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Brooklyn Law School, US has reference to this Academic Journal, THE EFFECT OF HIGH SALT CONCENTRATION ON PROTEIN DYNAMICS: MOLECULAR DYNAMICS SIMULATIONS OF P.WOESEI TATA BOX-BINDING PROTEIN. Nina Pastor1 in addition to Harel Weinstein2 1Facultad de Ciencias, UAEM, Av. Universidad 1001, Col. Chamilpa, 62210 Cuernavaca, Morelos, M‚xico in addition to Dept. of Physiology in addition to Biophysics, 2Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, U.S.A. INTRODUCTION The TATA box-binding protein (TBP) is a general transcription factor present in archaea in addition to eukaryotes; it is required in consideration of transcription by the three nuclear RNA polymerases. TBP is conserved throughout evolution, both in structure in addition to in function. In transcription mediated by RNA polymerase II, TBP binds so that the TATA box, in addition to this binary complex recruits TFIIB. The structure of this ternary complex is also conserved throughout evolution. The structures of the C-terminal domain of TBPs from plants (A.thaliana ? ATH [1]), yeast (S.cerevisiae ? SCE [2]) in addition to a hyperthermophile (P.woesei ? PWO [3]) have been solved by X-ray crystallography, revealing practically the same structure in consideration of all, as expected from the ~40% sequence identity between these proteins. A distinguishing feature of PWO TBP is a disulfide bond, that is absent in the other two TBPs. TBP molecules in different organisms are subject so that a wide variety of environments. ATH in addition to SCE represent mesophile organisms; the optimal growth temperature in consideration of yeast is ~30?C. On the other hand, PWO grows optimally at 105?C, alongside an internal salt concentration of 0.8 M. The thermal stability of TBPs from different organisms varies widely, in addition to depends also on the salt concentration: SCE will denature at 60?C in 50 mM salt, but it will denature even at room temperature in salt concentrations over 200 mM. PWO TBP denatures at 101?C in 50 mM salt, but at 109?C in 800 mM salt. Under reducing conditions, the melting temperature decreases so that 97?C [3]. The thermal stability of PWO TBP has been rationalized [3] from an analysis of the crystal structure on the basis of the unique disulfide bond, an increase in ion pairs on the surface of TBP, an increase in buried surface area, in addition to a more compact packing. The halostability can be rationalized on the basis of an increase in the number of acidic residues, compared so that the mesophilic TBPs. Given the phylogenetic conservation in function of TBP, it is expected that structure in addition to dynamics that relate so that function will be common so that ATH, SCE in addition to PWO TBPs. structural in addition to dynamic features that are specific so that PWO TBP will indicate adaptation mechanisms so that extreme environments. APPROACH: We carried out MD simulations of ATH, SCE in addition to PWO monomers, at 300K, in TIP3 water, in consideration of 2 ns, alongside the CHARMM23 program in addition to potential. PWO was also simulated alongside TIP3 water in addition to 0.75 M NaCl (PWOSAL). FINDINGS 1: OVERALL DYNAMICS Backbone atomic fluctuations reveal similarities in addition to individual differences in the dynamic patterns of the protein. The highest mobility regions correspond so that the stirrups (residues 30-40 in addition to 125-135, approximately), in addition to also so that the loop connecting helix 2 so that strand 1? (see C? trace in addition to alignment). Global motions of the protein, monitored by the stirrup ? stirrup distance, in addition to the distances in addition to angles between helices 1 in addition to 1?, in addition to 2 in addition to 2?, show no evidence of collapse of the protein in any of the simulations. SUMMARY OF H-BOND AND WATER BRIDGE INTERACTIONS ATH2 ( 19) 1 SGIVPTLQNI VSTVNLDCKL DLKAIALQAR NAEYNPKRFA AVIMRIREPK SCE ( 61) 1 SGIVPTLQNI VATVTLGCRL DLKTVALHAR NAEYNPKRFA AVIMRIREPK PWO ( 5) 1 SKVKLRIENI VASVDLFAQL DLEKVLDLCP NSKYNPEEFP GIICHLDDPK | | | -| |-| | -| S1 H1 S2 S3 ATH2 51 TTALIFASGK MVCTGAKSED FSKMAARKYA RIVQKLGF-PA KFKDFKIQNI SCE 51 TTALIFASGK MVVTGAKSED DSKLASRKYA RIIQKIGF-AA KFTDFKIQNI PWO 51 VALLIFSSGK LVVTGAKSVQ DIERAVAKLA QKLKSIGVKFK RAPQIDVQNM | -| | | | -| | S4 S5 H2 ATH2 101 VGSCDVKFPI RLEGLAYSHA AFSSYEPELF PGLIYRMKVP KIVLLIFVSG SCE 101 VGSCDVKFPI RLEGLAFSHG TFSSYEPELF PGLIYRMVKP KIVLLIFVSG PWO 102 VFSGDIGREF NLDVVALTLP N-CEYEPEQF PGVIYRVKEP KSVILLFSSG -| | -||-| | -| | -| S1? H1? S2? S3? S4? ATH2 151 KIVITGAKMR DETYKAFENI YPVLSEFRKI SCE 151 KIVLTGAKQR EEIYQAFEAI YPVLSEFRKM PWO 151 KIVCSGAKSE ADAWEAVRKL LRELDKYGLL | -| | -| S5? H2? Secondary structure assignment shown below the sequences. Red: water bridges; teal: water bridges induced in salt; pink: water bridges lost in salt. Underlined: sc ? sc in addition to sc ? mc H-bonds. Wavy underline: H-bonds induced in salt. Double underline: H-bonds lost in salt. PROTEIN-PROTEIN HYDROGEN BONDS: ATH SCE PWO PWOSAL sc-sc 19.8 24.2 26.6 26.7 mc-mc 172.2 173.7 179.7 170.7 mc-sc 19.7 14.5 12.9 11.8 total 211.7 212.4 219.2 209.2 PROTEIN-WATER HYDROGEN BONDS: ATH SCE PWO PWOSAL Hbonds so that water 387.3 398.1 441.2 444.1 sc-sc water bridges 46.0 49.5 63.8 67.1 sc-mc water bridges 29.3 27.5 33.5 33.3 Total water bridges 75.2 77.0 97.3 100.4 SIDE CHAIN DYNAMICS: ATH SCE PWO PWOSAL sc entropy 175ñ9 166ñ3 180ñ7 188ñ3 sc TS 52ñ3 50ñ1 54ñ2 56ñ1 Entropy in cal/mol?K from S = -R?pilnpi, where pi is the probability of populating a particular rotamer; TS calculated at 300K, in addition to given in Kcal/mol. CONCLUSIONS Structure stabilization in TBPs The N-terminal subdomain seems so that be the weakest part of the structure. In mesophiles it is stabilized by small networks of H-bonds in addition to water bridges (linking, in consideration of example, helix 2 so that strand 4), while in PWO it is stabilized by a disulfide bond linking H1 so that the body of the protein. The stabilization of the C-terminal subdomain is accomplished through both water in addition to H-bond networks. These are larger in addition to more complex in PWO than in the mesophiles. The addition of salt results in a ?swelling? of the PWO structure, alongside the loss of some main chain H-bonds, but alongside a gain of water bridges between side chains (especially D). Halophilicity in this protein is reflected in better hydration, mediated principally by the larger number of D residues, but not by specific binding of ions. The increase in H-bond in addition to water bridge formation does not necessarily lead so that an entropic penalty. The H-bonds in addition to water bridges are under constant formation in addition to breaking, thus conferring an enthalpic in addition to an entropic advantage so that PWO in addition to PWOSAL. Functional inferences TBP does not collapse in the absence of DNA, as seen in a previous simulation [4]. The two subdomains display twisting in addition to compression-extension type motions, a flexibility which is probably related so that the ability of TBP so that bind DNA sequences alongside different degrees of bendability. The addition of salt changes the dynamics of PWO such as so that bring it closer so that the behavior of the mesophilic TBPs, in agreement alongside the ?corresponding states? hypothesis [5]. FINDINGS 2: H-BONDS AND HYDRATION The relative sizes of polar (46%) in addition to apolar (54%) surfaces in PWO are slightly different in consideration of mesophile TBPs (42% polar in addition to 58% apolar). The increase in polar solvent accessible area in PWO relative so that ATH in addition to SCE, is also reflected in a greater number of H-bonds so that water during the simulations. More water molecules are coordinated by PWO due so that the larger number of D in addition to E residues in PWO compared so that ATH in addition to SCE. D residues are ~20% better at forming water bridges than E residues. There is a common water bridge network in all the simulations, linking the C-terminus so that helix 1? in addition to so that the stirrup below. PWO is more densely packed than the mesophile TBPs (1.4% smaller volume). The amount of H-bonds between side chains is larger in PWO in addition to PWOSAL than in ATH in addition to SCE. PWO in addition to PWOSAL have more in addition to larger networks of H-bonded residues, bridging elements of secondary structure in addition to probably conferring structural stability. Mesophile TBPs tend so that have more H-bonds in the N-terminal subdomain than in the C-terminal subdomain. This pattern is reversed in PWO in addition to PWOSAL, probably due so that the presence of the disulfide bond in the N-terminal subdomain. H-BOND NETWORKS: ATH: K151 – D105 – K107 (S5?) (S1?) (S1?) loop S3-S4 | K78 – Y79 – T51 (H2) (H2) (S4) SCE: loop S3-S4 | K78 – Y79 – T51 | R45 – E33 (S3) (S2) PWO: K151 – D106 – R109 | C-ter (H2?) (S3?) E173 – Y135 || (S2?) (H1?) R136 – E124 = C123 – A117 || Y125 – D114 (loop S1?-H1?) PWOSAL: (S5?) (S1?) K151 – D106 – R109 S155 – N100 | || C-ter Q99 – S13 (S1) (H2?) (S3?) (H2?) (H2?) (H2?) E173 – Y135 – K169 – E165 – R168 || (S2?) R136 – E124 (S1) (S5) (loop S4-S5) D15 – K60 – S58 REFERENCES: [1] Nikolov, D.B. in addition to Burley, S.K. (1994) Nat. Struct. Biol. 1:621-37. [2] Chasman, D.I., et al. (1993) Proc. Natl. Acad. Sci. 90:8174-8. [3] DeDecker, B. S., et al. (1996) J. Mol. Biol. 264:1072-84. [4] Miaskiewicz, K. in addition to Ornstein, R.L. (1996) J.Biomol. Struct. Dyn. 13:593 [5]Jaenicke, R. in addition to Bohm, G. (1998) Curr. Opin. Struct. Biol. 8:738-48. BACKBONE FLUCTUATIONS EFFECTS OF THE ADDITION OF SALT Increase in solvent accessible area, but not in a change of the relative amounts of polar (46%) in addition to apolar (54%) surfaces. No change in the number of coordinated water molecules, but an increase the number of water bridges between the side chains in PWOSAL. Slight increase in molecular volume, suggesting that salt makes the protein swell. Swelling in addition to increase in backbone fluctuations also evident from the decrease in the number of H-bonds between main chain atoms in PWOSAL compared so that PWO. Some of the regions alongside increased backbone fluctuations correspond so that the places where mc-mc H-bonds were either debilitated or lost. Overall decrease in the number of direct H-bonds in PWO. No change in the number of H-bonds between side chains. Nevertheless, there is a redistribution of the H-bonds. The actual H-bond networks are different in the two simulations, in addition to are spread over larger areas in PWOSAL. This is probably due so that the fact that the internal water molecule in PWO tried so that escape so that the bulk solution, causing an increase in the distance between strands 1? in addition to 5? (see also the stirrup ? stirrup distance distribution), in addition to disrupting the network underlining the bottom face of PWO. EFFECTS OF THE ADDITION OF SALT The dynamic behavior: PWO becomes closer so that the mesophile TBPs, except in consideration of the H1 ? H1? distance distribution. Increase in backbone fluctuations: general increase, except at the N-terminus in addition to the C-caps of helices 2 in addition to 2? (c.f. PWO in addition to PWOSAL). The most affected regions correspond so that surface exposed loops, a region in the middle of helix 2 (A80-L83), in addition to strand 4? (S148-K151). Side chain entropy: (estimated by monitoring the population of the different rotamers during the simulations) an apparent increase in side chain entropy, mainly of the hydrophobic residues. Most of the charged amino acids experience a decrease in conformational entropy. Salt condensation: As expected in consideration of a macromolecule alongside a total charge of +1e, PWOSAL shows no condensation. 18% of Na in addition to Cl atoms were found within 5.5  of the surface of PWOSAL, in addition to no particular association lasted more than 250 ps, suggesting that salt stabilization is not due so that specific binding so that the TBP.

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