-ray crystallographic analysis of the structural basis for the interactions of pokeweed antiviral protein with its active site inhibitor and ribosomal RNA substrate analogs.

The pokeweed antiviral protein (PAP) belongs to a family of ribosome-inactivating proteins (RIP), which depurinate ribosomal RNA through their site-specific N-glycosidase activity. We report low temperature, three-dimensional structures of PAP co-crystallized with adenyl-guanosine (ApG) and adenyl-cytosine-cytosine (ApCpC). Crystal structures of 2.0-2.1 A resolution revealed that both ApG or ApCpC nucleotides are cleaved by PAP, leaving only the adenine base clearly visible in the active site pocket of PAP. ApCpC does not resemble any known natural substrate for any ribosome-inactivating proteins and its cleavage by PAP provides unprecedented evidence for a broad spectrum N-glycosidase activity of PAP toward adenine-containing single stranded RNA. We also report the analysis of a 2.1 A crystal structure of PAP complexed with the RIP inhibitor pteoric acid. The pterin ring is strongly bound in the active site, forming four hydrogen bonds with active site residues and one hydrogen bond with the coordinated water molecule. The second 180 degrees rotation conformation of pterin ring can form only three hydrogen bonds in the active site and is less energetically favorable. The benzoate moiety is parallel to the protein surface of PAP and forms only one hydrogen bond with the guanido group of Arg135.

Deguanylation of human immunodeficiency virus (HIV-1) RNA by recombinant pokeweed antiviral protein.

Modeling studies, combined with the molecular docking of the trinucleotide GGG into the active site of the deadenylating RNA N-glycosidase pokeweed antiviral protein (PAP), indicated that a guanine base can fit into the active site pocket of PAP without disturbing its unique geometry and is sandwiched between residues Tyr(72) and Tyr(123) very much like an adenine base. The guanine base can form two specific hydrogen bonds with the active site residues Ser(121) and Val(73) and the attached negatively charged phosphate groups can entertain stabilizing electrostatic interactions with two clusters of positively charged patches on the PAP surface formed by Lys(210) and Arg(179) from one side and Arg(122) and Arg(135) from the other side of the active site. These observations prompted the hypothesis that the RNA depurinating activity of PAP may not be restricted to adenine residues and PAP should be capable of deguanylating ribosomal and viral RNA as well. This hypothesis was experimentally confirmed by direct demonstration that guanine base is released from both ribosomal and HIV-1 RNA after treatment with purified recombinant PAP using quantitative high performance liquid chromatography. Recombinant PAP released adenine and guanine residues at a 1:1 ratio from HIV-1 RNA and at an approximately 3:1 (adenine:guanine) ratio from Escherichia coli ribosomal RNA. At a concentration of 5 microM, recombinant PAP released 263 +/- 10 pmol of adenine and 100 +/- 11 pmol of guanine from 1 microgram of E. coli ribosomal RNA (16S + 23S) within 4 h of treatment. By comparison, 138 +/- 12 pmol of adenine and 143 +/- 10 pmol of guanine were released from 1 microgram of HIV-1 RNA under identical treatment conditions (5 microM recombinant PAP, 4 h treatment). The deguanylation of the ribosomal and viral RNA targets by recombinant PAP was concentration-dependent and is abolished by alanine substitutions of the catalytic active site residues Tyr(72) and Tyr(123). To our knowledge, these findings provide the first evidence that PAP can deguanylate both ribosomal and viral RNA.

[Comparison of dynamic properties of various globular proteins and polyglutamic acid in alpha-helical and coil states. Rayleigh scattering of Mossbauer radiation data]. / Sravnenie dinamicheskikh svoistv razlichnykh globuliarnykh belkov i poliglutaminovoi kisloty v sostoianii alpha-spirali i klubka. Dannye réleevskogo rasseianiia Messbauérovskogo izlucheniia.

Classical model system: Poly-L-glutamic acid (Poly-Glu) was investigated in a disordered coil state (at pH-7.0) and in helix state (at pH 2.0) by Rayleigh scattering of Moessbauer radiation technique. Consider that the coil state of poly-Glu models unfolded (random coil) state and alpha-helix state models the fluctuating secondary structure (during consequent folding of protein) comparative analysis of dynamical properties of poly-Glu in different states with dynamical properties of different proteins in native state (alpha-helical myoglobin and HSA, partially beta-sheet lysozyme) and in intermediate (molten globule) state (alpha-lactalbumin) was performed. This comparison bring some surprising results: native alpha-helical proteins behave itself close to random coil, native partially beta-sheet protein behaves close to fluctuating secondary structure (alpha-helix) and the dynamic behaviour of molten globule state (partially beta-sheet alpha-lactalbumin) is not different from those behaviour of lysozyme and much more rigid than native alpha-helical proteins. As a result one cannot exclude the possibility that folding process and dynamical properties at different steps of the folding are very different for alpha-helical and beta-sheet proteins.

[Study of the dynamics of human serum albumin by coherent Rayleigh dispersion of Mossbauer radiation]. / Izuchenie dinamiki syvorotochnogo al'bumina cheloveka metodom kogerentnogo réleevskogo rasseianiia Messbauérovskogo izlucheniia.

The measurements of angle dependencies of total and elastic Rayleigh scattering of Mossbauer radiation intensities have been performed for human serum albumin (HSA) with hydration degrees h = 0.13 and h = 0.4. The extended model was developed for calculating the inelastic intensity of Rayleigh scattering. Original data for HSA and published data on met-Mb were fitted within the frame of this model. The best agreement with experiment was obtained when two types of intraglobular motions were taken into account: individual motions of small side-chain groups and cooperative (mechanical) motions of segments (most probable alpha-helices). Long-range correlated motions are essential at low hydration degree. The possibilities of application of the coherent version of RSMS technique are described.

[Evaluation of the contribution of various types of movement of protein globules into effects observed using the Rayleigh scattering of Mössbauer radiation or the Mössbauer absorption spectroscopy]. / Otsenka vkladov raznykh vidov dvizheniia globul belka v éffekty, nabliudaemye s pomoshch'iu releevskogo rasseianiia messbauérovskogo izlucheniia i messbauérovskoi absorbtsionnoi spektroskopii.

Conditions (regions of hydration degrees and temperatures) are considered at which effects observed in Rayleigh Scattering of Mössbauer Radiation and Mössbauer Absorption Spectroscopy can be attributed to changes in intramolecular mobility, rather than contribution of different types of motions of macromolecules as a whole.

[A study of the effect of solvent composition and viscosity on the molecular dynamics of human serum albumin using Rayleigh scattering of Mössbauer radiation]. / Izuchenie vliianiia sostava i viazkosti rastvoritelia na molekuliarnuiu dinamiku syvorotochnogo al'bumina cheloveka metodom releevskogo rasseianiia messbauérovskogo izlucheniia.

By means of RSMR changes of human serum albumen intramolecular mobility by addition of 1.5% and 7.5% of glutar dialdehyde (GD) in concentrated protein solution, heat denaturation of a protein or substitution of water by water-glycerol solvent with amount of water to glycerol: 1 to 2 were studied. It is shown that the elastic fraction for HSA is changed much less addition of GD or by heat denaturation than by substitution of water solution by water-glycerol. It seems that the observed strong influence of glycerol on intramolecular mobility of HSA is connected mostly with effective dehydration of protein (by substitution of the part of a water solvent by glycerol) and with a small volume decrease of protein (due to preference hydration effect) rather than with the increase of the solvent viscosity.

[Study of the effect of hydration on the dynamics of various globular proteins by Rayleigh scattering of Mössbauer radiation]. / Izuchenie vliianiia gidratatsii na dinamiku nekotorykh globuliarnykh belkov metodom réleevskogo rasseianiia messbauérovskogo izlucheniia.

Hydration relationships of the elastic scattering fraction of Mössbauer radiation were studied for human serum albumin (HSA), pancreatic trypsin inhibitor and lysozyme within hydration degrees 0 less than or equal to h less than or equal to 0.75 g/g (at T = 295 degrees K) and temperatures 100K less than or equal to T less than or equal to 320 K (for HSA only at h = 0.03; 0.25; 0.41; 0.65). It is shown that the increase of both hydration degree above h greater than 0.1 and temperature above T greater than 200K leads to the appearance of intramolecular mobility in these proteins.