data_6639 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; On the Importance of Carbohydrate-Aromatic Interactions for the Molecular Recognition of Chitooligosaccharides by Hevein Domains. NMR Studies of the Structure and Binding Affinity of AcAMP2-Like Peptides with non Natural Napthyl and Fluoroaromatic Residues ; _BMRB_accession_number 6639 _BMRB_flat_file_name bmr6639.str _Entry_type original _Submission_date 2005-05-16 _Accession_date 2005-05-16 _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 Chavez M. Isabel . 2 Andreu Cecilia . . 3 Vidal Paloma . . 4 Freire Felix . . 5 Aboitiz Nuria . . 6 Groves Patrick . . 7 Asensio Juan L. . 8 Asensio Gregorio . . 9 Muraki Michiro . . 10 Canada F. Javier . 11 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" 148 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2005-05-25 original author 'original release' 2005-12-16 update author 'update the chemical shifts' stop_ loop_ _Related_BMRB_accession_number _Relationship 6591 AcAMP2F18Pff/Y20Pff 6637 'AcAMP2F18Nalb mutant' 6647 AcAMP2F18Wb 6656 AcAMP2F18Pff/F20Pfff 6657 'AcAMP2F18Nal mutant' stop_ _Original_release_date 2005-05-18 save_ ############################# # Citation for this entry # ############################# save_entry_citation _Saveframe_category entry_citation _Citation_full . _Citation_title ; On the Importance of Carbohydrate-Aromatic Interactions for the Molecular Recognition of Oligosaccharides by Proteins: NMR Studies of the Structure and Binding Affinity of AcAMP2-Like Peptides with Non-Natural Napthyl and Fluoroaromatic Residues. ; _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 16220560 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Chavez M. Isabel . 2 Andreu Cecilia . . 3 Vidal Paloma . . 4 Aboitiz Nuria . . 5 Freire Felix . . 6 Groves Patrick . . 7 Asensio Juan L. . 8 Asensio Gregorio . . 9 Muraki Michiro . . 10 Canada F. Javier . 11 Jimenez-Barbero Jesus . . stop_ _Journal_abbreviation 'Chem. Eur. J.' _Journal_volume 11 _Journal_issue 23 _Journal_CSD . _Book_chapter_title . _Book_volume . _Book_series . _Book_ISBN . _Conference_state_province . _Conference_abstract_number . _Page_first 7060 _Page_last 7074 _Year 2005 _Details . loop_ _Keyword AcAMP2 Chitin 'Molecular Dynamics' NMR 'carbohydrate binding' hevein 'molecular recognition' protein stop_ save_ ####################################### # Cited references within the entry # ####################################### save_reference-1 _Saveframe_category citation _Citation_full ; Martins JC, Maes D, Loris R, Pepermans HA, Wyns L, Willem R, Verheyden P. H NMR study of the solution structure of Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus. 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.' C. . 2 Maes D. . . 3 Loris R. . . 4 Pepermans 'H. A.' 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-2 _Saveframe_category citation _Citation_full ; Muraki M. The importance of CH/pi interactions to the function of carbohydrate binding proteins. Protein Pept Lett. 2002 Jun;9(3):195-209. ; _Citation_title ; The importance of CH/pi interactions to the function of carbohydrate binding proteins. ; _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 12144516 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Muraki Michiro . . stop_ _Journal_abbreviation 'Protein Pept. Lett.' _Journal_name_full 'Protein and peptide letters' _Journal_volume 9 _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 195 _Page_last 209 _Year 2002 _Details ; It is suggested that the interactions between the hydrophobic C-H groups of carbohydrate residues and the pi-electron systems of aromatic amino-acid residues play an important role in the ligand-recognition function of carbohydrate-binding proteins. This review focuses on our recent structural and functional studies of human lysozyme and hevein-domain type lectins (wheat-germ agglutinin and Ac-AMP2) aimed at understanding how CH/pi interactions are involved in the actual binding events. ; save_ save_reference-3 _Saveframe_category citation _Citation_full ; Aboitiz N, Vila-Perello M, Groves P, Asensio JL, Andreu D, Canada FJ, Jimenez-Barbero J. 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. Chembiochem. 2004 Sep 6;5(9):1245-55. ; _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 Luis' L. . 5 Andreu David . . 6 Canada 'Francisco Javier' J. . 7 Jimenez-Barbero Jesus . . stop_ _Journal_abbreviation Chembiochem _Journal_name_full 'Chembiochem : a European journal of chemical 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 1245 _Page_last 1255 _Year 2004 _Details ; HEV32, a 32-residue, truncated hevein lacking eleven C-terminal amino acids, was synthesized by solid-phase methodology and correctly folded with three cysteine bridge pairs. The affinities of HEV32 for small chitin fragments--in the forms of N,N',N"-triacetylchitotriose ((GlcNAc)3) (millimolar) and N,N',N",N"',N"",N""'-hexaacetylchitohexaose ((GlcNAc)6) (micromolar)--as measured by NMR and fluorescence methods, are comparable with those of native hevein. The HEV32 ligand-binding process is enthalpy driven, while entropy opposes binding. The NMR structure of ligand-bound HEV32 in aqueous solution was determined to be highly similar to the NMR structure of ligand-bound hevein. Solvated molecular-dynamics simulations were performed in order to monitor the changes in side-chain conformation of the binding site of HEV32 and hevein upon interaction with ligands. The calculations suggest that the Trp21 side-chain orientation of HEV32 in the free form differs from that in the bound state; this agrees with fluorescence and thermodynamic data. HEV32 provides a simple molecular model for studying protein-carbohydrate interactions and for understanding the physiological relevance of small native hevein domains lacking C-terminal residues. ; 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. 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. 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.' L. . 2 Siebert 'H. C.' C. . 3 'von Der Lieth' 'C. W.' W. . 4 Laynez J. . . 5 Bruix M. . . 6 Soedjanaamadja 'U. M.' M. . 7 Beintema 'J. J.' J. . 8 Canada 'F. J.' J. . 9 Gabius 'H. J.' 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. Structural basis for chitin recognition by defense proteins: GlcNAc residues are bound in a multivalent fashion by extended binding sites in hevein domains. 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.' L. . 2 Canada 'F. J.' J. . 3 Siebert 'H. C.' C. . 4 Laynez J. . . 5 Poveda A. . . 6 Nieto 'P. M.' M. . 7 Soedjanaamadja 'U. M.' M. . 8 Gabius 'H. J.' 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_AcAMP2F18W _Saveframe_category molecular_system _Mol_system_name 'AcAMP2F18W mutant' _Abbreviation_common AcAMP2F18W _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label AcAMP2F18W $AcAMP2F18W 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_AcAMP2F18W _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class protein _Name_common AcAMP2 _Name_variant 'AcAMP2F18W mutant' _Abbreviation_common AcAMP2F18W _Molecular_mass 3223 _Mol_thiol_state 'all disulfide bound' _Details ; The secondary structure of this protein presents beta-sheet on residues over residues 13 to 23. This protein is a hevein domain, and has high homology with AcAMP2 antimicrobial peptide. Carbohydrate binding site involves residues S16, W18, Y20 and Y27. ; ############################## # Polymer residue sequence # ############################## _Residue_count 30 _Mol_residue_sequence ; VGECVRGRCPSGMCCSQWGY CGKGPKYCGR ; loop_ _Residue_seq_code _Residue_label 1 VAL 2 GLY 3 GLU 4 CYS 5 VAL 6 ARG 7 GLY 8 ARG 9 CYS 10 PRO 11 SER 12 GLY 13 MET 14 CYS 15 CYS 16 SER 17 GLN 18 TRP 19 GLY 20 TYR 21 CYS 22 GLY 23 LYS 24 GLY 25 PRO 26 LYS 27 TYR 28 CYS 29 GLY 30 ARG stop_ _Sequence_homology_query_date 2008-08-19 _Sequence_homology_query_revised_last_date 2008-08-19 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 6647 AcAMP2 100.00 30 100.00 100.00 3.90e-08 PDB 1ZUV '24 Nmr Structures Of Acamp2-Like Peptide With Phenylalanine 18 Mutated To Tryptophan' 96.67 30 100.00 100.00 1.30e-07 stop_ save_ ############# # Ligands # ############# save_CTO _Saveframe_category ligand _Mol_type "non-polymer (D-sacchride)" _Name_common "CTO (TRIACETYLCHITOTRIOSE)" _BMRB_code . _PDB_code CTO _Molecular_mass 627.593 _Mol_charge 0 _Mol_paramagnetic . _Mol_aromatic no _Details ; Information obtained from PDB's Chemical Component Dictionary at http://wwpdb-remediation.rutgers.edu/downloads.html Downloaded on Mon Aug 8 12:37:48 2011 ; loop_ _Atom_name _PDB_atom_name _Atom_type _Atom_chirality _Atom_charge _Atom_oxidation_number _Atom_unpaired_electrons C11 C11 C . 0 . ? C21 C21 C . 0 . ? N21 N21 N . 0 . ? C71 C71 C . 0 . ? O71 O71 O . 0 . ? C81 C81 C . 0 . ? C31 C31 C . 0 . ? O31 O31 O . 0 . ? C41 C41 C . 0 . ? O41 O41 O . 0 . ? C51 C51 C . 0 . ? O51 O51 O . 0 . ? C61 C61 C . 0 . ? O61 O61 O . 0 . ? C12 C12 C . 0 . ? C22 C22 C . 0 . ? N22 N22 N . 0 . ? C72 C72 C . 0 . ? O72 O72 O . 0 . ? C82 C82 C . 0 . ? C32 C32 C . 0 . ? O32 O32 O . 0 . ? C42 C42 C . 0 . ? O42 O42 O . 0 . ? C52 C52 C . 0 . ? O52 O52 O . 0 . ? C62 C62 C . 0 . ? O62 O62 O . 0 . ? C13 C13 C . 0 . ? O13 O13 O . 0 . ? C23 C23 C . 0 . ? N23 N23 N . 0 . ? C73 C73 C . 0 . ? O73 O73 O . 0 . ? C83 C83 C . 0 . ? C33 C33 C . 0 . ? O33 O33 O . 0 . ? C43 C43 C . 0 . ? O43 O43 O . 0 . ? C53 C53 C . 0 . ? O53 O53 O . 0 . ? C63 C63 C . 0 . ? O63 O63 O . 0 . ? H11 H11 H . 0 . ? H21 H21 H . 0 . ? HNL HNL H . 0 . ? H811 H811 H . 0 . ? H812 H812 H . 0 . ? H813 H813 H . 0 . ? H31 H31 H . 0 . ? HOV HOV H . 0 . ? H41 H41 H . 0 . ? HO4 HO4 H . 0 . ? H51 H51 H . 0 . ? H611 H611 H . 0 . ? H612 H612 H . 0 . ? HO6 HO6 H . 0 . ? H12 H12 H . 0 . ? H22 H22 H . 0 . ? HNM HNM H . 0 . ? H821 H821 H . 0 . ? H822 H822 H . 0 . ? H823 H823 H . 0 . ? H32 H32 H . 0 . ? HOW HOW H . 0 . ? H42 H42 H . 0 . ? H52 H52 H . 0 . ? H621 H621 H . 0 . ? H622 H622 H . 0 . ? HO2 HO2 H . 0 . ? H13 H13 H . 0 . ? HOD HOD H . 0 . ? H23 H23 H . 0 . ? HNN HNN H . 0 . ? H831 H831 H . 0 . ? H832 H832 H . 0 . ? H833 H833 H . 0 . ? H33 H33 H . 0 . ? HOX HOX H . 0 . ? H43 H43 H . 0 . ? H53 H53 H . 0 . ? H631 H631 H . 0 . ? H632 H632 H . 0 . ? HO3 HO3 H . 0 . ? stop_ loop_ _Bond_order _Bond_atom_one_atom_name _Bond_atom_two_atom_name _PDB_bond_atom_one_atom_name _PDB_bond_atom_two_atom_name SING C11 C21 ? ? SING C11 O51 ? ? SING C11 O42 ? ? SING C11 H11 ? ? SING C21 N21 ? ? SING C21 C31 ? ? SING C21 H21 ? ? SING N21 C71 ? ? SING N21 HNL ? ? DOUB C71 O71 ? ? SING C71 C81 ? ? SING C81 H811 ? ? SING C81 H812 ? ? SING C81 H813 ? ? SING C31 O31 ? ? SING C31 C41 ? ? SING C31 H31 ? ? SING O31 HOV ? ? SING C41 O41 ? ? SING C41 C51 ? ? SING C41 H41 ? ? SING O41 HO4 ? ? SING C51 O51 ? ? SING C51 C61 ? ? SING C51 H51 ? ? SING C61 O61 ? ? SING C61 H611 ? ? SING C61 H612 ? ? SING O61 HO6 ? ? SING C12 C22 ? ? SING C12 O52 ? ? SING C12 O43 ? ? SING C12 H12 ? ? SING C22 N22 ? ? SING C22 C32 ? ? SING C22 H22 ? ? SING N22 C72 ? ? SING N22 HNM ? ? DOUB C72 O72 ? ? SING C72 C82 ? ? SING C82 H821 ? ? SING C82 H822 ? ? SING C82 H823 ? ? SING C32 O32 ? ? SING C32 C42 ? ? SING C32 H32 ? ? SING O32 HOW ? ? SING C42 O42 ? ? SING C42 C52 ? ? SING C42 H42 ? ? SING C52 O52 ? ? SING C52 C62 ? ? SING C52 H52 ? ? SING C62 O62 ? ? SING C62 H621 ? ? SING C62 H622 ? ? SING O62 HO2 ? ? SING C13 O13 ? ? SING C13 C23 ? ? SING C13 O53 ? ? SING C13 H13 ? ? SING O13 HOD ? ? SING C23 N23 ? ? SING C23 C33 ? ? SING C23 H23 ? ? SING N23 C73 ? ? SING N23 HNN ? ? DOUB C73 O73 ? ? SING C73 C83 ? ? SING C83 H831 ? ? SING C83 H832 ? ? SING C83 H833 ? ? SING C33 O33 ? ? SING C33 C43 ? ? SING C33 H33 ? ? SING O33 HOX ? ? SING C43 O43 ? ? SING C43 C53 ? ? SING C43 H43 ? ? SING C53 O53 ? ? SING C53 C63 ? ? SING C53 H53 ? ? SING C63 O63 ? ? SING C63 H631 ? ? SING C63 H632 ? ? SING O63 HO3 ? ? stop_ _Mol_thiol_state . _Sequence_homology_query_date . save_ #################### # Natural source # #################### save_natural_source _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species _Tissue $AcAMP2F18W 'inca wheat, love-lies bleendig, tassel flower' 3567 Eukaryota Viridiplantae Amaranthus caudatus seed 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 $AcAMP2F18W 'chemical synthesis' . . . . . ; The residue Phe18 of native AcAMP2 was mutated to Tryptophan. The aminoacid was manually assembled by solid phase synthesis using Fmoc chemistry according to standard 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 $AcAMP2F18W 2.8 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 from Bruker 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 ; This program helps visualization and assignment of 2D NMR spectra. 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. ; save_ save_DYANA _Saveframe_category software _Name DYANA _Version 1.5 loop_ _Task 'Structure solution' stop_ _Details ; This program yields a collection of protein structures that fit the 1H-1H distance constraints experimentally obtained. 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. ; save_ save_AMBER _Saveframe_category software _Name AMBER _Version 5.0 loop_ _Task 'Structure refinement' stop_ _Details ; The programs used from this package perform molecular dynamics calculations on protein structures. They also convert dyana-format pdb files into amber-format pdb files. 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. ; save_ ######################### # Experimental detail # ######################### ################################## # NMR Spectrometer definitions # ################################## save_NMR_spectrometer_1 _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model DRX _Field_strength 500 _Details . save_ save_NMR_spectrometer_2 _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model AMX _Field_strength 500 _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' 0.15 0.02 M pH 5.6 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_chemical_shifts_AcAMP2F18W _Saveframe_category assigned_chemical_shifts _Details . loop_ _Experiment_label TOCSY NOESY stop_ loop_ _Sample_label $sample-1 stop_ _Sample_conditions_label $conditions-1 _Chem_shift_reference_set_label $chemical_shift_reference _Mol_system_component_name AcAMP2F18W _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 VAL HA H 3.868 0.003 1 2 . 1 VAL HB H 2.191 0.002 1 3 . 1 VAL HG2 H 1.013 0.002 2 4 . 2 GLY H H 8.912 0.002 1 5 . 2 GLY HA2 H 3.977 0.004 2 6 . 2 GLY HA3 H 4.101 0.003 2 7 . 3 GLU H H 8.686 0.002 1 8 . 3 GLU HA H 4.276 0.007 1 9 . 3 GLU HB2 H 1.882 0.005 2 10 . 3 GLU HB3 H 1.967 0.006 2 11 . 3 GLU HG2 H 2.179 0.003 2 12 . 3 GLU HG3 H 2.294 0.007 2 13 . 4 CYS H H 7.753 0.002 1 14 . 4 CYS HA H 4.417 0.005 1 15 . 4 CYS HB2 H 2.755 0.005 2 16 . 4 CYS HB3 H 3.093 0.003 2 17 . 5 VAL H H 8.343 0.004 1 18 . 5 VAL HA H 4.083 0.006 1 19 . 5 VAL HB H 1.863 0.008 1 20 . 5 VAL HG1 H 0.841 0.004 2 21 . 6 ARG H H 9.303 0.006 1 22 . 6 ARG HA H 3.845 0.004 1 23 . 6 ARG HB2 H 1.790 0.005 2 24 . 6 ARG HB3 H 1.883 0.004 2 25 . 6 ARG HG2 H 1.560 0.007 2 26 . 6 ARG HD2 H 3.154 0.007 2 27 . 6 ARG HH21 H 6.691 0.002 2 28 . 6 ARG HH22 H 7.079 0.002 2 29 . 7 GLY H H 8.287 0.003 1 30 . 7 GLY HA2 H 3.692 0.005 2 31 . 7 GLY HA3 H 4.081 0.004 2 32 . 8 ARG H H 7.843 0.003 1 33 . 8 ARG HA H 4.628 0.005 1 34 . 8 ARG HB2 H 1.816 0.013 2 35 . 8 ARG HB3 H 1.870 0.004 2 36 . 8 ARG HG2 H 1.526 0.001 2 37 . 8 ARG HG3 H 1.614 0.001 2 38 . 8 ARG HD2 H 3.133 0.005 2 39 . 9 CYS H H 8.578 0.001 1 40 . 9 CYS HA H 5.172 0.008 1 41 . 9 CYS HB2 H 2.253 0.004 2 42 . 9 CYS HB3 H 2.805 0.006 2 43 . 10 PRO HA H 4.405 0.004 1 44 . 10 PRO HB2 H 1.677 0.007 2 45 . 10 PRO HB3 H 2.346 0.003 2 46 . 10 PRO HG2 H 1.937 0.008 2 47 . 10 PRO HG3 H 1.938 0.002 2 48 . 10 PRO HD2 H 3.424 0.002 2 49 . 10 PRO HD3 H 3.913 0.005 2 50 . 11 SER H H 8.311 0.002 1 51 . 11 SER HA H 4.111 0.004 1 52 . 11 SER HB2 H 3.793 0.004 2 53 . 12 GLY H H 8.862 0.003 1 54 . 12 GLY HA2 H 3.632 0.004 2 55 . 12 GLY HA3 H 4.187 0.008 2 56 . 13 MET H H 7.907 0.005 1 57 . 13 MET HA H 4.630 0.003 1 58 . 13 MET HB2 H 1.710 0.004 2 59 . 13 MET HB3 H 1.989 0.002 2 60 . 13 MET HG2 H 2.295 0.003 2 61 . 14 CYS H H 9.205 0.003 1 62 . 14 CYS HA H 4.569 0.003 1 63 . 14 CYS HB2 H 2.359 0.003 2 64 . 14 CYS HB3 H 3.798 0.004 2 65 . 15 CYS H H 8.759 0.003 1 66 . 15 CYS HA H 4.718 0.004 1 67 . 15 CYS HB2 H 2.842 0.003 2 68 . 16 SER H H 9.634 0.002 1 69 . 16 SER HA H 4.815 0.002 1 70 . 16 SER HB2 H 4.065 0.004 2 71 . 16 SER HB3 H 4.200 0.003 2 72 . 17 GLN H H 8.991 0.001 1 73 . 17 GLN HA H 3.936 0.003 1 74 . 17 GLN HB2 H 1.474 0.001 2 75 . 17 GLN HB3 H 1.642 0.002 2 76 . 17 GLN HG2 H 1.380 0.005 2 77 . 17 GLN HG3 H 1.573 0.002 2 78 . 17 GLN HE21 H 7.591 0.001 2 79 . 17 GLN HE22 H 7.047 0.001 2 80 . 18 TRP H H 7.276 0.004 1 81 . 18 TRP HA H 4.802 0.001 1 82 . 18 TRP HB2 H 3.153 0.003 2 83 . 18 TRP HB3 H 3.622 0.004 2 84 . 18 TRP HD1 H 7.255 0.002 1 85 . 18 TRP HE3 H 7.644 0.001 2 86 . 18 TRP HE1 H 10.167 0.001 1 87 . 18 TRP HZ3 H 7.121 0.000 1 88 . 18 TRP HZ2 H 7.299 0.004 1 89 . 18 TRP HH2 H 7.041 0.004 1 90 . 19 GLY H H 7.792 0.005 1 91 . 19 GLY HA2 H 3.531 0.008 2 92 . 19 GLY HA3 H 4.001 0.001 2 93 . 20 TYR H H 7.391 0.003 1 94 . 20 TYR HA H 5.025 0.005 1 95 . 20 TYR HB2 H 2.830 0.003 2 96 . 20 TYR HB3 H 3.228 0.003 2 97 . 20 TYR HD1 H 6.846 0.003 3 98 . 20 TYR HE1 H 6.988 0.003 3 99 . 21 CYS H H 8.801 0.003 1 100 . 21 CYS HA H 5.478 0.003 1 101 . 21 CYS HB2 H 2.771 0.008 2 102 . 22 GLY H H 8.431 0.004 1 103 . 22 GLY HA2 H 1.870 0.005 2 104 . 22 GLY HA3 H 3.644 0.002 2 105 . 23 LYS H H 8.032 0.002 1 106 . 23 LYS HA H 4.896 0.004 1 107 . 23 LYS HB2 H 1.621 0.004 2 108 . 23 LYS HB3 H 1.831 0.001 2 109 . 23 LYS HG2 H 1.304 0.001 2 110 . 23 LYS HG3 H 1.409 0.006 2 111 . 23 LYS HD2 H 1.629 0.005 2 112 . 23 LYS HE2 H 2.864 0.005 2 113 . 24 GLY H H 8.333 0.001 1 114 . 24 GLY HA2 H 3.936 0.001 2 115 . 24 GLY HA3 H 4.549 0.002 2 116 . 25 PRO HA H 4.250 0.005 1 117 . 25 PRO HB2 H 1.924 0.005 2 118 . 25 PRO HB3 H 2.262 0.065 2 119 . 25 PRO HG2 H 2.036 0.006 2 120 . 25 PRO HG3 H 2.038 0.001 2 121 . 25 PRO HD2 H 3.620 0.004 2 122 . 25 PRO HD3 H 3.813 0.002 2 123 . 26 LYS H H 8.794 0.002 1 124 . 26 LYS HA H 4.027 0.004 1 125 . 26 LYS HB3 H 1.809 0.005 2 126 . 26 LYS HG2 H 1.129 0.031 2 127 . 26 LYS HG3 H 1.388 0.003 2 128 . 26 LYS HD2 H 1.600 0.005 2 129 . 26 LYS HE2 H 2.863 0.004 2 130 . 27 TYR H H 7.547 0.003 1 131 . 27 TYR HA H 4.055 0.005 1 132 . 27 TYR HB2 H 2.504 0.006 2 133 . 27 TYR HB3 H 2.899 0.002 2 134 . 27 TYR HD1 H 7.154 0.003 2 135 . 27 TYR HE1 H 6.714 0.004 2 136 . 28 CYS H H 8.448 0.003 1 137 . 28 CYS HA H 4.509 0.006 1 138 . 28 CYS HB2 H 2.714 0.005 2 139 . 28 CYS HB3 H 3.200 0.008 2 140 . 29 GLY H H 7.977 0.004 1 141 . 29 GLY HA2 H 3.946 0.006 2 142 . 29 GLY HA3 H 3.985 0.013 2 143 . 30 ARG H H 8.280 0.005 1 144 . 30 ARG HA H 4.254 0.002 1 145 . 30 ARG HB2 H 1.683 0.001 2 146 . 30 ARG HB3 H 1.846 0.012 2 147 . 30 ARG HG2 H 1.579 0.010 2 148 . 30 ARG HD2 H 3.143 0.003 2 stop_ save_