Complete Sequence-specific Assignments Research Articles

Comparative strutural analysis of the calcium free and bound states of the calcium regulatory protein calbindin D 9K

The solution structure of apo calbindin D9K, a member of the calmodulin superfamily of calcium-binding regulatory proteins, has been investigated by 1H nuclear magnetic resonance spectroscopy and the results compared with a corresponding study of the calcium-loaded protein. On the basis of complete sequence-specific assignments, characteristic patterns of short proton-proton distances have been identified in two-dimensional nuclear Overhauser effect spectra, allowing the elements of secondary structure to be determined. It is found that four helices and a short section of antiparallel beta-sheet are present regardless of the calcium content of the protein. In addition, a preliminary analysis of the long-range nuclear Overhauser effects shows that the global folding patterns are the same and that the tertiary structures of the apo protein is very similar to that of the calcium-loaded protein. These results are in stark contrast to a number of very substantial changes in 1H chemical shift. Preliminary studies of protein dynamics show some very large differences in flexibility and internal mobility. This suggests that protein dynamics may play a role more important than was initially realized in the function of calbindin D9K and other homologous calcium-binding regulatory proteins.

The rate and structural consequences of proline cis-trans isomerization in calbindin D9k: NMR studies of the minor (cis-Pro43) isoform and the Pro43Gly mutant

The EF-hand calcium-binding protein, calbindin D9k, exists in solution in the calcium-loaded state, as a 1:3 equilibrium mixture of two isoforms, the result of cis-trans isomerism at the Gly42-Pro43 peptide bond [Chazin et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2195-2198]. Nuclear magnetic resonance (NMR) studies of the minor (cis-Pro43) isoform and the Pro43----Gly mutant are reported here. The rate of cis----trans isomerization at the Pro43 peptide bond in the wild-type protein was determined by line-shape analysis at elevated temperatures, using a sample in which all amino acids, except Ser and Val, were deuterated. The cis----trans rate is calculated to be 0.2 s-1 at 25 degrees C, corresponding to a free energy of activation, delta G, of 77 kJ/mol. The complete sequence-specific 1H NMR assignments of the cis-Pro43 isoform and the Pro43----Gly mutant in the calcium-loaded state have been obtained by using standard methods combined with comparisons to the previously assigned major (trans-Pro43) isoform. This has permitted detailed comparative analysis of 1H NMR chemical shifts, backbone scalar coupling constants, and nuclear Overhauser effects. The minor isoform has a global fold that is identical with that of the major isoform. Structural changes imposed by cis-trans isomerization at Pro43 are highly localized to the linker loop (containing Pro43) that joins the two EF hands. The Pro43----Gly mutant has a global fold that is identical with the wild-type protein, but does not exhibit conformational heterogeneity. Only very limited structural differences are observed between mutant and wild-type protein, and these are also highly localized to the linker loop. The ion-binding properties of the mutant, as determined by 43Ca and 113Cd NMR, are found to be very similar to the wild-type protein. These results provide crucial evidence that justifies the calculation of high-resolution three-dimensional structures of the Pro43Gly mutant, rather than of the conformationally heterogeneous wild-type protein.

Multinuclear magnetic resonance studies of the 2Fe.cntdot.2S* ferredoxin from Anabaena species strain PCC 7120. 1. Sequence-specific hydrogen-1 resonance assignments and secondary structure in solution of the oxidized form

Complete sequence-specific assignments were determined for the diamagnetic 1H resonances from Anabaena 7120 ferredoxin (Mr = 11,000). A novel assignment procedure was followed whose first step was the identification of the 13C spin systems of the amino acids by a 13C(13C) double quantum correlation experiment [Oh, B.-H., Westler, M. W., Darba, P., & Markley, J. L. (1988) Science 240, 908-911]. Then, the 1H spin systems of the amino acids were identified from the 13C spin systems by means of direct and relayed 1H(13C) single-bond correlations [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085]. The sequential resonance assignments were based mainly on conventional interresidue 1H alpha i-1HNi + 1 NOE connectivities. Resonances from 18 residues were not resolved in two-dimensional 1H NMR spectra. When these residues were mapped onto the X-ray crystal structure of the homologous ferredoxin from Spirulina platensis [Fukuyama, K., Hase, T., Matsumoto, S., Tsukihara, T., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., & Matsubara, H. (1980) Nature 286, 522-524], it was found that they correspond to amino acids close to the paramagnetic 2Fe.2S* cluster. Cross peaks in two-dimensional homonuclear 1H NMR spectra were not observed for any protons closer than about 7.8 A to both iron atoms. Secondary structural features identified in solution include two antiparallel beta-sheets, one parallel beta-sheet, and one alpha-helix.

Complete resonance assignment for the polypeptide backbone of interleukin 1.beta. using three-dimensional heteronuclear NMR spectroscopy

The complete sequence-specific assignment of the 15N and 1H backbone resonances of the NMR spectrum of recombinant human interleukin 1 beta (153 residues, Mr = 17,400) has been obtained by using primarily 15N-1H heteronuclear three-dimensional (3D) NMR techniques in combination with 15N-1H heteronuclear and 1H homonuclear two-dimensional NMR. The fingerprint region of the spectrum was analyzed by using a combination of 3D heteronuclear 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopies. We show that the problems of amide NH and C alpha H chemical shift degeneracy that are prevalent for proteins of this size are readily overcome by using the 3D heteronuclear NMR technique. A doubling of some peaks in the spectrum was found to be due to N-terminal heterogeneity of the 15N-labeled protein, corresponding to a mixture of wild-type and des-Ala-1-interleukin 1 beta. The complete list of 15N and 1H assignments is given for all the amide NH and C alpha H resonances of all non-proline residues, as well as the 1H assignments for some of the amino acid side chains. This first example of the sequence-specific assignment of a protein using heteronuclear 3D NMR provides a basis for further conformational and dynamic studies of interleukin 1 beta.

Complete sequence-specific proton NMR assignments for human insulin

Solvent conditions where human insulin could be studied by high-resolution NMR were determined. Both low pH and addition of acetonitrile were required to overcome the protein's self-association and to obtain useful spectra. Two hundred eighty-six 1H resonances were located and assigned to specific sites on the protein by using two-dimensional NMR methods. The presence and position of numerous dNN sequential NOE's indicate that the insulin conformation seen in crystallographic studies is largely retained under these solution conditions. Slowly exchanging protons were observed for seven backbone amide protons and were assigned to positions A15 and A16 and to positions B15-B19. These amides all occur within helical regions of the protein [Chawdhury, S.A., Dodson, E.J., Dodson, G.G., Reynolds, C.D., Tolley, S.P., Blundell, T.L., Cleasby, A., Pitts, J.E., Tickle, I.J., & Wood, S.P. (1983) Diabetologia 25, 460-464].

Structural studies of cytochrome b5: complete sequence-specific resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from pig and calf

We report complete sequence-specific proton resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from calf liver. In addition, sequence-specific resonance assignments for the main-chain amino acid protons (i.e., C alpha, C beta, and amide protons) are also reported for the porcine cytochrome b5. Assignment of the majority of the main-chain resonances was rapidly accomplished by automated procedures that used COSY and HOHAHA peak coordinates as input. Long side chain amino acid spin system identification was facilitated by long-range coherence-transfer experiments (HOHAHA). Problems with resonance overlap were resolved by examining differences between the two-dimensional 500-MHz NMR spectra of rabbit, pig, and calf proteins and by examining the temperature-dependent variation of amide proton resonances. Calculations of the aromatic ring-current shifts for protons that the X-ray crystal structure indicated were proximal to aromatic residues were found to be useful in corroborating assignments, especially those due to the large shifts induced by the heme. Assignment of NOESY cross peaks was greatly facilitated by a prediction of intensities using a complete relaxation matrix analysis based on the crystal structure. These results suggest that the single-crystal X-ray structure closely resembles that of the solution structure although there is evidence that the solution structure has a more dynamic character.

Solution conformation of leiurotoxin I (scyllatoxin) by 1H nuclear magnetic resonance : Resonance assignment and secondary structure

A proton NMR study at 500 MHz of leiurotoxin I in water is presented. Nearly complete sequence-specific assignments of the individual backbone and side-chain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of this toxin is inferred from a combination of short-range nuclear Overhauser enhancements, scalar couplings and proton/deuteron exchange rates. Three disulflde bridges locate the N-terminal part (that is α-helical from residue 6 to 16) on one side of a C-terminal two stranded antiparallel β sheet (from Leu 18 to Val 29). The latter features a tight turn at Gly 23-Asp 24.

Sequential resonance assignment and secondary structure determination of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors

The solution conformation of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors, has been investigated by nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments of the 1H NMR spectrum have been obtained by using a number of two-dimensional techniques for identifying through-bond and through-space (less than 5-A) connectivities. Elements of regular secondary structure have been identified on the basis of a qualitative interpretation of the nuclear Overhauser enhancement, coupling constant, and amide exchange data. These are two beta-sheet regions. One double-stranded antiparallel beta-sheet comprises residues 11-14 (strand 1) and 37-39 (strand 2). The other triple-stranded sheet is formed by two antiparallel strands comprising residues 45-49 (strand 4) and 53-57 (strand 5) connected by a turn (residues 50-52), and a small strand consisting of residues 20-22 (strand 3) that is parallel to strand 4.

NMR studies of the Escherichia coli trp aporepressor. Sequence-specific assignment of the aromatic proton resonances.

The resonances in the aromatic region of the 1H-NMR spectrum of the Escherichia coli trp aporepressor have been assigned to amino acid type by two-dimensional correlated spectroscopy (COSY), homonuclear Hartmann-Hahn (HOHAHA) spectroscopy and nuclear Overhauser enhancement spectroscopy (NOESY) techniques and studies of the pH dependence of the chemical shifts, in combination with selective deuteration of the protein. Complete sequence-specific assignments of the aromatic resonances have been made by comparing the observed inter-residue NOEs with those expected on the basis of the crystal structure of the protein [Zhang, R.-G., Joachimiak, A., Lawson, C.L., Shevitz, R.W., Otwinowski, Z. & Sigler, P.B. (1987) Nature 327, 591-597]. The latter experiments have also permitted the sequence-specific assignment of some of the high-field methyl resonances. The complete assignment of the aromatic region of the spectrum, in particular of resonances from residues at the dimer interface, opens the way to detailed studies of the conformational effects of corepressor and operator binding.

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.

Complete sequence-specific 1H nuclear magnetic resonance assignments for mouse epidermal growth factor.

The 1H NMR spectrum of the mouse epidermal growth factor (53 residues) was analyzed with the use of two-dimensional NMR techniques. All the observable 296 proton resonances were completely assigned in a sequential manner. For the spin system identification, two-dimensional homonuclear Hartmann-Hahn spectrum was useful, especially for arginine and proline residues. The easy spin system identification of these long-side-chain-bearing amino acid residues greatly facilitated the sequence-specific resonance assignment of the epidermal growth factor.

Sequence specific assignment of the proton nuclear magnetic resonance spectrum of barley serine proteinase inhibitor 2

The sequence specific assignment of the1H NMR spectrum of barley serine proteinase inhibitor 2 is described. The sequential assignment procedure has been applied to the residues 22 to 83. The 21 residues in the N-terminal part of the structure were shown to be a random coil for which sequential assignment was not immediately possible. For the C-terminal part of the protein starting at Thr-22 and ending with the C-terminal residue Gly-83, sequence specific assignment of all the HN, Hα, Hβ′, Hβ″ resonances was achieved and complete sequence specific assignment of the non labile protons of the amino acid residue types of glycine, alanine, threonine, serine, valine, isoleucine, leucine, asparagine, aspartic acid, glutamine, glutamic acid, phenylalanine, tyrosine and tryptophan is reported.

Complete sequence-specific 1H nuclear magnetic resonance assignments for the α-amylase polypeptide inhibitor tendamistat from Streptomyces tendae

The 1H nuclear magnetic resonance (n.m.r.) spectrum of the α-amylase inhibitor Tendamistat was completely assigned with the use of phase-sensitive homonuclear twodimensional n.m.r. The assignments include the non-labile protons of the 74 amino acid residues as well as the labile protons which exchange sufficiently slowly to be observed in H 2O solution. The proton chemical shifts are listed at 50 °C and pH 3.2, which coincides with the conditions used for the determination of the three-dimensional structure of Tendamistat.

The three-dimensional structure of α1-purothionin in solution: combined use of nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The determination of the three-dimensional solution structure of alpha1-purothionin using a combination of metric matrix distance geometry and restrained molecular dynamics calculations based on n.m.r. data is presented. The experimental data comprise complete sequence-specific proton resonance assignments, a set of 310 approximate interproton distance restraints derived from nuclear Overhauser effects, 27 Ø backbone torsion angle restraints derived from vicinal coupling constants, 4 distance restraints from hydrogen bonds and 12 distance restraints from disulphide bridges. The average atomic rms difference between the final nine converged structures and the mean structure obtained by averaging their coordinates is 1.5 +/- 0.1 å for the backbone atoms and 2.0 +/- 0.1 å for all atoms. The overall shape of alpha1-purothionin is that of the capital letter L, similar to that of crambin, with the longer arm comprising two approximately parallel alpha-helices and the shorter arm a strand and a mini anti-parallel beta sheet.

Sequence-specific 1H-NMR assignments in rabbit-liver metallothionein-2.

The complete sequence-specific assignment of the 1H nuclear magnetic resonance spectrum of a major subform of rabbit liver metallothionein-2 is presented. The sequential assignment procedures revealed a number of differences with regard to results obtained by earlier partial chemical sequencing of a preparation now known to be microheterogeneous. In particular, the present data indicate a polypeptide chain length of 62 amino acid residues as compared to the occurrence of 61 amino acids in all other known mammalian metallothioneins. In the new sequence, which was also fully confirmed by chemical means, the additional amino acid residue was identified as Ala8' inserted between Ala8 and Ala9 of the standard amino acid numeration. In addition to the predominant protein species all preparations contained a minor component, for which the two-dimensional 1H-nuclear magnetic resonance features are compatible with a chemically different, homologous metallothionein.

Comparative studies of conformation and internal mobility in native and circular basic pancreatic trypsin inhibitor by 1H nuclear magnetic resonance in solution

A circular derivative of bovine pancreatic trypsin inhibitor (c-BPTI) with the C- and N-termini covalently linked with a peptide bond, has been studied in solution by spectroscopic techniques, principally two-dimensional 1H NMR. Near complete sequence specific assignment of the 1H NMR spectrum of c-BPTI has been obtained in a highly efficient manner, using recently developed experimental techniques. The assignments serve as the necessary background for detailed examination of the effects of the chemical modification on global and local features of the protein conformation and on the internal mobility. Analysis of chemical shifts, spin-spin coupling constants, amide proton exchange and proton-proton Overhauser enhancements indicates that perturbations of the native BPTI conformation in c-BPTI involve exclusively residues adjacent to the modification site. The strict localization and small magnitude of the conformation changes relative to native BPTI emphasize the important role of the salt bridge between the chain ends in determining the solution conformation and dynamics in the chain terminal regions of the native protein.

Secondary structure of the α-amylase polypeptide inhibitor Tendamistat from Streptomyces tendae determined in solution by 1H nuclear magnetic resonance

Complete sequence-specific 1H nuclear magnetic resonance assignments were obtained for the backbone hydrogen atoms in Tendamistat, a protein with 74 residues. From NOESY observation of 1H- 1H short distance constraints, measurements of the spin-spin couplings 3J HNα and a qualitative identification of slowly exchanging amide protons, two antiparallel β-sheets containing three and four strands, respectively, were identified. The peptide segments outside the β-sheets do not form regular secondary structure. Preliminary data were obtained on the relative spatial arrangements of the two β-sheets.