1H, 13C and 15N NMR backbone assignments and secondary structure of the 269-residue protease subtilisin 309 from Bacillus lentus

Summary ¹H, ¹³C and ¹⁵N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the -protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, ¹⁵N TOCSY-HMQC and ¹⁵N NOESY-HMQC experiments provided the H chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020–9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the C and H frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C chemical shifts and TOCSY ladders. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D ¹⁵N NOESY-HMQC data and C secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427–445] shows no major differences.Supplementary material available from F.J.M. van de Ven: the source code (PASCAL) for the computer program described in this paper.     

Sequential assignment of 1H, 15N, 13C resonances and secondary structure of human calmodulin-like protein determined by NMR spectroscopy.

Human calmodulin-like protein (CLP) is closely related to vertebrate calmodulin, yet its unique cell specific expression pattern, overlapping but divergent biochemical properties, and specific target proteins suggest that it is not an isoform of calmodulin. To gain insight into the structural differences that may underlie the difference target specificities and biochemical properties of CLP when compared to calmodulin, we determined the sequential backbone assignment and associated secondary structure of 144 out of the 148 residues of Ca2+-CLP by using multinuclear multidimensional NMR spectroscopy. Despite a very high overall degree of structural similarity between CLP and calmodulin, a number of significant differences were found mainly in the length of alpha-helices and in the central nonhelical flexible region. Interestingly, the regions of greatest primary sequence divergence between CLP and calmodulin in helices III and VIII displayed only minor secondary structure differences. The data suggest that the distinct differences in target specificity and biochemical properties of CLP and calmodulin result from the sum of several minor structural and side-chain changes spread over multiple domains in these proteins.     

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly ¹⁵N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping ¹H^N chemical shifts were resolved by a three-dimensional ¹H-¹⁵N HMQC-NOESY-HMQC spectrum. Medium-and long-range NOEs, ³J_NH coupling constants, and ¹H^N exchange data indicate a secondary structure consisting of five parallel -strands and four -helices with a topology similar to that of Desulfovibrio vulgaris [Hidenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal -helices.Abbreviations CSI chemical shift index - DQF-COSY double-quantum-filtered correlation spectroscopy - DIPSI decoupling in the presence of scalar interactions - FMN flavin mononucleotide - GARP globally optimized alternating phase rectangular pulse - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - TPPI time-proportional phase increments - TSP 3-(trimethylsilyl)propionic-2,2,3,3-d ₄ acid, sodium salt     

NMR secondary structure of the plasminogen activator protein staphylokinase

Staphylokinase (Sak) is a 15.5 kDa protein secreted by several strains of Staphylococcusaureus. Due to its ability to convert plasminogen, the inactive proenzyme of the fibrinolyticsystem, into plasmin, Sak is presently undergoing clinical trials for blood clot lysis in thetreatment of thrombovascular disorders. With a view to developing a better understanding ofthe mode of action of Sak, we have initiated a structural investigation of Sak viamultidimensional heteronuclear NMR spectroscopy employing uniformly 15N- and 15N,13C-labelled Sak. Sequence-specific resonance assignments have been made employing 15N-editedTOCSY and NOE experiments and from HNCACB, CBCA(CO)NH, HBHA(CBCACO)NHand CC(CO)NH sets of experiments. From an analysis of the chemical shifts,3JHNH scalar coupling constants, NOEs and HN exchange data, the secondary structural elements of Sakhave been characterized.     

The dynein stalk head,the microtubule binding-domain of dynein: NMR assignment and ligand binding

Dynein is a motor ATPase, and the C-terminal two-thirds of its heavy chain form a ring structure. One of protrudings from this ring structure is a stalk whose tip, the dynein stalk head (DSH), is thought to be the microtubule-binding domain. As a first step toward elucidating the functional mechanisms of DSH, we aimed at the NMR structural analysis of an isolated DSH from mouse cytoplasmic dynein. The DSH expressed in bacteria and purified was coprecipitated with microtubules, suggesting its proper folding. Chemical shifts of the DSH were obtained from NMR measurements, and backbone assignment identified 94% of the main-chain N-H signals. Secondary structural prediction programs showed that about 60% of the residues formed alpha-helices. A region with cationic residues K58 and R61 (and possibly R66 as well), and another with R86, K88, K90, and K91, were found to form alpha-helices. Both of these regions may be important in the formation of the DSH-binding site to a microtubule that has a low pI with a number of acidic residues. Two synthetic peptides containing the sequence of the alpha-helix 12 of beta-tubulin, considered to be important in binding to DSH, were investigated. Of these two peptides, the one with higher helix-formation propensity appeared to bind to DSH, since it precipitated with DSH in a nearly stoichiometric manner. This suggested that the alpha-helicity of this region would be important in its binding to DSH.     

1H NMR studies of plastocyanin from Scenedesmus obliquus: complete sequence-specific assignment, secondary structure analysis, and global fold

Two-dimensional 1H NMR methods have been used to make sequence-specific resonance assignments for the 97 amino acid residues of the plastocyanin from the green alga Scenedesmus obliquus. Assignments were obtained for all backbone protons and the majority of the side-chain protons. Spin system identification relied heavily on the observation of relayed connectivities to the backbone amide proton. Sequence-specific assignments were made by using the sequential assignment procedure. During this process, an extra valine residue was identified that had not been detected in the original amino acid sequence. Elements of regular secondary structure were identified from characteristic NOE connectivities between backbone protons, 3JHN alpha coupling constant values, and the observation of slowly exchanging amide protons. The protein in solution contains eight beta-strands, one short segment of helix, five reverse turns, and five loops. The beta-strands may be arranged into two beta-sheets on the basis of extensive cross-strand NOE connectivities. The chain-folding topology determined from the NMR experiments is that of a Greek key beta-barrel and is similar to that observed for French bean plastocyanin in solution and poplar plastocyanin in the crystalline state. While the overall structures are similar, several differences in local structure between the S. obliquus and higher plant plastocyanins have been identified.     

Backbone assignment,secondary structure and Protein A binding of an isolated,human antibody VH domain

Summary Antibody heavy chain variable domains (VH) lacking their light chain domain (VL) partner are prime candidates for the design of minimum-size immunoreagents. To obtain structural information about isolated VH domains, a human VH was labelled with ¹⁵N or doubly labelled with both ¹⁵N and ¹³C and was studied by heteronuclear nuclear magnetic resonance spectroscopy. Most (90%) of the ¹H and ¹⁵N main-chain signals were assigned through two-dimensional TOCSY and NOESY experiments on the unlabelled VH and three-dimensional heteronuclear multiple quantum correlation TOCSY and NOESY experiments on the ¹⁵N-labelled VH. Four short stretches of the polypeptide chain could only be assigned on the basis of three-dimensional HNCA and HN(CO)CA experiments on the ¹³C-/¹⁵N-labelled protein. Long-range interstrand backbone NOEs suggest the presence of two adjacent -sheets formed by altogether nine antiparallel -strands. ³J_H ^NHC coupling constants and the location of slowly exchanging backbone amides support this interpretation. The secondary structure of the isolated VH is identical to that of heavy chain variable domains in intact antibodies, where VH domains are packed against a VL domain. The backbone assignments of the VH made it possible to locate its Protein A binding site. Chemical shift movements after complexing with the IgG binding fragment of Protein A indicate binding through one of the two -sheets of the VH. This -sheet is solvent exposed in intact antibodies. The Protein A binding site obviously differs from that on the Fc portion of immunoglobulins and is unique to members of the human VH_III gene subgroup.Abbreviations CDR complementarity determining region - CHAPS [(cholamidopropyl)-dimethylammonio]-1-propanesulfonate - DQF-COSY double-quantum-filtered correlated spectroscopy - Fab antigen binding antibody fragment - Fc crystallisable antibody fragment - Fv heterodimer of VH and VL - H1 (2, 3) hypervariable loop 1 (2, 3) - IgG immunoglobulin G - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - HMQC heteronuclear multiple quantum correlation spectroscopy - HSQC heteronuclear single quantum correlation spectroscopy - scFv single chain Fv - TOCSY total correlation spectroscopy - TPPI time-proportional phase incrementation - VH antibody heavy chain variable region - VL antibody light chain variable region. Mutants are denoted by the wild-type amino acid (one-letter code), follwed by the residue number and the new amino acid     

Graph-theoretical assignment of secondary structure in multidimensional protein NMR spectra: Application to the lac repressor headpiece

Summary A novel procedure is presented for the automatic identification of secondary structures in proteins from their corresponding NOE data. The method uses a branch of mathematics known as graph theory to identify prescribed NOE connectivity patterns characteristic of the regular secondary structures. Resonance assignment is achieved by connecting these patterns of secondary structure together, thereby matching the connected spin systems to specific segments of the protein sequence. The method known as SERENDIPITY refers to a set of routines developed in a modular fashion, where each program has one or several well-defined tasks. NOE templates for several secondary structure motifs have been developed and the method has been successfully applied to data obtained from NOESY-type spectra. The present report describes the application of the SERENDIPITY protocol to a 3D NOESY-HMQC spectrum of the ¹⁵N-labelled lac repressor headpiece protein. The application demonstrates that, under favourable conditions, fully automated identification of secondary structures and semi-automated assignment are feasible.Abbreviations 2D, 3D two-, three-dimensional - NOESY nuclear Overhauser enhancement spectroscopy - HMQC heteronuclear multiple quantum coherence - SSE secondary structure element - SERENDIPITY SEcondary structuRE ideNtification in multiDImensional ProteIn specTra analYsis Supplementary Material available from the authors: Two tables containing the total number of mappings resulting from the graph search procedure for simulated and experimental NOE data.     

1H, 15N and 13C NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450

The FMN-binding domain of human NADPH-cytochrome P450 reductase,corresponding to exons 3-;7, has been expressed at high level in anactive form and labelled with ¹³C and ¹⁵N. Mostof the backbone and aliphatic side-chain ¹H, ¹⁵Nand ¹³C resonances have been assigned using heteronucleardouble- and triple-resonance methods, together with a semiautomaticassignment strategy. The secondary structure as estimated from the chemicalshift index and NOE connectivities consists of six -helices and five-strands. The global fold was deduced from the long-range NOEsunambiguously assigned in a 4D ¹³C-resolved HMQC-NOESY-HMQCspectrum. The fold is of the alternating / type, with the five-strands arranged into a parallel -sheet. The secondarystructure and global fold are very similar to those of the bacterialflavodoxins, but the FMN-binding domain has an extra short helix in place ofa loop, and an extra helix at the N-terminus (leading to the membrane anchordomain in the intact P450 reductase). The experimental constraints werecombined with homology modelling to obtain a structure of the FMN-bindingdomain satisfying the observed NOE constraints. Chemical shift comparisonsshowed that the effects of FMN binding and of FMN reduction are largelylocalised at the binding site.     

Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling

Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific ¹⁵N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A.     

1H NMR studies of porcine calbindin D9k in solution: sequential resonance assignment, secondary structure, and global fold

The 1H nuclear magnetic resonance (NMR) spectrum of Ca2+-saturated porcine calbindin D9k (78 amino acids, Mr 8800) has been assigned. Greater than 98% of the 1H resonances, including spin systems for each amino acid residue, have been identified by using an approach that integrates data from a wide range of two-dimensional scalar correlated NMR experiments [Chazin, Rance, & Wright (1988) J. Mol. Biol. 202, 603-626]. Due to the limited quantity of sample and conformational heterogeneity of the protein, two-dimensional nuclear Overhauser effect (NOE) experiments also played an essential role in the identification of spin systems. On the basis of the pattern of scalar connectivities, 43 of the 78 spin systems could be directly assigned to the appropriate residue type. This provided an ample basis for obtaining the sequence-specific resonance assignments. The elements of secondary structure are identified from sequential and medium-range NOEs, values of 3JNH alpha, and the location of slowly exchanging backbone amide protons. Four well-defined helices and a mini beta-sheet between the two calcium binding loops are present in solution. These elements of secondary structure and a few key long-range NOEs provided sufficient information to define the global fold of the protein in solution. Generally good agreement is found between the crystal structure of the minor A form of bovine calbindin D9k and the solution structure of intact porcine calbindin D9k. The only significant difference is a short one-turn helix in the loop between helices II and III in the bovine crystal structure, which is clearly absent in the porcine solution structure.     

NMR assignments, secondary structure, and global fold of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea

Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.     

1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Summary Sequence-specific backbone ¹H and ¹⁵N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D ¹H-¹⁵N-correlated spectra obtained from uniformly ¹⁵N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.     

1H, 13C and 15N NMR backbone assignments of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of the backbone of serine protease PB92 have been assigned. This 269-residue protein is one of the largest monomeric proteins assigned so far. The amount and quality of information available suggest that even larger proteins could be assigned with present methods. Measured chemical shifts show excellent agreement with the secondary structure.Abbreviations 2D/3D two-/three-dimensional - HSQC Heteronuclear Single Quantum Coherence - TOCSY total correlation spectroscopy - NOE nuclear Overhauser effect Supplementary material available from the authors: One table containing the backbone ¹⁵N, ¹H^N, ¹³CO, ¹³CO and ¹H assignments for serine protease PB92.     

1H and 15N resonance assignments and secondary structure of cellular retinoic acid-binding protein with and without bound ligand

Summary Sequence-specific assignments for the ¹H and ¹⁵N backbone resonances of cellular retinoic acid-binding protein (CRABP), with and without the bound ligand, have been obtained. Most of the side-chain resonances of both apo- and holo-CRABP have also been assigned. The assignments have been obtained using two-dimensional homonuclear and heteronuclear NMR data, and three-dimensional ¹H-¹⁵N TOCSY-HMQC and NOESY-HMQC experiments. The secondary structure, deduced from nuclear Overhauser effects, amide H/D exchange rates and H chemical shifts, is analogous in both forms of the protein and is completely consistent with a model of CRABP that had been constructed by homology with the crystal structure of myelin P2 protein [Zhang et al. (1992) Protein Struct. Funct. Genet., 13, 87–99]. This model comprises two five-stranded -sheets that form a sandwich or -clam structure, and a short N-terminal helix-turn-helix motif that closes the binding cavity between the two sheets. Comparison of the data obtained for apo- and holo-CRABP indicates that a region around the C-terminus of the second helix is much more flexible in the apo-protein. Our data provide experimental evidence for the hypothesis that the ligand-binding mechanism of CRABP, and of other homologous proteins that bind hydrophobic ligands in the cytoplasm, involves opening of a portal to allow entry of the ligand into the cavity.     

Relationship between1H and13C NMR chemical shifts and the secondary and tertiary structure of a zinc finger peptide

Summary Essentially complete assignments have been obtained for the¹H and protonated¹³C NMR spectra of the zinc finger peptide Xfin-31 in the presence and absence of zinc. The patterns observed for the¹H and¹³C chemical shifts of the peptide in the presence of zinc, relative to the shifts in the absence of zinc, reflect the zinc-mediated folding of the unstructured peptide into a compact globular structure with distinct elements of secondary structure. Chemical shifts calculated from the 3D solution structure of the peptide in the presence of zinc and the observed shifts agree to within ca. 0.2 and 0.6 ppm for the backbone C^aH and NH protons, respectively. In addition, homologous zinc finger proteins exhibit similar correlations between their¹H chemical shifts and secondary structure.     

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure,backbone dynamics and protein association

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of¹H-¹⁵N-correlated 2D HSQC,¹⁵N-edited 3D TOCSY-HMQC, and¹⁵N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T₁ and T₂ relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH Src homology - GST glutathione-S-transferase - IPTG isopropyl--D-galactopyranoside - DTT dithiothreitol - PMSF phenyl-methyl-sulphonyl-fluoride - TBS 50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0 - MWCO molecular weight cut off - NMR nuclear magnetic resonance - HSQC heteronuclear single-quantum correlation - NOESY nuclear Overhauser effect spectroscopy