data_5908 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; Backbone 1HN and 15N Chemical Shift Assignments for Sac7d V30I ; _BMRB_accession_number 5908 _BMRB_flat_file_name bmr5908.str _Entry_type original _Submission_date 2003-08-19 _Accession_date 2003-08-19 _Entry_origination author _NMR_STAR_version 2.1.1 _Experimental_method NMR _Details . loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Clark Andrew T. . 2 McCrary Bradford S. . 3 Edmondson Stephen P. . 4 Shriver John W. . stop_ loop_ _Saveframe_category_type _Saveframe_category_type_count assigned_chemical_shifts 1 stop_ loop_ _Data_type _Data_type_count "1H chemical shifts" 67 "15N chemical shifts" 66 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2003-10-06 original author . stop_ loop_ _Related_BMRB_accession_number _Relationship 5905 'Sac7d monomer' 5909 'Sso7d monomer' 5910 'Sso7d monomer mutant' stop_ _Original_release_date 2003-10-06 save_ ############################# # Citation for this entry # ############################# save_entry_citation _Saveframe_category entry_citation _Citation_full . _Citation_title ; Thermodynamics of Hydrophobic Core Packing in the Thermophile Proteins Sac7d and Sso7d ; _Citation_status 'in preparation' _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID ? loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Clark Andrew T. . 2 McCrary Bradford S. . 3 Edmondson Stephen P. . 4 Shriver John W. . stop_ _Journal_abbreviation 'J. Mol. Biol.' _Journal_volume . _Journal_issue . _Journal_CSD . _Book_chapter_title . _Book_volume . _Book_series . _Book_ISBN . _Conference_state_province . _Conference_abstract_number . _Page_first . _Page_last . _Year . _Details . loop_ _Keyword hyperthermophile Sulfolobus calorimetry 'nuclear magnetic resonance' stop_ save_ ####################################### # Cited references within the entry # ####################################### save_references_1 _Saveframe_category citation _Citation_full ; Bosch D, Campillo M, Pardo L. J Comput Chem. 2003 Apr 30;24(6):682-91 ; _Citation_title 'Binding of proteins to the minor groove of DNA: what are the structural and energetic determinants for kinking a basepair step?' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 12666159 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Bosch David . . 2 Campillo Mercedes . . 3 Pardo Leonardo . . stop_ _Journal_abbreviation . _Journal_name_full 'Journal of computational chemistry' _Journal_volume 24 _Journal_issue 6 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 682 _Page_last 691 _Year 2003 _Details ; The structural and energetic determinants for kinking a basepair step by minor groove-insertion of the protein side chains of PurR, LacI, LEF-1, IHF, Sac7d, and Sso7d, have been calculated by molecular dynamics/potential of mean force simulations. The structural determinants of the kinked structures are: two contiguous furanose rings achieve different conformations, in the region of C3'endo (A-DNA) and C2'endo (B-DNA); the chi torsion angle always takes values characteristic of the C2'endo conformation of B-DNA, independently of sugar puckering; and protein side chain insertion increases slide (from negative to positive values), rise, and roll, and decreases twist. The energetic determinants of DNA kinking are: the conformational transition of the sugar-phosphate backbone is not energetically demanding; the relative importance of the interbase parameters in the free energy penalty is slide, followed by twist and rise, and concluding with shift and roll; and the characteristic increase of roll and decrease of twist, upon side chain insertion, tends to stabilize the process of DNA kinking. ; save_ save_references_2 _Saveframe_category citation _Citation_full ; Napoli A, Zivanovic Y, Bocs C, Buhler C, Rossi M, Forterre P, Ciaramella M. Nucleic Acids Res. 2002 Jun 15;30(12):2656-62. ; _Citation_title 'DNA bending, compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 12060682 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Napoli Alessandra . . 2 Zivanovic Yvan . . 3 Bocs Chantal . . 4 Buhler Cyril . . 5 Rossi Mose' . . 6 Forterre Patrick . . 7 Ciaramella Maria . . stop_ _Journal_abbreviation 'Nucleic Acids Res.' _Journal_name_full 'Nucleic acids research' _Journal_volume 30 _Journal_issue 12 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 2656 _Page_last 2662 _Year 2002 _Details ; Members of the Sso7d/Sac7d family are small, abundant, non-specific DNA-binding proteins of the hyperthermophilic Archaea SULFOLOBUS: Crystal structures of these proteins in complex with oligonucleotides showed that they induce changes in the helical twist and marked DNA bending. On this basis they have been suggested to play a role in organising chromatin structures in these prokaryotes, which lack histones. We report functional in vitro assays to investigate the effects of the observed Sso7d-induced structural modifications on DNA geometry and topology. We show that binding of multiple Sso7d molecules to short DNA fragments induces significant curvature and reduces the stiffness of the complex. Sso7d induces negative supercoiling of DNA molecules of any topology (relaxed, positively or negatively supercoiled) and in physiological conditions of temperature and template topology. Binding of Sso7d induces compaction of positively supercoiled and relaxed DNA molecules, but not of negatively supercoiled ones. Finally, Sso7d inhibits the positive supercoiling activity of the thermophile-specific enzyme reverse gyrase. The proposed biological relevance of these observations is that these proteins might model the behaviour of DNA in constrained chromatin environments. ; save_ save_references_3 _Saveframe_category citation _Citation_full ; Edmondson SP, Shriver JW. Methods Enzymol. 2001;334:129-45. ; _Citation_title 'DNA binding proteins Sac7d and Sso7d from Sulfolobus.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 11398456 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Edmondson 'S P' P. . 2 Shriver 'J W' W. . stop_ _Journal_abbreviation 'Meth. Enzymol.' _Journal_name_full 'Methods in enzymology' _Journal_volume 334 _Journal_issue . _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 129 _Page_last 145 _Year 2001 _Details . save_ save_references_4 _Saveframe_category citation _Citation_full ; Bedell JL, McCrary BS, Edmondson SP, Shriver JW. Protein Sci. 2000 Oct;9(10):1878-88. ; _Citation_title 'The acid-induced folded state of Sac7d is the native state.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 11106160 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Bedell 'J L' L. . 2 McCrary 'B S' S. . 3 Edmondson 'S P' P. . 4 Shriver 'J W' W. . stop_ _Journal_abbreviation 'Protein Sci.' _Journal_name_full 'Protein science : a publication of the Protein Society' _Journal_volume 9 _Journal_issue 10 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 1878 _Page_last 1888 _Year 2000 _Details ; Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J. Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H,15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid. ; save_ save_references_5 _Saveframe_category citation _Citation_full ; Su S, Gao YG, Robinson H, Liaw YC, Edmondson SP, Shriver JW, Wang AH. J Mol Biol. 2000 Oct 27;303(3):395-403. ; _Citation_title 'Crystal structures of the chromosomal proteins Sso7d/Sac7d bound to DNA containing T-G mismatched base-pairs.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 11031116 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Su S . . 2 Gao 'Y G' G. . 3 Robinson H . . 4 Liaw 'Y C' C. . 5 Edmondson 'S P' P. . 6 Shriver 'J W' W. . 7 Wang 'A H' H. . stop_ _Journal_abbreviation 'J. Mol. Biol.' _Journal_name_full 'Journal of molecular biology' _Journal_volume 303 _Journal_issue 3 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 395 _Page_last 403 _Year 2000 _Details ; Sso7d and Sac7d are two small chromatin proteins from the hyperthermophilic archaeabacterium Sulfolobus solfataricus and Sulfolobus acidocaldarius, respectively. The crystal structures of Sso7d-GTGATCGC, Sac7d-GTGATCGC and Sac7d-GTGATCAC have been determined and refined at 1.45 A, 2.2 A and 2.2 A, respectively, to investigate the DNA binding property of Sso7d/Sac7d in the presence of a T-G mismatch base-pair. Detailed structural analysis revealed that the intercalation site includes the T-G mismatch base-pair and Sso7d/Sac7d bind to that mismatch base-pair in a manner similar to regular DNA. In the Sso7d-GTGATCGC complex, a new inter-strand hydrogen bond between T2O4 and C14N4 is formed and well-order bridging water molecules are found. The results suggest that the less stable DNA stacking site involving a T-G mismatch may be a preferred site for protein side-chain intercalation. ; save_ save_references_6 _Saveframe_category citation _Citation_full ; Delaglio, F. et al. J. Biomol. NMR 6, 277-293 (1995). ; _Citation_title 'NMRPipe: a multidimensional spectral processing system based on UNIX pipes.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 8520220 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Delaglio F . . 2 Grzesiek S . . 3 Vuister 'G W' W. . 4 Zhu G . . 5 Pfeifer J . . 6 Bax A . . stop_ _Journal_abbreviation 'J. Biomol. NMR' _Journal_name_full 'Journal of biomolecular NMR' _Journal_volume 6 _Journal_issue 3 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 277 _Page_last 293 _Year 1995 _Details ; The NMRPipe system is a UNIX software environment of processing, graphics, and analysis tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks. ; save_ save_references_7 _Saveframe_category citation _Citation_full ; Hunenberger PH, McCammon JA. Biophys Chem. 1999 Apr 5;78(1-2):69-88. ; _Citation_title 'Effect of artificial periodicity in simulations of biomolecules under Ewald boundary conditions: a continuum electrostatics study.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 10343384 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Hunenberger 'P H' H. . 2 McCammon 'J A' A. . stop_ _Journal_abbreviation 'Biophys. Chem.' _Journal_name_full 'Biophysical chemistry' _Journal_volume 78 _Journal_issue 1-2 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 69 _Page_last 88 _Year 1999 _Details ; Ewald and related methods are nowadays routinely used in explicit-solvent simulations of biomolecules, although they impose an artificial periodicity in systems which are inherently non-periodic. The consequences of this approximation should be assessed, since they may crucially affect the reliability of computer simulations under Ewald boundary conditions. In the present study we use a method based on continuum electrostatics to investigate the nature and magnitude of possible periodicity-induced artifacts on the potentials of mean force for conformational equilibria in biomolecules. Three model systems and pathways are considered: polyalanine oligopeptides (unfolding), a DNA tetranucleotide (separation of the strands), and the protein Sac7d (conformations from a molecular dynamics simulation). Artificial periodicity may significantly affect these conformational equilibria, in each case stabilizing the most compact conformation of the biomolecule. Three factors enhance periodicity-induced artifacts: (i) a solvent of low dielectric permittivity; (ii) a solute size which is non-negligible compared to the size of the unit cell; and (iii) a non-neutral solute. Neither the neutrality of the solute nor the absence of charge pairs at distances exceeding half the edge of the unit cell do guarantee the absence of artifacts. ; save_ save_references_8 _Saveframe_category citation _Citation_full ; de Bakker PI, Hunenberger PH, McCammon JA. J Mol Biol. 1999 Jan 29;285(4):1811-30. ; _Citation_title 'Molecular dynamics simulations of the hyperthermophilic protein sac7d from Sulfolobus acidocaldarius: contribution of salt bridges to thermostability.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 9917414 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 'de Bakker' 'P I' I. . 2 Hunenberger 'P H' H. . 3 McCammon 'J A' A. . stop_ _Journal_abbreviation 'J. Mol. Biol.' _Journal_name_full 'Journal of molecular biology' _Journal_volume 285 _Journal_issue 4 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 1811 _Page_last 1830 _Year 1999 _Details ; Hyperthermophilic proteins often possess an increased number of surface salt bridges compared with their mesophilic homologues. However, salt bridges are generally thought to be of minor importance in protein stability at room temperature. In an effort to understand why this may no longer be true at elevated temperatures, we performed molecular dynamics simulations of the hyperthermophilic protein Sac7d at 300 K, 360 K, and 550 K. The three trajectories are stable on the nanosecond timescale, as evidenced by the analysis of several time-resolved properties. The simulations at 300 K and (to a lesser extent) 360 K are also compatible with nuclear Overhauser effect-derived distances. Raising the temperature from 300 K to 360 K results in a less favourable protein-solvent interaction energy, and a more favourable intraprotein interaction energy. Both effects are almost exclusively electrostatic in nature and dominated by contributions due to charged side-chains. The reduced solvation is due to a loss of spatial and orientational structure of water around charged side-chains, which is a consequence of the increased thermal motion in the solvent. The favourable change in the intraprotein Coulombic interaction energy is essentially due to the tightening of salt bridges. Assuming that charged side-chains are on average more distant from one another in the unfolded state than in the folded state, it follows that salt bridges may contribute to protein stability at elevated temperatures because (i) the solvation free energy of charged side-chains is more adversely affected in the unfolded state than in the folded state by an increase in temperature, and (ii) due to the tightening of salt bridges, unfolding implies a larger unfavourable increase in the intraprotein Coulombic energy at higher temperature. Possible causes for the unexpected stability of the protein at 550 K are also discussed. ; save_ save_references_10 _Saveframe_category citation _Citation_full ; Gao YG, Su SY, Robinson H, Padmanabhan S, Lim L, McCrary BS, Edmondson SP, Shriver JW, Wang AH. Nat Struct Biol. 1998 Sep;5(9):782-6. ; _Citation_title 'The crystal structure of the hyperthermophile chromosomal protein Sso7d bound to DNA.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 9731772 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Gao 'Y G' G. . 2 Su 'S Y' Y. . 3 Robinson H . . 4 Padmanabhan S . . 5 Lim L . . 6 McCrary 'B S' S. . 7 Edmondson 'S P' P. . 8 Shriver 'J W' W. . 9 Wang 'A H' H. . stop_ _Journal_abbreviation 'Nat. Struct. Biol.' _Journal_name_full 'Nature structural biology' _Journal_volume 5 _Journal_issue 9 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 782 _Page_last 786 _Year 1998 _Details ; Sso7d and Sac7d are two small (approximately 7,000 Mr), but abundant, chromosomal proteins from the hyperthermophilic archaeabacteria Sulfolobus solfataricus and S. acidocaldarius respectively. These proteins have high thermal, acid and chemical stability. They bind DNA without marked sequence preference and increase the Tm of DNA by approximately 40 degrees C. Sso7d in complex with GTAATTAC and GCGT(iU)CGC + GCGAACGC was crystallized in different crystal lattices and the crystal structures were solved at high resolution. Sso7d binds in the minor groove of DNA and causes a single-step sharp kink in DNA (approximately 60 degrees) by the intercalation of the hydrophobic side chains of Val 26 and Met 29. The intercalation sites are different in the two complexes. Observations of this novel DNA binding mode in three independent crystal lattices indicate that it is not a function of crystal packing. ; save_ save_references_11 _Saveframe_category citation _Citation_full ; Robinson H, Gao YG, McCrary BS, Edmondson SP, Shriver JW, Wang AH. Nature. 1998 Mar 12;392(6672):202-5. ; _Citation_title 'The hyperthermophile chromosomal protein Sac7d sharply kinks DNA.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 9515968 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Robinson H . . 2 Gao 'Y G' G. . 3 McCrary 'B S' S. . 4 Edmondson 'S P' P. . 5 Shriver 'J W' W. . 6 Wang 'A H' H. . stop_ _Journal_abbreviation Nature _Journal_name_full Nature _Journal_volume 392 _Journal_issue 6672 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 202 _Page_last 205 _Year 1998 _Details ; The proteins Sac7d and Sso7d belong to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius and S. solfactaricus, respectively. These proteins are extremely stable to heat, acid and chemical agents. Sac7d binds to DNA without any particular sequence preference and thereby increases its melting temperature by approximately 40 degrees C. We have now solved and refined the crystal structure of Sac7d in complex with two DNA sequences to high resolution. The structures are examples of a nonspecific DNA-binding protein bound to DNA, and reveal that Sac7d binds in the minor groove, causing a sharp kinking of the DNA helix that is more marked than that induced by any sequence-specific DNA-binding proteins. The kink results from the intercalation of specific hydrophobic side chains of Sac7d into the DNA structure, but without causing any significant distortion of the protein structure relative to the uncomplexed protein in solution. ; save_ save_references_12 _Saveframe_category citation _Citation_full ; McCrary BS, Bedell J, Edmondson SP, Shriver JW. J Mol Biol. 1998 Feb 13;276(1):203-24. ; _Citation_title 'Linkage of protonation and anion binding to the folding of Sac7d.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 9514720 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 McCrary 'B S' S. . 2 Bedell J . . 3 Edmondson 'S P' P. . 4 Shriver 'J W' W. . stop_ _Journal_abbreviation 'J. Mol. Biol.' _Journal_name_full 'Journal of molecular biology' _Journal_volume 276 _Journal_issue 1 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 203 _Page_last 224 _Year 1998 _Details ; The temperature, pH, and salt dependence of the folding of recombinant Sac7d from the hyperthermophile Sulfolobus acidocaldarius is mapped using multi-dimensional differential scanning calorimetry (DSC) and folding progress surfaces followed by circular dichroism. Linkage relations are derived to explain the observed dependencies, and it is shown that the data can be explained by the linkage of at least two protonation reactions and two anion binding sites to a two-state unfolding process. Circular dichroism spectra indicate that a native-like fold is stabilized at acid pH by anion binding. An apparent binding isotherm surface (folding progress versus pH and salt) is used to obtain intrinsic chloride binding constants as a function of pH for both sites. A saddle is predicted in the folding progress surface (progress versus temperature and pH) at low salt with a minimum near pH 2 and 20 degrees C with approximately 25% of the protein folded. The position of the saddle is sensitive to the intrinsic delta C degrees of unfolding and provides a third measure of delta C degrees independent of that obtained by a Kirchoff plot of DSC data and chemical denaturation. The observed enthalpy of unfolding approaches zero near the saddle making the unfolding largely invisible to DSC under these conditions. The linkage analysis demonstrates that the delta C degrees for unfolding obtained from a Kirchoff plot of DSC data should be distinguished from the intrinsic delta C degrees of unfolding. It is shown that the discrepancy between the free energy of unfolding for Sac7d obtained by DSC and that obtained by chemical denaturation may be explained by the linkage of protonation and anion binding to protein folding. The linkage analysis demonstrates the limitations of using the delta Hcal/ delta Hvh ratio an indication of two-state unfolding. ; save_ save_references_13 _Saveframe_category citation _Citation_full ; Kulms D, Schafer G, Hahn U. Biol Chem. 1997 Jun;378(6):545-51. ; _Citation_title 'Overproduction of Sac7d and Sac7e reveals only Sac7e to be a DNA-binding protein with ribonuclease activity from the extremophilic archaeon Sulfolobus acidocaldarius.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 9224936 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Kulms D . . 2 Schafer G . . 3 Hahn U . . stop_ _Journal_abbreviation 'Biol. Chem.' _Journal_name_full 'Biological chemistry' _Journal_volume 378 _Journal_issue 6 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 545 _Page_last 551 _Year 1997 _Details ; Genomic DNA from Sulfolobus acidocaldarius was screened using a degenerate oligodeoxyribonucleotide, derived from the sequence of 16 N-terminal amino acids from SaRD protein. SaRD protein was previously isolated in our laboratory and identified as a protein from S. acidocaldarius exhibiting ribonuclease activity as well as DNA-binding properties. On the basis of Southern hybridization analysis two genes from S. acidocaldarius have been cloned, sequenced and overproduced in Escherichia coli. The deduced amino acid sequences revealed that one gene encodes Sac7d and the other one Sac7e; two small, previously described basic proteins from S. acidocaldarius, and furthermore the N-termini of Sac7e and SaRD are identical. Northern blot analysis demonstrated that the genes are transcribed separately. After expression of sac7d and sac7e genes in E. coli it was shown that only recombinant Sac7e protein exhibits RNase activity and is catalytically indistinguishable from SaRD protein. Western blot analysis using a polyclonal antiserum raised against purified SaRD protein further confirmed that Sac7e and SaRD are identical proteins endowed with RNase activity and DNA-binding properties. A new RNA cleavage mechanism has to be postulated for Sac7e since, in contrast to common RNases (e.g. RNase A and T1), no histidines are present in the amino acid sequence. Differences between the very closely related 7 kDa proteins from two Sulfolobus strains converting DNA-binding proteins into RNases are pointed out and discussed, whereas substitutions of Glu by Gln (S. solfataricus) or by Lys (S. acidocaldarius) seem to be crucial. ; save_ save_references_14 _Saveframe_category citation _Citation_full ; McCrary BS, Edmondson SP, Shriver JW. J Mol Biol. 1996 Dec 13;264(4):784-805. ; _Citation_title 'Hyperthermophile protein folding thermodynamics: differential scanning calorimetry and chemical denaturation of Sac7d.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 8980686 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 McCrary 'B S' S. . 2 Edmondson 'S P' P. . 3 Shriver 'J W' W. . stop_ _Journal_abbreviation 'J. Mol. Biol.' _Journal_name_full 'Journal of molecular biology' _Journal_volume 264 _Journal_issue 4 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 784 _Page_last 805 _Year 1996 _Details ; Recombinant Sac7d protein from the thermoacidophile Sulfolobus acidocaldarius is shown to be stable towards acid, thermal and chemical denaturation. The protein maintains a compact native fold between pH 0 and 10 in 0.3 M KCl and 25 degrees C as indicated by near and far UV circular dichroism spectra. Thermal unfolding followed by differential scanning calorimetry (DSC) occurs as a reversible, two-state transition from pH 0 to 10, with a maximal Tm of 90.7 degrees C between pH 5 and 9. At pH 0 the protein unfolds with a Tm of 63.3 degrees C. Plots of the enthalpy of unfolding as a function of Tm are linear and yield an anomalously low delta Cp of 497 (+/-20) cal deg-1 mol-1 using the Kirchhoff relation. Guanidine hydrochloride and urea-induced chemical denaturation of Sac7d occur reversibly and can be followed by circular dichroism. Global non-linear regression of the chemical denaturation data constrained by DSC determined values for delta Hm and Tm yields a delta Cp of unfolding of 858 (+/-21) cal deg-1 mol-1. The higher delta Cp is in good agreement with that predicted from the buried polar and apolar surface areas using the NMR solution structure. It is similar to values reported for mesophile proteins of comparable size, indicating that the packing and change in solvent-accessible surface area on unfolding are not unusual. Similarly, guanidine hydrochloride and urea m-values are in good agreement with those expected for a protein of 66 residues. Possible explanations for the difference in delta Cp determined by application of the Kirchhoff relation to DSC data and that determined by the global fit are discussed. Protein stability curves defined by either delta Cp values are similar to those observed for small mesophile proteins. Although the protein is thermally stable, it is marginally stable thermodynamically with a free energy of unfolding of 1.6 (+/-0.1) kcal mol-1 at the growth temperature of 80 degrees C. The large number of potential ion pairs on the surface of this hyperthermophile protein do not result in an inordinate increase in stability. Post-translational modification, possibly lysine monomethylation, appears to be the single most important stabilizing factor that distinguishes the native hyperthermophile protein from small mesophile proteins. Additional stabilization in vivo is expected from compatible osmolytes (polyamines) and DNA-binding. ; save_ save_references_15 _Saveframe_category citation _Citation_full ; McAfee JG, Edmondson SP, Zegar I, Shriver JW. Biochemistry. 1996 Apr 2;35(13):4034-45. ; _Citation_title 'Equilibrium DNA binding of Sac7d protein from the hyperthermophile Sulfolobus acidocaldarius: fluorescence and circular dichroism studies.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 8672437 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 McAfee 'J G' G. . 2 Edmondson 'S P' P. . 3 Zegar I . . 4 Shriver 'J W' W. . stop_ _Journal_abbreviation Biochemistry _Journal_name_full Biochemistry _Journal_volume 35 _Journal_issue 13 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 4034 _Page_last 4045 _Year 1996 _Details ; The thermodynamics of the binding of the Sac7d protein of Sulfolobus acidocaldarius to double-stranded DNA has been characterized using spectroscopic signals arising from both the protein and the DNA. Ligand binding density function analysis has been used to demonstrate that the fractional change in protein intrinsic tryptophan fluorescence quenching that occurs upon DNA binding is equal to the fraction of protein bound. Reverse titration data have been fit directly to the McGhee-von Hippel model [McGhee, J., & von Hippel, P. (1974) J. Mol. Biol. 86, 469-489] using nonlinear regression. Sac7d binds noncooperatively to poly(dGdC) x poly(dGdC) with an intrinsic affinity of 6.5 x 10(6) M(-1) and a site size of 4 base pairs in 1 mM KH2PO4 and 50 mM KC1 (pH 6.8). Some binding sequence preference is noted, with the binding to poly(dIdC) x poly(dIdC) over 10-fold stronger than to poly(DAdT) x poly(dAdT). The binding is largely driven by the polyelectrolyte effect and is consistent with a release of 4.4 monovalent cations from DNA upon complex formation or the formation of 5 ion pairs at the protein-DNA interface. Extrapolation of salt back-titration data to 1 M KC1 indicates a -2.2 kcal/mol nonelectrostatic contribution to the binding free energy. A van't Hoff analysis of poly(dGdC) x poly(dGdC) binding shows that the binding enthalpy is approximately zero and the process is entropically driven. The affinity decreases slightly between pH 5.4 and 8.0. There is no significant difference between the binding parameters of recombinant Sac7d and native Sac7 proteins, indicating that methylation of the native protein has no effect on the DNA binding function. The binding of Sac7d to various DNAs leads to a significant increase in the DNA long-wavelength circular dichroism (CD) band, the intensity of which shows a sigmoidal dependence on Sac7d concentration. The sigmoidal CD binding isotherm can be quantitatively modeled by a conformational transition in the DNA that is cooperatively induced when protein monomers are bound within a given number of base pairs, ranging from zero for poly(dIdC) x poly(dIdC) to 8 or less for poly(dAdG) x poly(dCdT). ; save_ save_references_16 _Saveframe_category citation _Citation_full ; Edmondson SP, Qiu L, Shriver JW. Biochemistry. 1995 Oct 17;34(41):13289-304. ; _Citation_title 'Solution structure of the DNA-binding protein Sac7d from the hyperthermophile Sulfolobus acidocaldarius.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 7577913 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Edmondson 'S P' P. . 2 Qiu L . . 3 Shriver 'J W' W. . stop_ _Journal_abbreviation Biochemistry _Journal_name_full Biochemistry _Journal_volume 34 _Journal_issue 41 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 13289 _Page_last 13304 _Year 1995 _Details ; The Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius are a heterogeneous mixture of small, thermostable, nonspecific DNA-binding proteins. One of these proteins, Sac7d, has been overexpressed in Escherichia coli to provide a homogeneous preparation for structure, stability, and function studies. We present here essentially complete sequence-specific 1H NMR assignments for Sac7d, a delineation of secondary structural elements, and the high-resolution solution structure obtained from a full relaxation matrix refinement. The final structure provides an excellent fit to the NMR data with an NOE R-factor of 0.27 for backbone NOEs. The structure has a compact globular fold with 82% of the sequence involved in regular secondary structure: an antiparallel two-stranded beta-ribbon with a tight turn, followed by a short 3(10) helix, an antiparallel three-stranded beta-sheet, another short 3(10) helix, and finally four turns of alpha-helix. The amphipathic alpha-helix packs across the hydrophobic face of the three-stranded beta-sheet in an open-faced sandwich arrangement with at least one turn of the helix exposed beyond the sheet. The hydrophobic face of the beta-ribbon packs against a corner of the twisted beta-sheet. The single tryptophan responsible for the 88% fluorescence quenching upon DNA binding is exposed on the surface of the three-stranded beta-sheet. Lysines 5 and 7, whose monomethylation may be associated with enhanced thermostability, are highly solvent exposed along the inner edge of the two-stranded ribbon. The structure of Sac7d differs in many respects from that reported for the homologous native Sso7d [Baumann et al. (1994) Nature Struct. Biol. 1, 808] with a backbone RMSD greater than 3.0 A, largely due to the packing and length of the C-terminal alpha-helix which may be important in Sac7d DNA binding. ; save_ save_references_17 _Saveframe_category citation _Citation_full ; McAfee JG, Edmondson SP, Datta PK, Shriver JW, Gupta R. Biochemistry. 1995 Aug 8;34(31):10063-77. ; _Citation_title 'Gene cloning, expression, and characterization of the Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 7632679 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 McAfee 'J G' G. . 2 Edmondson 'S P' P. . 3 Datta 'P K' K. . 4 Shriver 'J W' W. . 5 Gupta R . . stop_ _Journal_abbreviation Biochemistry _Journal_name_full Biochemistry _Journal_volume 34 _Journal_issue 31 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 10063 _Page_last 10077 _Year 1995 _Details ; The genes for two Sac7 DNA-binding proteins, Sac7d and Sac7e, from the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned into Escherichia coli and sequenced. The sac7d and sac7e open reading frames encode 66 amino acid (7608 Da) and 65 amino acid (7469 Da) proteins, respectively. Southern blots indicate that these are the only two Sac7 protein genes in S. acidocaldarius, each present as a single copy. Sac7a, b, and c proteins appear to be carboxy-terminal modified Sac7d species. The transcription initiation and termination regions of the sac7d and sac7e genes have been identified along with the promoter elements. Potential ribosome binding sites have been identified downstream of the initiator codons. The sac7d gene has been expressed in E. coli, and various physical properties of the recombinant protein have been compared with those of native Sac7. The UV absorbance spectra and extinction coefficients, the fluorescence excitation and emission spectra, the circular dichroism, and the two-dimensional double-quantum filtered 1H NMR spectra of the native and recombinant species are essentially identical, indicating essentially identical local and global folds. The recombinant and native proteins bind and stabilize double-stranded DNA with a site size of 3.5 base pairs and an intrinsic binding constant of 2 x 10(7) M-1 for poly[dGdC].poly[dGdC] in 0.01 M KH2PO4 at pH 7.0. The availability of the recombinant protein permits a direct comparison of the thermal stabilities of the methylated and unmethylated forms of the protein. Differential scanning calorimetry demonstrates that the native protein is extremely thermostable and unfolds reversibly at pH 6.0 with a Tm of approximately 100 degrees C, while the recombinant protein unfolds at 92.7 degrees C. ; save_ ################################## # Molecular system description # ################################## save_system_Sac7d _Saveframe_category molecular_system _Mol_system_name 'Sac7d V30I monomer' _Abbreviation_common Sac7d _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label 'Sac7d V30I subunit 1' $Sac7d stop_ _System_molecular_weight . _System_physical_state native _System_oligomer_state monomer _System_paramagnetic no _System_thiol_state 'not present' loop_ _Biological_function 'DNA-binding protein' 'chromatin protein' 'function unknown' stop_ _Database_query_date . _Details . save_ ######################## # Monomeric polymers # ######################## save_Sac7d _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class protein _Name_common Sac7d _Abbreviation_common Sac7d _Molecular_mass 7623 _Mol_thiol_state 'not present' _Details . ############################## # Polymer residue sequence # ############################## _Residue_count 66 _Mol_residue_sequence ; MVKVKFKYKGEEKEVDTSKI KKVWRVGKMISFTYDDNGKT GRGAVSEKDAPKELLDMLAR AEREKK ; loop_ _Residue_seq_code _Residue_label 1 MET 2 VAL 3 LYS 4 VAL 5 LYS 6 PHE 7 LYS 8 TYR 9 LYS 10 GLY 11 GLU 12 GLU 13 LYS 14 GLU 15 VAL 16 ASP 17 THR 18 SER 19 LYS 20 ILE 21 LYS 22 LYS 23 VAL 24 TRP 25 ARG 26 VAL 27 GLY 28 LYS 29 MET 30 ILE 31 SER 32 PHE 33 THR 34 TYR 35 ASP 36 ASP 37 ASN 38 GLY 39 LYS 40 THR 41 GLY 42 ARG 43 GLY 44 ALA 45 VAL 46 SER 47 GLU 48 LYS 49 ASP 50 ALA 51 PRO 52 LYS 53 GLU 54 LEU 55 LEU 56 ASP 57 MET 58 LEU 59 ALA 60 ARG 61 ALA 62 GLU 63 ARG 64 GLU 65 LYS 66 LYS stop_ _Sequence_homology_query_date . _Sequence_homology_query_revised_last_date 2015-01-21 loop_ _Database_name _Database_accession_code _Database_entry_mol_name _Sequence_query_to_submitted_percentage _Sequence_subject_length _Sequence_identity _Sequence_positive _Sequence_homology_expectation_value BMRB 5905 Sac7d 100.00 66 98.48 100.00 3.85e-37 PDB 1AZP "Hyperthermophile Chromosomal Protein Sac7d Bound With Kinked Dna Duplex" 100.00 66 98.48 100.00 3.85e-37 PDB 1AZQ "Hyperthermophile Chromosomal Protein Sac7d Bound With Kinked Dna Duplex" 100.00 66 98.48 100.00 3.85e-37 PDB 1CA5 "Intercalation Site Of Hyperthermophile Chromosomal Protein Sso7dSAC7D Bound To Dna" 100.00 66 98.48 100.00 3.85e-37 PDB 1CA6 "Intercalation Site Of Hyperthermophile Chromosomal Protein Sso7dSAC7D Bound To Dna" 100.00 66 98.48 100.00 3.85e-37 PDB 1SAP "Hyperthermophile Protein, Relaxation Matrix Refinement Structure" 98.48 66 98.46 100.00 2.69e-36 PDB 1WD0 "Crystal Structures Of The Hyperthermophilic Chromosomal Protein Sac7d In Complex With Dna Decamers" 100.00 66 98.48 100.00 3.85e-37 PDB 1WD1 "Crystal Structures Of The Hyperthermophilic Chromosomal Protein Sac7d In Complex With Dna Decamers" 100.00 66 98.48 100.00 3.85e-37 PDB 1WTP "Hyperthermophile Chromosomal Protein Sac7d Single Mutant M29f In Complex With Dna Gcga(Ubr)cgc" 100.00 66 96.97 98.48 2.85e-36 PDB 1WTQ "Hyperthermophile Chromosomal Protein Sac7d Single Mutant M29f In Complex With Dna Gtaattac" 100.00 66 96.97 98.48 2.85e-36 PDB 1WTR "Hyperthermophile Chromosomal Protein Sac7d Single Mutant M29a In Complex With Dna Gcgatcgc" 100.00 66 96.97 98.48 3.24e-36 PDB 1WTV "Hyperthermophile Chromosomal Protein Sac7d Single Mutant M29a In Complex With Dna Gtaattac" 100.00 66 96.97 98.48 3.24e-36 PDB 1WTW "Hyperthermophile Chromosomal Protein Sac7d Single Mutant V26a In Complex With Dna Gcgatcgc" 100.00 66 96.97 98.48 1.39e-36 PDB 1WTX "Hyperthermophile Chromosomal Protein Sac7d Single Mutant V26a In Complex With Dna Gtaattac" 100.00 66 96.97 98.48 1.39e-36 PDB 1WVL "Crystal Structure Of Multimeric Dna-Binding Protein Sac7d- Gcn4 With Dna Decamer" 100.00 80 98.48 100.00 2.87e-37 PDB 1XX8 "Nmr Structure Of The W24a Mutant Of The Hyperthermophile Sac7d Protein" 100.00 66 96.97 98.48 8.07e-36 GB AAA80315 "DNA-binding protein [Sulfolobus sp.]" 100.00 66 98.48 100.00 3.85e-37 GB AAY79492 "conserved DNA-binding protein 7e [Sulfolobus acidocaldarius DSM 639]" 100.00 66 98.48 100.00 3.85e-37 GB AGE70041 "DNA-binding protein 7e [Sulfolobus acidocaldarius N8]" 100.00 66 98.48 100.00 3.85e-37 GB AGE72316 "DNA-binding protein 7e [Sulfolobus acidocaldarius Ron12/I]" 100.00 66 98.48 100.00 3.85e-37 GB AHC50588 "DNA-binding protein [Sulfolobus acidocaldarius SUSAZ]" 100.00 66 98.48 100.00 3.85e-37 REF WP_011276993 "DNA-binding protein [Sulfolobus acidocaldarius]" 100.00 66 98.48 100.00 3.85e-37 REF YP_007433189 "DNA-binding protein 7e [Sulfolobus acidocaldarius N8]" 100.00 66 98.48 100.00 3.85e-37 REF YP_007435464 "DNA-binding protein 7e [Sulfolobus acidocaldarius Ron12/I]" 100.00 66 98.48 100.00 3.85e-37 REF YP_254785 "DNA-binding protein 7e [Sulfolobus acidocaldarius DSM 639]" 100.00 66 98.48 100.00 3.85e-37 SP P13123 "RecName: Full=DNA-binding protein 7d; AltName: Full=7 kDa DNA-binding protein d; AltName: Full=Sac7d; Contains: RecName: Full=D" 100.00 66 98.48 100.00 3.85e-37 stop_ save_ #################### # Natural source # #################### save_natural_source _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species $Sac7d 'Sulfolobus acidocaldarius Brock et al.' 2285 Archaea . Sulfolobus acidocaldarius stop_ save_ ######################### # Experimental source # ######################### save_experimental_source _Saveframe_category experimental_source loop_ _Mol_label _Production_method _Host_organism_name_common _Genus _Species _Strain _Vector_name $Sac7d 'recombinant technology' . . . . . stop_ save_ ##################################### # Sample contents and methodology # ##################################### ######################## # Sample description # ######################## save_Sac7d_V30I _Saveframe_category sample _Sample_type solution _Details . loop_ _Mol_label _Concentration_value _Concentration_value_units _Concentration_min_value _Concentration_max_value _Isotopic_labeling $Sac7d 1.18 mM 1 1.5 '[U-98% 15N]' KCl 0.3 M . . . H2O 90 % . . . D2O 10 % . . . DSS 100 uM . . . stop_ save_ ############################ # Computer software used # ############################ save_NMRPipe _Saveframe_category software _Name NMRPipe _Version . loop_ _Task 'data processing' transformation referencing stop_ _Details . save_ save_NMRView _Saveframe_category software _Name NMRView _Version 5 loop_ _Task 'sequential assignments' stop_ _Details . save_ ######################### # Experimental detail # ######################### ################################## # NMR Spectrometer definitions # ################################## save_NMR_spectrometer_1 _Saveframe_category NMR_spectrometer _Manufacturer Varian _Model VXR-S _Field_strength 500 _Details . save_ save_NMR_spectrometer_2 _Saveframe_category NMR_spectrometer _Manufacturer Varian _Model INOVA _Field_strength 500 _Details . save_ save_NMR_spectrometer_3 _Saveframe_category NMR_spectrometer _Manufacturer Varian _Model INOVA _Field_strength 800 _Details . save_ ############################# # NMR applied experiments # ############################# save_1H,15N_HSQC_1 _Saveframe_category NMR_applied_experiment _Experiment_name '1H,15N HSQC' _Sample_label $Sac7d_V30I save_ save_3D_HSQC-TOCSY_2 _Saveframe_category NMR_applied_experiment _Experiment_name '3D HSQC-TOCSY' _Sample_label $Sac7d_V30I save_ save_3D_HSQC-NOESY_3 _Saveframe_category NMR_applied_experiment _Experiment_name '3D HSQC-NOESY' _Sample_label $Sac7d_V30I save_ save_NMR_spec_expt__0_1 _Saveframe_category NMR_applied_experiment _Experiment_name '1H,15N HSQC' _BMRB_pulse_sequence_accession_number . _Details . save_ save_NMR_spec_expt__0_2 _Saveframe_category NMR_applied_experiment _Experiment_name '3D HSQC-TOCSY' _BMRB_pulse_sequence_accession_number . _Details . save_ save_NMR_spec_expt__0_3 _Saveframe_category NMR_applied_experiment _Experiment_name '3D HSQC-NOESY' _BMRB_pulse_sequence_accession_number . _Details . save_ ####################### # Sample conditions # ####################### save_sample_conds _Saveframe_category sample_conditions _Details . loop_ _Variable_type _Variable_value _Variable_value_error _Variable_value_units pH 4.06 0.1 n/a temperature 303 1 K stop_ save_ #################### # NMR parameters # #################### ############################## # Assigned chemical shifts # ############################## ################################ # Chemical shift referencing # ################################ save_chemical_shift_reference _Saveframe_category chemical_shift_reference _Details . loop_ _Mol_common_name _Atom_type _Atom_isotope_number _Atom_group _Chem_shift_units _Chem_shift_value _Reference_method _Reference_type _External_reference_sample_geometry _External_reference_location _External_reference_axis _Indirect_shift_ratio DSS H 1 'methyl protons' ppm 0.0 internal direct . . . 1.0 DSS N 15 'methyl protons' ppm 0.0 . indirect . . . 0.101329118 stop_ save_ ################################### # Assigned chemical shift lists # ################################### ################################################################### # Chemical Shift Ambiguity Index Value Definitions # # # # The values other than 1 are used for those atoms with different # # chemical shifts that cannot be assigned to stereospecific atoms # # or to specific residues or chains. # # # # Index Value Definition # # # # 1 Unique (including isolated methyl protons, # # geminal atoms, and geminal methyl # # groups with identical chemical shifts) # # (e.g. ILE HD11, HD12, HD13 protons) # # 2 Ambiguity of geminal atoms or geminal methyl # # proton groups (e.g. ASP HB2 and HB3 # # protons, LEU CD1 and CD2 carbons, or # # LEU HD11, HD12, HD13 and HD21, HD22, # # HD23 methyl protons) # # 3 Aromatic atoms on opposite sides of # # symmetrical rings (e.g. TYR HE1 and HE2 # # protons) # # 4 Intraresidue ambiguities (e.g. LYS HG and # # HD protons or TRP HZ2 and HZ3 protons) # # 5 Interresidue ambiguities (LYS 12 vs. LYS 27) # # 6 Intermolecular ambiguities (e.g. ASP 31 CA # # in monomer 1 and ASP 31 CA in monomer 2 # # of an asymmetrical homodimer, duplex # # DNA assignments, or other assignments # # that may apply to atoms in one or more # # molecule in the molecular assembly) # # 9 Ambiguous, specific ambiguity not defined # # # ################################################################### save_V30I_shift_set_1 _Saveframe_category assigned_chemical_shifts _Details . loop_ _Sample_label $Sac7d_V30I stop_ _Sample_conditions_label $sample_conds _Chem_shift_reference_set_label $chemical_shift_reference _Mol_system_component_name 'Sac7d V30I subunit 1' _Text_data_format . _Text_data . loop_ _Atom_shift_assign_ID _Residue_author_seq_code _Residue_seq_code _Residue_label _Atom_name _Atom_type _Chem_shift_value _Chem_shift_value_error _Chem_shift_ambiguity_code 1 . 2 VAL H H 8.675 0.01 1 2 . 2 VAL N N 123.971 0.05 1 3 . 3 LYS H H 8.424 0.01 1 4 . 3 LYS N N 123.876 0.05 1 5 . 4 VAL H H 8.876 0.01 1 6 . 4 VAL N N 121.556 0.05 1 7 . 5 LYS H H 8.494 0.01 1 8 . 5 LYS N N 127.835 0.05 1 9 . 6 PHE H H 8.675 0.01 1 10 . 6 PHE N N 120.258 0.05 1 11 . 7 LYS H H 8.708 0.01 1 12 . 7 LYS N N 119.294 0.05 1 13 . 8 TYR H H 8.989 0.01 1 14 . 8 TYR N N 124.941 0.05 1 15 . 9 LYS H H 9.155 0.01 1 16 . 9 LYS N N 127.976 0.05 1 17 . 10 GLY H H 8.476 0.01 1 18 . 10 GLY N N 103.500 0.05 1 19 . 11 GLU H H 7.868 0.01 1 20 . 11 GLU N N 119.996 0.05 1 21 . 12 GLU H H 8.689 0.01 1 22 . 12 GLU N N 123.474 0.05 1 23 . 13 LYS H H 8.848 0.01 1 24 . 13 LYS N N 126.533 0.05 1 25 . 14 GLU H H 7.932 0.01 1 26 . 14 GLU N N 116.671 0.05 1 27 . 15 VAL H H 8.651 0.01 1 28 . 15 VAL N N 121.011 0.05 1 29 . 16 ASP H H 8.540 0.01 1 30 . 16 ASP N N 127.101 0.05 1 31 . 17 THR H H 8.790 0.01 1 32 . 17 THR N N 116.111 0.05 1 33 . 18 SER H H 8.619 0.01 1 34 . 18 SER N N 116.507 0.05 1 35 . 19 LYS H H 7.971 0.01 1 36 . 19 LYS N N 119.267 0.05 1 37 . 20 ILE H H 7.274 0.01 1 38 . 20 ILE N N 120.854 0.05 1 39 . 21 LYS H H 8.866 0.01 1 40 . 21 LYS N N 127.832 0.05 1 41 . 22 LYS H H 7.470 0.01 1 42 . 22 LYS N N 119.896 0.05 1 43 . 23 VAL H H 8.179 0.01 1 44 . 23 VAL N N 118.804 0.05 1 45 . 24 TRP H H 9.261 0.01 1 46 . 24 TRP N N 125.193 0.05 1 47 . 24 TRP NE1 N 129.279 0.05 1 48 . 24 TRP HE1 H 10.074 0.01 2 49 . 25 ARG H H 8.784 0.01 1 50 . 25 ARG N N 120.386 0.05 1 51 . 26 VAL H H 8.615 0.01 1 52 . 26 VAL N N 126.037 0.05 1 53 . 27 GLY H H 9.121 0.01 1 54 . 27 GLY N N 118.323 0.05 1 55 . 28 LYS H H 8.884 0.01 1 56 . 28 LYS N N 126.621 0.05 1 57 . 29 MET H H 8.175 0.01 1 58 . 29 MET N N 118.050 0.05 1 59 . 30 ILE H H 9.280 0.01 1 60 . 30 ILE N N 123.644 0.05 1 61 . 31 SER H H 8.717 0.01 1 62 . 31 SER N N 122.907 0.05 1 63 . 32 PHE H H 7.586 0.01 1 64 . 32 PHE N N 114.592 0.05 1 65 . 33 THR H H 8.926 0.01 1 66 . 33 THR N N 109.064 0.05 1 67 . 34 TYR H H 8.612 0.01 1 68 . 34 TYR N N 116.158 0.05 1 69 . 35 ASP H H 8.572 0.01 1 70 . 35 ASP N N 119.438 0.05 1 71 . 36 ASP H H 8.918 0.01 1 72 . 36 ASP N N 127.105 0.05 1 73 . 37 ASN H H 8.689 0.01 1 74 . 37 ASN N N 120.447 0.05 1 75 . 37 ASN ND2 N 113.132 0.05 1 76 . 37 ASN HD21 H 7.590 0.01 2 77 . 37 ASN HD22 H 6.905 0.01 2 78 . 38 GLY H H 8.795 0.01 1 79 . 38 GLY N N 106.152 0.05 1 80 . 39 LYS H H 7.489 0.01 1 81 . 39 LYS N N 120.117 0.05 1 82 . 40 THR H H 8.612 0.01 1 83 . 40 THR N N 118.351 0.05 1 84 . 41 GLY H H 8.528 0.01 1 85 . 41 GLY N N 115.315 0.05 1 86 . 42 ARG H H 7.720 0.01 1 87 . 42 ARG N N 117.967 0.05 1 88 . 43 GLY H H 7.725 0.01 1 89 . 43 GLY N N 107.320 0.05 1 90 . 44 ALA H H 6.731 0.01 1 91 . 44 ALA N N 117.474 0.05 1 92 . 45 VAL H H 9.040 0.01 1 93 . 45 VAL N N 116.438 0.05 1 94 . 46 SER H H 9.026 0.01 1 95 . 46 SER N N 121.484 0.05 1 96 . 47 GLU H H 8.803 0.01 1 97 . 47 GLU N N 123.859 0.05 1 98 . 48 LYS H H 8.109 0.01 1 99 . 48 LYS N N 116.159 0.05 1 100 . 49 ASP H H 7.562 0.01 1 101 . 49 ASP N N 116.636 0.05 1 102 . 50 ALA H H 7.430 0.01 1 103 . 50 ALA N N 124.408 0.05 1 104 . 52 LYS H H 8.987 0.01 1 105 . 52 LYS N N 125.234 0.05 1 106 . 53 GLU H H 9.543 0.01 1 107 . 53 GLU N N 116.264 0.05 1 108 . 54 LEU H H 7.282 0.01 1 109 . 54 LEU N N 117.000 0.05 1 110 . 55 LEU H H 7.419 0.01 1 111 . 55 LEU N N 119.179 0.05 1 112 . 56 ASP H H 8.783 0.01 1 113 . 56 ASP N N 120.603 0.05 1 114 . 57 MET H H 7.627 0.01 1 115 . 57 MET N N 119.752 0.05 1 116 . 58 LEU H H 7.966 0.01 1 117 . 58 LEU N N 121.198 0.05 1 118 . 59 ALA H H 8.044 0.01 1 119 . 59 ALA N N 120.974 0.05 1 120 . 60 ARG H H 7.831 0.01 1 121 . 60 ARG N N 117.114 0.05 1 122 . 61 ALA H H 7.959 0.01 1 123 . 61 ALA N N 122.278 0.05 1 124 . 62 GLU H H 8.237 0.01 1 125 . 62 GLU N N 116.639 0.05 1 126 . 63 ARG H H 7.804 0.01 1 127 . 63 ARG N N 119.045 0.05 1 128 . 64 GLU H H 7.904 0.01 1 129 . 64 GLU N N 119.274 0.05 1 130 . 65 LYS H H 8.014 0.01 1 131 . 65 LYS N N 121.726 0.05 1 132 . 66 LYS H H 7.983 0.01 1 133 . 66 LYS N N 127.350 0.05 1 stop_ save_