data_6123 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; 1H chemical shift assignments for truncated hevein of 32 aa bound to N,N',N''-triacetylchitotriose ; _BMRB_accession_number 6123 _BMRB_flat_file_name bmr6123.str _Entry_type original _Submission_date 2004-03-02 _Accession_date 2004-03-02 _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 Aboitiz Nuria . . 2 Vila-Perello Miquel . . 3 Groves Patrick . . 4 Asensio Juan L. . 5 Andreu David . . 6 Canada F. J. . 7 Jimenez-Barbero Jesus . . stop_ loop_ _Saveframe_category_type _Saveframe_category_type_count assigned_chemical_shifts 1 stop_ loop_ _Data_type _Data_type_count "1H chemical shifts" 165 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2004-10-13 original author . stop_ _Original_release_date 2004-10-13 save_ ############################# # Citation for this entry # ############################# save_entry_citation _Saveframe_category entry_citation _Citation_full . _Citation_title 'NMR and modeling studies of protein-carbohydrate interactions: synthesis, three-dimensional structure, and recognition properties of a minimum hevein domain with binding affinity for chitooligosaccharides' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 15368576 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Aboitiz Nuria . . 2 Vila-Perello Miquel . . 3 Groves Patrick . . 4 Asensio Juan L. . 5 Andreu David . . 6 Canada F. Javier . 7 Jimenez-Barbero Jesus . . stop_ _Journal_abbreviation ChemBioChem _Journal_volume 5 _Journal_issue 9 _Journal_CSD . _Book_chapter_title . _Book_volume . _Book_series . _Book_ISBN . _Conference_state_province . _Conference_abstract_number . _Page_first 1245 _Page_last 1245 _Year 2004 _Details . loop_ _Keyword carbohydrate hevein 'Molecular Dynamics' 'molecular recognition' NMR protein stop_ save_ ####################################### # Cited references within the entry # ####################################### save_reference-1 _Saveframe_category citation _Citation_full ; Asensio JL, Canada FJ, Bruix M, Rodriguez-Romero A, Jimenez-Barbero J. Eur J Biochem. 1995 Jun 1;230(2):621-33. ; _Citation_title 'The interaction of hevein with N-acetylglucosamine-containing oligosaccharides. Solution structure of hevein complexed to chitobiose.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 7607237 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Asensio J L. . 2 Canada F J. . 3 Bruix M . . 4 Rodriguez-Romero A . . 5 Jimenez-Barbero J . . stop_ _Journal_abbreviation 'Eur. J. Biochem.' _Journal_name_full 'European journal of biochemistry / FEBS' _Journal_volume 230 _Journal_issue 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 621 _Page_last 633 _Year 1995 _Details ; The three-dimensional structure of hevein, a small protein isolated from the latex of Hevea brasiliensis (rubber tree), in water solution has been obtained by using 1H-NMR spectroscopy and dynamic simulated annealing calculations. The average root-mean-square deviation (rmsd) of the best 20 refined structures generated using DIANA prior to simulated annealing was 0.092 nm for the backbone atoms and 0.163 nm for all heavy atoms (residues 3-41). The specific interaction of hevein with N-acetylglucosamine-containing oligosaccharides has also been analyzed by 1H-NMR. The association constants, Ka, for the binding of hevein to GlcNAc, chitobiose [GlcNAc-beta(1-->4)-GlcNAc], chitotriose [GlcNAc-beta(1-->4)-GlcNAc-beta(1-->4)-GlcNAc], and GlcNAc-alpha(1-->6)-Man have been estimated from 1H-NMR titration experiments. Since the measured Ka values for chitobiose binding are almost identical with and without calcium ions, it is shown that these cations are not required for sugar binding. The association increases in the order GlcNAc-alpha(1-->6)-Man < or = GlcNAc < chitobiose < chitotriose. The equilibrium thermodynamic parameters entropy and enthalpy of binding, delta S0 and delta H0, for the hevein-chitobiose and hevein-chitotriose associations have been obtained from van't Hoff analysis of the temperature dependence of the Ka values between 25-40 degrees C. The driving force for the binding process is provided for a negative delta H0 which is partially compensated by a negative delta S0. These negative signs seem to indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. Protein-carbohydrate nuclear Overhauser enhancements have allowed a three-dimensional model of the hevein-chitobiose complex to be built. From inspection of this model, a hydrogen bond between Ser19 and the non-reducing N-acetyl carbonyl group is suggested, as well as between Tyr30 and HO-3 of the same sugar residue. The N-acetyl methyl group of the non-reducing GlcNAc displays non-polar contacts to the aromatic Tyr30 and Trp21 residues. In addition, the higher affinities deduced for the beta-linked oligosaccharides with respect to GlcNAc and GlcNAc-alpha(1-->6)-Man can be explained by favourable stacking of the second beta-linked GlcNAc moiety and Trp21. ; save_ save_reference-2 _Saveframe_category citation _Citation_full ; Martins JC, Maes D, Loris R, Pepermans HA, Wyns L, Willem R, Verheyden P. J Mol Biol. 1996 May 3;258(2):322-33. ; _Citation_title 'H NMR study of the solution structure of Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 8627629 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Martins J C. . 2 Maes D . . 3 Loris R . . 4 Pepermans H A. . 5 Wyns L . . 6 Willem R . . 7 Verheyden P . . stop_ _Journal_abbreviation 'J. Mol. Biol.' _Journal_name_full 'Journal of molecular biology' _Journal_volume 258 _Journal_issue 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 322 _Page_last 333 _Year 1996 _Details ; The conformation in water of antimicrobial protein 2 from Amaranthus caudatus (Ac-AMP2) was determined using 1H NMR, DIANA and restrained molecular modeling. Ac-AMP2 is a 30 amino acid residue, lectin-like protein that specifically binds to chitin, a polymer of beta-1,4-N-acetyl-D-glucosamine. After sequence specific resonance assignments, a total of 198 distance restraints were collected from 2D NOESY buildup spectra at 500 MHz at pH 2, supplemented by a 2D NOESY spectrum at 600 MHz. The location of the three previously unassigned disulfide bridges was determined from preliminary DIANA structures, using a statistical analysis of intercystinyl distances. The solution structure of Ac-AMP2 is presented as a set of 26 DIANA structures, further refined by restrained molecular dynamics using a simulated annealing protocol in the AMBER force field, with a backbone r.m.s.d. for the well defined Glu3-Cys28 segment of 0.69(+/-0.12) angstroms. The main structural element is an antiparallel beta-sheet from Met13 to Lys23 including a betaI-turn over Gln17-Phel8 with a beta bulge at Gly19. In addition, a beta'I turn over Arg6-Gly7, a beta'III turn over Ser11-Gly12 and a helical turn from Gly24 to Cys28 are identified. This structure is very similar to the equivalent regions of the X-ray structure of wheat germ agglutinin and the NMR structure of hevein. ; save_ save_reference-3 _Saveframe_category citation _Citation_full ; Martins JC, Maes D, Loris R, Pepermans HA, Wyns L, Willem R, Verheyden P. J Mol Biol. 1996 May 3;258(2):322-33. ; _Citation_title 'NMR investigations of protein-carbohydrate interactions: refined three-dimensional structure of the complex between hevein and methyl beta-chitobioside.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 9592123 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Asensio J L. . 2 Canada F J. . 3 Bruix M . . 4 Gonzalez C . . 5 Khiar N . . 6 Rodriguez-Romero A . . 7 Jimenez-Barbero J . . stop_ _Journal_abbreviation Glycobiology _Journal_name_full Glycobiology _Journal_volume 8 _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 569 _Page_last 577 _Year 1998 _Details ; The specific interaction of hevein with GlcNAc-containing oligosaccharides has been analyzed by1H-NMR spectroscopy. The association constants for the binding of hevein to a variety of ligands have been estimated from1H-NMR titration experiments. The association constants increase in the order GlcNAc-alpha(1-->6)-Man < GlcNAc < benzyl-beta-GlcNAc < p-nitrophenyl-beta-GlcNAc < chitobiose < p-nitrophenyl-beta-chitobioside < methyl-beta-chitobioside < chitotriose. Entropy and enthalpy of binding for different complexes have been obtained from van't Hoff analysis. The driving force for the binding process is provided by a negative DeltaH0which is partially compensated by negative DeltaS0. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 475 accurate protein proton-proton distance constraints after employing the MARDIGRAS program. In addition, 15 unambiguous protein/carbohydrate NOEs were detected. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the highly refined solution conformation of this protein-carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein nOe's were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 20 refined structures was 0.055 nm, while the heavy atom rmsd was 0.116 nm. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to the complex. A comparison of the three-dimensional structure of hevein in solution to those reported for wheat germ agglutinin (WGA) and hevein itself in the solid state has also been performed. The polypeptide conformation has also been compared to the NMR-derived structure of a smaller antifungical peptide, Ac-AMP2. ; save_ save_reference-4 _Saveframe_category citation _Citation_full ; Asensio JL, Siebert HC, von Der Lieth CW, Laynez J, Bruix M, Soedjanaamadja UM, Beintema JJ, Canada FJ, Gabius HJ, Jimenez-Barbero J. Proteins. 2000 Aug 1;40(2):218-36. ; _Citation_title 'NMR investigations of protein-carbohydrate interactions: studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N',N"-triacetylchitotriose.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 10842338 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Asensio J L. . 2 Siebert H C. . 3 'von Der Lieth' C W. . 4 Laynez J . . 5 Bruix M . . 6 Soedjanaamadja U M. . 7 Beintema J J. . 8 Canada F J. . 9 Gabius H J. . 10 Jimenez-Barbero J . . stop_ _Journal_abbreviation Proteins _Journal_name_full Proteins _Journal_volume 40 _Journal_issue 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 218 _Page_last 236 _Year 2000 _Details ; Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N', N"-triacetylchitotriose has been analyzed by (1)H-NMR spectroscopy. Because of its small size, with a chain length of 45 amino acids, this lectin is a prime target to solution-structure determination by NOESY NMR experiments in water. The NMR-analysis was extended to assessment of the topology of the complex between pseudohevein and N, N',N"-triacetylchitotriose. NOESY experiments in water solution provided 342 protein proton-proton distance constraints. Binding of the ligand did not affect the pattern of the protein nuclear Overhauser effect signal noticeably, what would otherwise be indicative of a ligand-induced conformational change. The average backbone (residues 3-41) RMSD of the 20 refined structures was 1.14 A, whereas the heavy atom RMSD was 2.18 A. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectin's capacity to target chitin. In both cases, hydrogen bonds and van der Waals contacts confer stability to the complexes. This conclusion is corroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpically driven. In relation to hevein, the Trp/Tyr-substitution in the binding pocket has only a small effect on the free energy of binding in contrast to engineered galectin-1 and a mammalian C-type lectin. A comparison of the three-dimensional structure of pseudohevein in solution to those reported for wheat germ agglutinin (WGA) in the solid state and for hevein and WGA-B in solution has been performed, providing a data source about structural variability of the hevein domains. The experimentally derived structures and the values of the solvent accessibilities for several key residues have also been compared with conformations obtained by molecular dynamics simulations, pointing to the necessity to further refine the programs to enhance their predictive reliability and, thus, underscoring the importance of this kind of combined analysis in model systems. ; save_ save_reference-5 _Saveframe_category citation _Citation_full ; Asensio JL, Canada FJ, Siebert HC, Laynez J, Poveda A, Nieto PM, Soedjanaamadja UM, Gabius HJ, Jimenez-Barbero J. Chem Biol. 2000 Jul;7(7):529-43. ; _Citation_title 'Structural basis for chitin recognition by defense proteins: GlcNAc residues are bound in a multivalent fashion by extended binding sites in hevein domains.' _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 10903932 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Asensio J L. . 2 Canada F J. . 3 Siebert H C. . 4 Laynez J . . 5 Poveda A . . 6 Nieto P M. . 7 Soedjanaamadja U M. . 8 Gabius H J. . 9 Jimenez-Barbero J . . stop_ _Journal_abbreviation 'Chem. Biol.' _Journal_name_full 'Chemistry & biology' _Journal_volume 7 _Journal_issue 7 _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 529 _Page_last 543 _Year 2000 _Details ; BACKGROUND: Many plants respond to pathogenic attack by producing defense proteins that are capable of reversible binding to chitin, a polysaccharide present in the cell wall of fungi and the exoskeleton of insects. Most of these chitin-binding proteins include a common structural motif of 30 to 43 residues organized around a conserved four-disulfide core, known as the 'hevein domain' or 'chitin-binding' motif. Although a number of structural and thermodynamic studies on hevein-type domains have been reported, these studies do not clarify how chitin recognition is achieved. RESULTS: The specific interaction of hevein with several (GlcNAc)(n) oligomers has been studied using nuclear magnetic resonance (NMR), analytical ultracentrifugation and isothermal titration microcalorimetry (ITC). The data demonstrate that hevein binds (GlcNAc)(2-4) in 1:1 stoichiometry with millimolar affinity. In contrast, for (GlcNAc)(5), a significant increase in binding affinity is observed. Analytical ultracentrifugation studies on the hevein-(GlcNAc)(5,8) interaction allowed detection of protein-carbohydrate complexes with a ratio of 2:1 in solution. NMR structural studies on the hevein-(GlcNAc)(5) complex showed the existence of an extended binding site with at least five GlcNAc units directly involved in protein-sugar contacts. CONCLUSIONS: The first detailed structural model for the hevein-chitin complex is presented on the basis of the analysis of NMR data. The resulting model, in combination with ITC and analytical ultracentrifugation data, conclusively shows that recognition of chitin by hevein domains is a dynamic process, which is not exclusively restricted to the binding of the nonreducing end of the polymer as previously thought. This allows chitin to bind with high affinity to a variable number of protein molecules, depending on the polysaccharide chain length. The biological process is multivalent. ; save_ ################################## # Molecular system description # ################################## save_system_HEV32 _Saveframe_category molecular_system _Mol_system_name 'Truncated hevein of 32 aa' _Abbreviation_common HEV32 _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label HEV32 $HEV32 N,N',N''-triacetylchitotriose $CTO stop_ _System_molecular_weight . _System_physical_state native _System_oligomer_state monomer _System_paramagnetic no _System_thiol_state 'all disulfide bound' loop_ _Biological_function 'chitin binding lectin' stop_ _Database_query_date . _Details . save_ ######################## # Monomeric polymers # ######################## save_HEV32 _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class protein _Name_common hevein _Name_variant 'truncated hevein of 32 aa' _Abbreviation_common HEV32 _Molecular_mass 3485 _Mol_thiol_state 'all disulfide bound' _Details ; The secondary structure of this protein presents an alfa-helix on residues 29 to 32 and an antiparallel beta-sheet on residues 17 to 25. This protein is a hevein domain, and has high homology with AcAMP2 antimicrobial peptide. Carbohydrate binding site involves residues S19, W21, W23 and Y30. ; ############################## # Polymer residue sequence # ############################## _Residue_count 32 _Mol_residue_sequence ; EQCGRQAGGKLCPNNLCCSQ WGWCGSTDEYCS ; loop_ _Residue_seq_code _Residue_label 1 GLU 2 GLN 3 CYS 4 GLY 5 ARG 6 GLN 7 ALA 8 GLY 9 GLY 10 LYS 11 LEU 12 CYS 13 PRO 14 ASN 15 ASN 16 LEU 17 CYS 18 CYS 19 SER 20 GLN 21 TRP 22 GLY 23 TRP 24 CYS 25 GLY 26 SER 27 THR 28 ASP 29 GLU 30 TYR 31 CYS 32 SER stop_ _Sequence_homology_query_date . _Sequence_homology_query_revised_last_date 2015-01-28 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 PDB 1HEV "Hevein: The Nmr Assignment And An Assessment Of Solution- State Folding For The Agglutinin-Toxin Motif" 100.00 43 100.00 100.00 3.01e-13 PDB 1Q9B "Crystal Structure Analysis Of Hev B 6.02 (Hevein) At 1.5 Angstroms Resolution" 100.00 43 100.00 100.00 3.01e-13 PDB 1T0W "25 Nmr Structures Of Truncated Hevein Of 32 Aa (Hevein-32) Complex With N,N,N-Triacetylglucosamina" 100.00 33 100.00 100.00 2.74e-13 EMBL CAA05978 "prohevein [Hevea brasiliensis]" 100.00 187 100.00 100.00 2.39e-13 GB AAA33357 "hevein (HEV1) precursor [Hevea brasiliensis]" 100.00 204 100.00 100.00 1.51e-13 GB AAG16225 "hevein [Hevea brasiliensis]" 100.00 50 100.00 100.00 4.38e-14 GB AAO63573 "HEV2.1 [Hevea brasiliensis]" 100.00 204 100.00 100.00 1.70e-13 GB AAO63574 "HEV2.2 [Hevea brasiliensis]" 100.00 204 100.00 100.00 1.70e-13 GB ABW34946 "hevein [Hevea brasiliensis]" 100.00 204 100.00 100.00 1.70e-13 SP P02877 "RecName: Full=Pro-hevein; AltName: Full=Major hevein; Contains: RecName: Full=Hevein; AltName: Allergen=Hev b 6; Contains: RecN" 100.00 204 100.00 100.00 1.51e-13 stop_ save_ #################### # Natural source # #################### save_natural_source _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species $HEV32 'Para rubber tree' 3981 Eukaryota Viridiplantae Hevea brasiliensis 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 _Details $HEV32 'chemical synthesis' . . . . . ; The sequence was prepared on a MBHA resin by standard solid phase peptide synthesis protocols ; stop_ save_ ##################################### # Sample contents and methodology # ##################################### ######################## # Sample description # ######################## save_sample-1 _Saveframe_category sample _Sample_type solution _Details . loop_ _Mol_label _Concentration_value _Concentration_value_units _Isotopic_labeling $HEV32 0.5 mM . $CTO 3 mM . 'Phosphate buffer' 20 mM . NaCl 100 mM . H2O 90 % . D2O 10 % . stop_ save_ ############################ # Computer software used # ############################ save_XWINNMR _Saveframe_category software _Name xwinnmr _Version 3.2 loop_ _Task 'Data collection' stop_ _Details 'The software performs acquisition and processing of NMR experiments' save_ save_XEASY _Saveframe_category software _Name XEASY _Version 1.3.13 loop_ _Task 'Data analysis' stop_ _Details ; Bartels C., Xia T., Billeter M., Guntert P. and Wuthrich K. (1995) J. Biol. NMR 6, 1-10. The program XEASY for computer-supported NMR spectral analysis of biological macromolecules." This program helps visualization and assignment of 2D NMR spectra ; save_ save_DYANA _Saveframe_category software _Name DYANA _Version 1.5 loop_ _Task 'Structure solution' stop_ _Details ; Guntert P., Mumenthaler C. and Wuthrich K. (1997) J. Mol. Biol. 273, 283-298. Torsion angle dynamics for NMR structure calculation with the new program DYANA.This program yields a collection of protein structures that fit the 1H-1H distance constraints experimentally obtained. ; save_ save_AMBER _Saveframe_category software _Name AMBER _Version 5.0 loop_ _Task 'Structure refinement' stop_ _Details ; Pearlman D. A., Case D. A., Caldwell J. W., Cheatham T. E., DeBolt S., Ross W. S., Ferguson D., Seibel G. L., and Kollman P. A. (1995) Comp. Phys. Commun. 91, 1-41. AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules. The programs used from this package perform molecular dynamics calculations on protein and carbohydrate structures and interactions. . They also convert dyana-format pdb files into amber-format pdb files. ; save_ ######################### # Experimental detail # ######################### ################################## # NMR Spectrometer definitions # ################################## save_NMR_spectrometer _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model AVANCE _Field_strength 800 _Details . save_ ############################# # NMR applied experiments # ############################# save_TOCSY_1 _Saveframe_category NMR_applied_experiment _Experiment_name TOCSY _Sample_label $sample-1 save_ save_NOESY_2 _Saveframe_category NMR_applied_experiment _Experiment_name NOESY _Sample_label $sample-1 save_ ####################### # Sample conditions # ####################### save_conditions-1 _Saveframe_category sample_conditions _Details . loop_ _Variable_type _Variable_value _Variable_value_error _Variable_value_units 'ionic strength' 0.24 0.02 M pH 5.8 0.2 pH temperature 298 0.2 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 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_HEV32_shfit _Saveframe_category assigned_chemical_shifts _Details ; An unassigned signal at 7.018 ppm was observed that may correspond protonated LYS10 (proton in position Z) The atom HG2 of GLN 20 shows up at 0.728 ppm (not in average range) due to near by TRP 21 which shifts it highfield ; loop_ _Sample_label $sample-1 stop_ _Sample_conditions_label $conditions-1 _Chem_shift_reference_set_label $chemical_shift_reference _Mol_system_component_name HEV32 _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 . 1 GLU HA H 2.424 0.02 1 2 . 1 GLU HB2 H 1.587 0.005 1 3 . 1 GLU HB3 H 1.960 0.002 1 4 . 1 GLU HG2 H 1.881 0.002 1 5 . 1 GLU HG3 H 2.499 0.001 1 6 . 2 GLN HA H 4.321 0.002 1 7 . 2 GLN HB2 H 1.903 0.005 1 8 . 2 GLN HB3 H 1.982 0.005 1 9 . 2 GLN HG2 H 2.201 0.02 1 10 . 2 GLN HE21 H 6.939 0.02 1 11 . 2 GLN HE22 H 7.531 0.02 1 12 . 3 CYS H H 7.910 0.02 1 13 . 3 CYS HA H 4.815 0.001 1 14 . 3 CYS HB2 H 2.725 0.002 1 15 . 3 CYS HB3 H 3.039 0.003 1 16 . 4 GLY H H 8.495 0.002 1 17 . 4 GLY HA2 H 3.827 0.002 1 18 . 4 GLY HA3 H 4.110 0.004 1 19 . 5 ARG H H 8.883 0.02 1 20 . 5 ARG HA H 3.966 0.004 1 21 . 5 ARG HB2 H 1.749 0.005 1 22 . 5 ARG HB3 H 1.830 0.004 1 23 . 5 ARG HG2 H 1.587 0.006 1 24 . 5 ARG HD2 H 3.178 0.007 1 25 . 5 ARG HE H 7.235 0.005 1 26 . 6 GLN H H 8.977 0.001 1 27 . 6 GLN HA H 4.192 0.002 1 28 . 6 GLN HB2 H 1.936 0.005 1 29 . 6 GLN HB3 H 2.056 0.003 1 30 . 6 GLN HG2 H 2.428 0.003 1 31 . 6 GLN HG3 H 2.620 0.004 1 32 . 6 GLN HE21 H 6.626 0.004 1 33 . 6 GLN HE22 H 7.633 0.002 1 34 . 7 ALA H H 7.627 0.004 1 35 . 7 ALA HA H 4.540 0.004 1 36 . 7 ALA HB H 0.826 0.002 1 37 . 8 GLY H H 8.044 0.001 1 38 . 8 GLY HA2 H 3.849 0.003 1 39 . 8 GLY HA3 H 3.860 0.003 1 40 . 9 GLY H H 8.298 0.001 1 41 . 9 GLY HA2 H 3.372 0.002 1 42 . 9 GLY HA3 H 4.043 0.001 1 43 . 10 LYS H H 6.941 0.002 1 44 . 10 LYS HA H 4.059 0.003 1 45 . 10 LYS HB2 H 1.774 0.005 1 46 . 10 LYS HG2 H 1.564 0.003 1 47 . 10 LYS HG3 H 1.611 0.02 1 48 . 10 LYS HD2 H 1.724 0.002 1 49 . 10 LYS HE2 H 3.026 0.003 1 50 . 10 LYS HZ H 6.751 0.02 1 51 . 11 LEU H H 8.391 0.004 1 52 . 11 LEU HA H 4.597 0.007 1 53 . 11 LEU HB2 H 1.641 0.003 1 54 . 11 LEU HD1 H 0.911 0.002 1 55 . 11 LEU HD2 H 0.964 0.002 1 56 . 11 LEU HG H 1.789 0.006 1 57 . 12 CYS H H 9.094 0.001 1 58 . 12 CYS HA H 4.705 0.003 1 59 . 12 CYS HB2 H 2.676 0.004 1 60 . 12 CYS HB3 H 2.836 0.002 1 61 . 13 PRO HA H 4.619 0.006 1 62 . 13 PRO HB2 H 1.919 0.005 1 63 . 13 PRO HB3 H 2.290 0.001 1 64 . 13 PRO HG2 H 1.972 0.001 1 65 . 13 PRO HG3 H 2.092 0.001 1 66 . 13 PRO HD2 H 3.613 0.004 1 67 . 13 PRO HD3 H 3.837 0.004 1 68 . 14 ASN H H 8.731 0.001 1 69 . 14 ASN HA H 4.296 0.005 1 70 . 14 ASN HB2 H 2.623 0.003 1 71 . 14 ASN HB3 H 2.904 0.003 1 72 . 14 ASN HD21 H 6.893 0.02 1 73 . 14 ASN HD22 H 7.500 0.02 1 74 . 15 ASN H H 8.677 0.003 1 75 . 15 ASN HA H 4.427 0.004 1 76 . 15 ASN HB2 H 2.881 0.004 1 77 . 15 ASN HB3 H 3.003 0.005 1 78 . 15 ASN HD21 H 6.878 0.003 1 79 . 15 ASN HD22 H 7.579 0.02 1 80 . 16 LEU H H 7.675 0.002 1 81 . 16 LEU HA H 4.295 0.002 1 82 . 16 LEU HB2 H 1.330 0.002 1 83 . 16 LEU HB3 H 1.757 0.001 1 84 . 16 LEU HD1 H 0.699 0.004 1 85 . 16 LEU HD2 H 0.791 0.002 1 86 . 16 LEU HG H 1.681 0.002 1 87 . 17 CYS H H 8.650 0.003 1 88 . 17 CYS HA H 4.419 0.002 1 89 . 17 CYS HB2 H 2.856 0.002 1 90 . 17 CYS HB3 H 3.742 0.003 1 91 . 18 CYS H H 8.838 0.002 1 92 . 18 CYS HA H 5.123 0.002 1 93 . 18 CYS HB2 H 2.978 0.003 1 94 . 18 CYS HB3 H 3.202 0.003 1 95 . 19 SER H H 9.971 0.001 1 96 . 19 SER HA H 5.158 0.002 1 97 . 19 SER HB2 H 4.565 0.001 1 98 . 19 SER HB3 H 4.666 0.004 1 99 . 20 GLN H H 9.168 0.003 1 100 . 20 GLN HA H 4.106 0.008 1 101 . 20 GLN HB2 H 1.416 0.008 1 102 . 20 GLN HB3 H 1.582 0.002 1 103 . 20 GLN HG2 H 0.728 0.003 1 104 . 20 GLN HG3 H 1.335 0.003 1 105 . 20 GLN HE21 H 7.154 0.02 1 106 . 20 GLN HE22 H 7.508 0.001 1 107 . 21 TRP H H 7.768 0.003 1 108 . 21 TRP HA H 4.726 0.001 1 109 . 21 TRP HB2 H 3.202 0.003 1 110 . 21 TRP HB3 H 3.782 0.007 1 111 . 21 TRP HD1 H 7.266 0.002 1 112 . 21 TRP HE1 H 10.353 0.005 1 113 . 21 TRP HE3 H 7.651 0.006 1 114 . 21 TRP HZ2 H 7.478 0.001 1 115 . 21 TRP HZ3 H 7.240 0.003 1 116 . 21 TRP HH2 H 7.267 0.002 1 117 . 22 GLY H H 7.965 0.002 1 118 . 22 GLY HA2 H 3.726 0.002 1 119 . 22 GLY HA3 H 3.866 0.001 1 120 . 23 TRP H H 7.521 0.002 1 121 . 23 TRP HA H 5.148 0.005 1 122 . 23 TRP HB2 H 3.288 0.004 1 123 . 23 TRP HB3 H 3.752 0.003 1 124 . 23 TRP HD1 H 7.206 0.003 1 125 . 23 TRP HE1 H 10.485 0.007 1 126 . 23 TRP HE3 H 7.894 0.002 1 127 . 23 TRP HZ2 H 7.592 0.005 1 128 . 23 TRP HZ3 H 7.268 0.001 1 129 . 23 TRP HH2 H 7.356 0.003 1 130 . 24 CYS H H 8.774 0.002 1 131 . 24 CYS HA H 5.916 0.003 1 132 . 24 CYS HB2 H 2.807 0.008 1 133 . 24 CYS HB3 H 3.036 0.003 1 134 . 25 GLY H H 9.169 0.001 1 135 . 25 GLY HA2 H 2.006 0.002 1 136 . 25 GLY HA3 H 3.743 0.004 1 137 . 26 SER H H 8.978 0.002 1 138 . 26 SER HA H 5.076 0.004 1 139 . 26 SER HB2 H 3.650 0.004 1 140 . 26 SER HB3 H 4.056 0.005 1 141 . 27 THR H H 7.144 0.002 1 142 . 27 THR HA H 4.770 0.005 1 143 . 27 THR HB H 4.919 0.005 1 144 . 27 THR HG2 H 1.414 0.004 1 145 . 28 ASP H H 9.119 0.002 1 146 . 28 ASP HA H 4.380 0.002 1 147 . 28 ASP HB2 H 2.755 0.001 1 148 . 29 GLU H H 8.728 0.004 1 149 . 29 GLU HA H 4.161 0.001 1 150 . 29 GLU HB2 H 1.837 0.003 1 151 . 29 GLU HG2 H 2.213 0.006 1 152 . 29 GLU HG3 H 2.219 0.02 1 153 . 30 TYR H H 7.899 0.004 1 154 . 30 TYR HA H 4.154 0.003 1 155 . 30 TYR HB2 H 2.978 0.003 1 156 . 30 TYR HB3 H 3.049 0.008 1 157 . 30 TYR HD1 H 7.522 0.001 2 158 . 30 TYR HE1 H 6.740 0.001 2 159 . 31 CYS H H 7.962 0.001 1 160 . 31 CYS HA H 4.576 0.002 1 161 . 31 CYS HB2 H 2.895 0.001 1 162 . 31 CYS HB3 H 3.345 0.003 1 163 . 32 SER H H 8.332 0.02 1 164 . 32 SER HA H 4.506 0.001 1 165 . 32 SER HB2 H 3.933 0.004 1 stop_ save_