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At least 91 records · Page 5Linked to original sources
PubMed1992

Structural effects induced by mutagenesis affected by crystal packing factors: the structure of a 30-51 disulfide mutant of basic pancreatic trypsin inhibitor.

The X-ray structure of the C30V/C51A disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) has been analyzed at 1.6 A resolution. The mutant crystallizes in a cell having two molecules in the asymmetric unit. The packing environments of these two molecules are quite different, allowing for an assessment of which among the observed structural changes result from the mutation and which are produced by lattice packing considerations. The removal of the 30-51 disulfide bridge has little apparent affect on the B-factors of segments of adjacent polypeptide chain, although there are distinct differences in the structure compared to wild-type BPTI crystal structures. Both of the two C30V/C51A molecules show differences at the mutation site when compared to another 30-51 disulfide mutant, C30A/C51A, presumably due to the larger steric bulk of a valine versus an alanine at residue 30. A comparison of the two independent C30V/C51A molecules indicates that there are significant differences between them even at the site of mutation. The description of the specific structural differences of each molecule differs in detail and suggests different conclusions about the nature of structural perturbation near 30-51. In addition, when these two molecules are compared to two different wild-type structures, which had been determined from different space groups, a somewhat different pattern of changes is observed. These findings indicate that crystal packing can influence the observed perturbations in mutant structures.

Alanine↗
PubMed1992

Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of x-ray and neutron diffraction data. III. Complete structure.

We present in this paper the complete structure of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the L alpha phase (66% RH, 23 degrees C) obtained by the joint refinement of neutron and x-ray lamellar diffraction data. The structural details obtained have previously required a large number of neutron diffraction experiments, using numerous specifically-deuterated phospholipid isomorphs (Büldt et al., 1978. Nature (Lond.). 271:182-184). The joint-refinement approach minimizes specific deuteration by utilizing independent neutron and x-ray data sets. The method yields a quasimolecular structure consisting of a series of multiatomic fragments that are each represented by one or several Gaussian distributions whose positions and widths can be determined to within 0.06 to 0.52 A exclusive of the methylene region. The image of DOPC at 66% RH (5.36 +/- 0.08 waters per lipid) is consistent with many aspects of bilayer structure previously determined by structural and spectroscopic studies. The most striking feature of the structure is the large amount of transbilayer thermal motion suggested by the widths and overlaps of the Gaussian envelopes of the quasimolecular fragments. We discuss the "dynamic bilayer thickness" which describes the minimum effective thickness of the hydrocarbon permeability barrier in terms of the thermal motion of the water. A gradient of thermal motion exists that increases in either direction away from the glycerol backbone which is the most constrained portion of the bilayer. The steric interactions between headgroups of apposed bilayers, expected at the hydration level of our experiments, are clearly revealed. A useful consequence of the quasimolecular structure is that average boundaries within bilayers calculated using composition and volumetric data and ad hoc assumptions can be related to the positions of the principal structural groups. Several measures of "bilayer thickness" in common use can be identified as the positions of the cholines for Luzzati's d1 (Luzzati and Husson. 1962. J. Cell Biol. 12:207-219) and the glycerols for Small's dL (Small. 1967. J. Lipid Res. 8:551-556). We do not know if these relations will be true at other hydrations or for other lipids. Of particular interest is the fact that the position of the carbonyl groups marks the average hydrocarbon/headgroup boundary. It must be emphasized, however, that this region of the bilayer must be generally characterized as one of tumultuous chemical heterogeneity because of the thermal motion of the bilayer.

Biophysical Phenomena↗
PubMed1995

Three-dimensional solution structure of bombyxin-II an insulin-like peptide of the silkmoth Bombyx mori: structural comparison with insulin and relaxin.

The three-dimensional solution structure of bombyxin-II, an insulin-like two-chain peptide produced by the brain of the silkworm Bombyx mori, has been determined by simulated annealing calculations based on 535 distance constraints and 24 torsion-angle constraints derived from NMR data and three distance constraints of the disulfide bonds. To our knowledge, this is the first three-dimensional structure determined for an invertebrate insulin-related peptide. The root-mean-square deviations between the best 10 structures and the mean structure are 0.58(+/- 0.15) A for the backbone heavy atoms (N, C alpha, C) and 1.03(+/- 0.18) A for all non-hydrogen atom if less well-defined N and C termini (A1, A20, B(-2) to B4 and B23 to B25) are excluded. The overall main-chain structure of bombyxin-II is similar to that of insulin. However, there are significant conformational and functional differences in their B-chain C-terminal parts. The B-chain C-terminal part of bombyxin-II adopts an extension of the B-chain central helix like that of relaxin and is not required for bombyxin activity, while the corresponding part of insulin adopts a sharp turn and a beta-strand and is essential for insulin activity. This structure demonstrates that bombyxin-II is more closely related to relaxin than to insulin, and suggests that insulin might have evolved the additional receptor-recognition site in the B-chain C-terminal beta-strand to distinguish itself from bombyxin and relaxin. The structure of bombyxin-II thus provides novel insights into the receptor recognition and divergent molecular evolution of insulin-superfamily peptides.

Amino Acid Sequence↗
PubMed1995

Mechanism of post-segregational killing: secondary structure analysis of the entire Hok mRNA from plasmid R1 suggests a fold-back structure that prevents translation and antisense RNA binding.

The hok/sok system of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The Hok mRNA is very stable and can be translated into Hok killer protein. Translation of the Hok mRNA is inhibited by the small unstable Sok antisense RNA. Translation of hok is coupled to an overlapping reading frame termed mok. Translation of mok is tightly regulated by Sok RNA, and Sok RNA thus regulates hok translation indirectly through mok. The rapid decay of Sok RNA explains the onset of Hok synthesis in newborn plasmid-free segregants. However, a second control level is superimposed on this simple induction scheme, since the full-length Hok mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was active. We have therefore previously suggested, that the 3'-terminal region of the full-length Hok mRNA encodes an element which prevents its translation. This element was termed fbi (fold-back inhibition). Here we describe the in vitro secondary structure of the entire Hok mRNA. Our results suggest a closed structure in which the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of mok. This structure explains why full-length Hok mRNA is translationally silent. The proposed structure was further supported by results obtained using mutations in the 3'-end fbi element. These "structure closing" mutations affected the structure much further upstream in the mok translational initiation region and concomitantly prevented antisense RNA binding to the same region of the mRNA. These results lend further support to the induction model that explains onset of Hok mRNA translation in plasmid-free segregants. The most important regulatory element in this model is the FBI structure formed between the 3'-end and the mok translational initiation region. This structure renders Hok mRNA translationally inactive and prevents antisense RNA binding, thus allowing the accumulation of a pool of mRNA which, by slow 3'-end processing, is activated in plasmid-free segregants, eventually leading to the elimination of these cells.

Bacterial Proteins↗
PubMed1992

Comparison of the NMR solution structure with the X-ray crystal structure of the activation domain from procarboxypeptidase B.

The NMR solution structure of the activation domain isolated from porcine procarboxypeptidase B is compared with the X-ray crystal structure of the corresponding segment in the intact proenzyme. For the region of the polypeptide chain that has a well-defined three-dimensional structure in solution, i.e., the backbone atoms of residues 11-76 and 25 amino acid side chains in this segment that form a hydrophobic core in the activation domain, the root-mean-square distance between the two structures is 1.1.A. There are no significant differences in average atom positions between the two structures, but only the NMR structure shows increased structural disorder in three outlying loops located along the same edge of the activation domain. These regions of increased structural disorder in the free domain coincide only partially with the interface to the enzyme domain in the proenzyme.

Amino Acid Sequence↗
PubMed1981

A computer method for construction of secondary structure from polynucleotide sequence. Possible structure of the bacterial replication origin.

A computer method to search the possible secondary structure of a long polynucleotide was developed. As a criterion for the stabilization of a secondary structure, free energy originating from base-pairing was employed, since the structure in solution would be at the free energy minimum. The method is summarized as follows: all possible helices are collected from a given nucleotide sequence under restrictions that the length of a helix is greater than N0 bases (e.g., four bases) and the free energy of the helix calculated according to free energies of two successive sequence-dependent basepairs is lower than E0 (e.g., -5 kcal/mol). The search of secondary structures of low free energy is performed by connecting one helix to another without allowing any base-pairing between loops. For connecting single-stranded regions, destabilizing free energy of 2--3 kcal/mol is added. The method was first applied to several tRNAs and the clover-leaf structure of tRNA was obtained as a free energy minimum. Then, possible secondary structures of the replication origin regions of the Escherichia coli and Salmonella typhimurium chromosomes were examined by the method, assuming that one of the strands in the origin region takes a specific secondary structure. The lowest-energy structure for the E. coli origin was found to be approximately identical to that for the S. typhimurium origin region.

Base Sequence↗
PubMed1986

Water structure in vitamin B12 coenzyme crystals. II. Structural characteristics of the solvent networks.

The geometrical details of the solvent structure in vitamin B12 coenzyme crystals with respect to hydrogen bonding and nonbonded contacts, are described. The individual H-bond geometries varied over wide ranges, similar to those observed in small molecule structures. Large deviations from tetrahedral coordination were found around a majority of the waters. The mutual positions and orientations of the water molecules could not be adequately explained in terms of the H-bonding relationships present in the structure. However, additional investigations, which focused on the short range nonbonded contacts around water positions in a variety of crystal hydrates, revealed several structural regularities (Savage, 1986b). These features relate to the nonbonded O...O, H...O, and H...H interactions, and give rise to a set of repulsive restrictions that are seen to be very much stronger stereochemical restraints than those associated with H-bonding. The short-range restrictions appear largely to govern the local orientational correlations and packing arrangements of the water structure within the coenzyme (and other hydrate) crystals. In more general terms, the inclusion of the nonbonding relationships as well as the attractive H-bonding interactions, leads to a significant increase in our understanding of water structure(s). The repulsive restrictions can be used as stereochemical restraints in the interpretation and refinement of solvent structures within larger hydrate systems, such as protein crystals. They may also be included in potential functions used to simulate solvent structures in aqueous solutions and hydrate systems.

Hydrogen Bonding↗
PubMed1991

Three-dimensional structure of murine anti-p-azophenylarsonate Fab 36-71. 2. Structural basis of hapten binding and idiotypy.

Comparison between the structures and solvent-accessible surfaces of the antigen-binding fragments of two murine anti-p-azophenylarsonate monoclonal antibodies, one bearing a major cross-reactive idiotype of A/J strain mice (36-71) and one lacking the idiotype (R19.9; Lascombe et al., 1989), highlight the structural basis for the determination of hapten affinity and idiotypy. Since the sequence of R 19.9 is identical with the germline-encoded sequence at 16 positions in both heavy-chain and light-chain variable regions where somatic mutations and junctional differences have occurred to produce the 36-71 sequence, the structure of R 19.9 can be used to model the structure of the germline-encoded antibody (36-65) in the regions around these sites. These 16 sequence differences exclude the third heavy-chain complementarity-determining region because R 19.9 utilizes a D gene segment not associated with the predominant idiotype, which is 4 residues longer than the canonical D gene segment utilized in the sequences of 36-71 and 36-65. This difference between the structures of R 19.9 and 36-71 does not affect the validity of using the structure of R 19.9 to model the structure of 36-65 since the third heavy-chain complementarity-determining region is highly solvent-exposed in both 36-71 and R 19.9, and does not interact with any of these 16 sites. Comparing the structures of 36-71 and R 19.9 suggests that only three of the differences in the heavy-chain sequences, and three of the differences in the light-chain sequences of 36-71 and 36-65, increase the affinity for hapten.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Three-dimensional structure of the kringle sequence: structure of prothrombin fragment 1.

The three-dimensional structure of bovine prothrombin fragment 1 has been solved at 2.8-A resolution. The electron density clearly reveals four disulfide bridges along with more than 80% of the side chains completely in density, which correspond faithfully to the kringle sequence, its preceding 30 residues, and the dodecapeptide carboxy terminal; the polysaccharide and the first 35 residues of the amino terminal of fragment 1 are disordered or about 40% of the structure. The folding of the kringle sequence is based upon close disulfide van der Waals contacts between Cys-87-Cys-127 and Cys-115-Cys-139 (4.1 A between midpoints of the bridges), two antiparallel strands of highly conserved (113-118, 124-129) beta-structure, and the stacking of some conserved aromatic residues, all near the center of the folded structure. Moreover, the overall folding appears to be duplicated as a pair of stacked duplex loops with an antiparallel open loop. The overall shape of the kringle structure approximates an eccentric oblate ellipsoid of dimensions 11 X 28 X 30 A. The residues immediately preceding the kringle are dominated by alpha-helical structure (Phe-41-Cys-48; Leu-56-Glu-63). Residues Phe-41-Trp-42 and Tyr-45, which are conserved in factor IX, factor X, protein C, and protein Z, form another aromatic stacked cluster while the Cys-48-Cys-61 disulfide loop corresponds to the well-known alpha/beta structural unit. The dodecapeptide carboxy-terminal interkringle chain extends along the periphery of the kringle in its plane and forms a beta-structure with the kringle-closing Ser-140-Val-143 tetrapeptide.

Amino Acid Sequence↗
PubMed1988

A study of the structure of human complement component factor H by Fourier transform infrared spectroscopy and secondary structure averaging methods.

Fourier transform infrared spectroscopy was used to investigate the secondary structure of human complement component factor H in H2O and 2H2O buffers. The spectra show a broad amide I band which after second-derivative calculations is shown to be composed of three components at 1645, 1663, and 1685 cm-1 in H2O and at 1638, 1661, and 1680 cm-1 in 2H2O. The frequencies of these components are consistent with the existence of an extensive antiparallel beta-strand secondary structure. The exchange properties of the amide protons of factor H as measured in 2H2O buffers are rapid and lead to an estimate of NH proton nonexchange that is comparable with those for small globular proteins. Human factor H is constructed from a linear sequence of 20 short consensus repeats with a mean of 61 residues in each one. To investigate the secondary structure further, secondary structure predictions were carried out on the basis of an alignment scheme for 101 sequences for these repeats as found in human factor H and 12 other proteins. These predictions were averaged in order to improve the reliability of the calculations. Both the Robson and the Chou-Fasman methods indicate significant beta-structural contents. Residues 21-51 in the 61-residue repeat show a clear prediction of four strands of beta-structure and four beta-turns. A structural model based on antiparallel beta-strands in the secondary structure is proposed and discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Secondary structure of the mammalian 70-kilodalton heat shock cognate protein analyzed by circular dichroism spectroscopy and secondary structure prediction.

Heat shock proteins are rapidly synthesized when cells are exposed to stressful agents that cause protein damage. The 70-kDa heat shock induced proteins and their closely related constitutively expressed cognate proteins bind to unfolded and aberrant polypeptides and to hydrophilic peptides. The structural features of the 70-kDa heat shock proteins that confer the ability to associate with diverse polypeptides are unknown. In this study, we have used circular dichroism (CD) spectroscopy and secondary structure prediction to analyze the secondary structure of the mammalian 70-kDa heat shock cognate protein (hsc 70). The far-ultraviolet CD spectrum of hsc 70 indicates a large fraction of alpha-helix in the protein and resembles the spectra one obtains from proteins of the alpha/beta structural class. Analysis of the CD spectra with deconvolution methods yielded estimates of secondary structure content. The results indicate about 40% alpha-helix and 20% aperiodic structure within hsc 70 and between 16-41% beta-sheet and 21-0% beta-turn. The Garnier-Osguthorpe-Robson method of secondary structure prediction was applied to the rat hsc 70 amino acid sequence. The predicted estimates of alpha-helix and aperiodic structure closely matched the values derived from the CD analysis, whereas the predicted estimates of beta-sheet and beta-turn were midway between the CD-derived values. Present evidence suggests that the polypeptide ligand binding domain of the 70-kDa heat shock protein resides within the C-terminal 160 amino acids [Milarski, K. L., & Morimoto, R. I. (1989) J. Cell Biol. 109, 1947-1962].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Structures of monosialyl oligosaccharides isolated from the respiratory mucins of a non-secretor (O, Lea+b-) patient suffering from chronic bronchitis. Characterization of a novel type of mucin carbohydrate core structure.

Mucin glycopeptides were prepared from the respiratory mucus of a non-secretor, chronic bronchitis patient with blood group O, Le(a)+b-. Oligosaccharides were released by alkaline borohydride treatment and purified by anion-exchange chromatography, size-exclusion chromatography and high-performance liquid chromatography on a silica-bonded alkylamine column. Structural studies employed 500-MHz 1H-NMR spectroscopy and fast atom bombardment-mass spectrometry. Twenty-six monosialyl oligosaccharides, ranging in size from di- to octasaccharide, were fully characterized in this study. The sialic acid occurs either alpha(2-->3)- or alpha(2-->6)-linked to a galactosyl residue, or alpha(2-->6)-linked to GalNAc-ol. In keeping with the non-secretor status of the patient, no structures with an alpha(-->2)-linked fucose residue were found. Nine of the structures had fucose present in alpha(-->3) linkage, either in the Le(x) determinant or in the sialyl-Le(x) determinant, or both (structure 11). Only one oligosaccharide (structure 3b) was seen with fucose alpha (1-->4)-linked in the Le(a) determinant. Eight structures isolated from the mucins of the non-secretor patient had not been found previously in the respiratory mucins of secretor individuals; they are listed below. Among these, structure 4 alpha represents a novel type of mucin carbohydrate core structure.

Bronchi↗
PubMed1988

Stability of RNA stem-loop structure and distribution of non-random structure in the human immunodeficiency virus (HIV-I).

The stability of potential RNA stem-loop structures in human immunodeficiency virus isolates, HTLV-III and ARV, has been calculated, and the relevance to the local significant secondary structures in the sequence has been tested statistically using a Monte Carlo simulation method. Potentially significant structures exist in the 5'non-coding region, the boundary regions between the protein coding frames, and the 3' non-coding region. The locally optimal secondary structure occurring in the 5' terminal region has been assessed using different overlapping segment sizes and the Monte Carlo method. The results show that the most favorable structure for the 5' mRNA leader sequence of HIV has two stem-loops folded at nucleotides 5-104 in the R region (stem-loop I, 5-54 and stem-loop II, 58-104). A large fluctuation of segment score of the local optimal secondary structure also occurs in the boundary between the exterior glycosylated protein or outer membrane protein and transmembrane protein coding region. This finding is surprising since no RNA signals or RNA processing are expected to occur at this site. In addition, regions of the genome predicted to have significantly more open structure at the RNA level correlate closely with hypervariable sites found in these viral genomes. The possible importance of local secondary structure to the biological function of the human immunodeficiency virus genome is discussed.

Base Sequence↗
PubMed1986

Structural-functional relationships along the distal nephron.

The distal tubule, which includes the thick ascending limb (TAL), the macula densa, and the distal convoluted tubule (DCT), and the collecting duct are structurally heterogeneous, thus reflecting the functional heterogeneity that is also present. As the TAL ascends from medulla to cortex, the surface area of the apical plasma membrane increases while that of the basolateral membrane decreases. The structure of the DCT resembles that of the medullary TAL. An excellent correlation exists between structure, Na-K-ATPase activity, and NaCl reabsorptive capacity in the distal tubule. The collecting duct is subdivided into the initial collecting tubule (ICT), and cortical (CCD), outer medullary (OMCD), and inner medullary (IMCD) collecting ducts. Between the distal tubule and the collecting duct is a transition region termed the connecting segment or connecting tubule (CNT). Considerable structural heterogeneity exists along the collecting duct within the two major cell populations, the intercalated cells and the principal cells. In the CNT, the ICT, and the CCD, potassium loading and mineralocorticoids stimulate Na-K-ATPase activity and cause proliferation of the basolateral membrane of CNT cells and principal cells, thus identifying the cells responsible for mineralocorticoid-stimulated potassium secretion in these regions. Finally, at least two morphologically distinct populations of intercalated cells exist, types A and B. In the rat, type A predominates in the CNT and the OMCD and is believed to be responsible for H+ secretion, at least in the OMCD. Type B predominates in the CCD, where it may be involved in bicarbonate secretion.

Animals↗
PubMed1990

Crystal structures of subtilisin BPN' variants containing disulfide bonds and cavities: concerted structural rearrangements induced by mutagenesis.

The X-ray structures of four genetically engineered disulfide variants of subtilisin have been analyzed to determine the energetic and structural constraints involved in inserting disulfide bonds into proteins. Each of the engineered disulfides exhibited atypical sets of dihedral angles compared with known structures of natural disulfide bridges in proteins and affected its local structural environment to a different extent. The disulfides located in buried regions, Cys26-Cys232 and Cys29-Cys119, induced larger changes than did Cys24-Cys87 and Cys22-Cys87, which are located on the surface of the molecule. An analysis of the concerted changes in secondary structure units such as alpha-helices and beta-sheets indicated systematic long-range effects. The observed changes in the mutants were largely distributed asymmetrically around the inserted disulfides, reflecting different degrees of inherent flexibility of neighboring secondary structure types. The disulfide substitution in each variant molecule created some invaginations or cavities, causing a reorganization of the surrounding water structure. These changes are described, as well as the changes in side chain positions of groups that border the cavities.

Base Sequence↗
PubMed1991

Structure determination of the Antp (C39----S) homeodomain from nuclear magnetic resonance data in solution using a novel strategy for the structure calculation with the programs DIANA, CALIBA, HABAS and GLOMSA.

The structure of a mutant Antennapedia homeodomain, Antp(C39----S), from Drosophila melanogaster was determined using a set of new programs introduced in the accompanying paper. An input dataset of 957 distance constraints and 171 dihedral angle constraints was collected using two-dimensional n.m.r. experiments with the 15N-labeled protein. The resulting high quality structure for Antp(C39----S), with an average root-mean-square deviation of 0.53 A between the backbone atoms of residues 7 to 59 in 20 energy-refined distance geometry structures and the mean structure, is nearly identical to the previously reported structure of the wild-type Antp homeodomain. The only significant difference is in the connection between helices III and IV, which was found to be less kinked than was indicated by the structure determination for Antp. The main emphasis of the presentation in this paper is on a detailed account of the practical use of a novel strategy for the computation of nuclear magnetic resonance structures of proteins with the combined use of the programs DIANA, CALIBA, HABAS and GLOMSA.

Algorithms↗
PubMed1994

Principles determining the structure of beta-sheet barrels in proteins. II. The observed structures.

In the accompanying paper we derived a set of principles that, we argue, govern the structure of beta-sheet barrels. Barrel structures are classified in terms of two integral parameters: the number of strands in the beta-sheet, n, and a measure of the stagger in the beta-sheet, S. We derived a set of equations that show how the (n, S) values of a barrel structure determine the arrangement of its strands; its general shape; the twist and coiling of the beta-sheet, and the arrangement of residues in the barrel interior. This work suggested that there are ten different combinations of n and S that form barrels with good beta-sheet geometries and interiors close packed by beta-sheet residues. In this paper we demonstrate the validity of these principles. We analyse in detail the observed structures of 39 different beta-sheet barrels. These structures include representatives of all the different barrel structures currently known and for which atomic co-ordinates are available. We show that the observed arrangement of the strands, and the extent of the twist and coiling of the beta-sheets, are very close to those calculated from the (n, S) values for the barrel. Of the 39 structures, 34 have one of the ten (n, S) values that we expect to form barrels with good beta-sheet geometries and interiors close packed by beta-sheet residues. The other five have one of two (n, S) values that give good beta-sheet geometries but radii so large the beta-sheet residues leave cavities at the centre of the barrels. In at least four of these cavities have a functional role.

Amino Acid Sequence↗
PubMed1995

Solution structure of the cytoplasmic domain of phopholamban: phosphorylation leads to a local perturbation in secondary structure.

Peptides representing the N-terminal domain (Ia) of the cardiac sarcoplasmic reticulum protein phospholamban (residues 1-25 [PLB(1-25)] and a phosphorylated form [pPLB(1-25)]) were synthesized and their conformations examined using circular dichroism and nuclear magnetic resonance spectroscopy. In aqueous solution, both PLB(1-25) and pPLB(1-25) adopt a primarily disordered conformation. In 30% trifluoroethanol/10 mM phosphate, PLB(1-25) exhibits a CD spectrum consistent with 60% helical structure. This value decreases to 27% for the phosphorylated peptide. CD spectra in 2% SDS indicate 40% alpha-helix for PLB(1-25) and 20% for pPLB(1-25). Full chemical shift assignments were obtained by conventional homonuclear NMR methodologies for both PLB(1-25) and pPLB(1-25) in 30% trifluoroethanol/water and 300 mM SDS. The solution structure of PLB(1-25) in 30% TFE/water was determined from distance geometry calculations using 54 NOE distance constraints and 17 torsion angle constraints. In the family of 20 calculated conformers, the root mean square deviation from the mean structure is 0.79 A for backbone heavy atoms of residues 1-17. The structure comprises a regular alpha-helix extending from M1 to S16 with the remaining C-terminal residues disordered. The calculated structure is supported by analysis of C alpha H secondary shifts which are significantly negative for residues 1-16. Chemical shift degeneracy is substantially more extensive in the phospho form and precludes a direct comparison of calculated structures. However, the magnitudes of upfield secondary shifts are decreased by 20% in residues 1-11 and are not significantly helical for residues 12-16 according to the criteria of Wishart et al. [(1992) Biochemistry 31, 1647-1651]. 3JHN alpha coupling constants measured for I12, R13, A15, and S16 also suggest that residues 12-16 undergo a local unwinding of the helix upon phosphorylation. Similar results are obtained for PLB(1-25) and pPLB(1-25) in 300 mM perdeuterated sodium dodecyl sulfate except that differences in backbone dynamics for the helical and nonhelical regions of the peptide are evident in the DQF-COSY line shapes for fingerprint cross-peaks. This disruption of structure at the C-terminus of the helix suggests a model for phosphorylation-induced dissociation of the PLB/Ca(2+)-ATPase complex.

Amino Acid Sequence↗