Methyl dynamics of the amyloid-beta peptides Abeta40 and Abeta42

To probe the role of side chain dynamics in Abeta aggregation, we studied the methyl dynamics of native Abeta40 and Abeta42 by measuring cross relaxation rates with interleaved data collection. The methyl groups in the C-terminus are in general more rigid in Abeta42 than in Abeta40, consistent with previous results from backbone (15)N dynamics. This lends support to the hypothesis that a rigid C-terminus in Abeta42 may serve as an internal aggregation seed. Interestingly, two methyl groups of V18 located in the central hydrophobic cluster are more mobile in Abeta42 than in Abeta40, most likely due to the paucity of V18 intra-molecular interactions in Abeta42. V18 may then be more available for inter-molecular interactions to form Abeta42 aggregates. Thus, the side chain mobility of the central hydrophobic cluster may play an important role in Abeta aggregation and may contribute to the difference in aggregation propensity between Abeta40 and Abeta42.     

Isomerization and/or racemization at Asp23 of Abeta42 do not increase its aggregative ability, neurotoxicity, and radical productivity in vitro

Aggregation of the 42-mer amyloid β peptide (Aβ42) plays a pivotal role in the pathogenesis of Alzheimer’s disease. Recent investigations suggested the isomerization and/or racemization of Asp at position 1, 7, or 23 to be associated with the pathological role of Aβ42. Our previous study indicated that the turn at positions 22 and 23 of Aβ42 is closely related to its neurotoxicity through the formation of radicals. To clarify the contribution of these modifications at Asp23 to the pathology, three isomerized and/or racemized Aβ42 mutants were prepared. l-isoAsp23- and d-Asp23-Aβ42 showed moderate aggregative ability similar to the wild type. However, d-Asp23-Aβ42 was less neurotoxic than the wild type, while l-isoAsp23-Aβ42 was as toxic as the wild type. In contrast, d-isoAsp23-Aβ42 showed weak aggregative ability without neurotoxicity. These results suggest the isomerization and/or racemization of Asp23 not to be related to the pathogenesis, but to be a consequence of chemical reactions during the long-term deposition of fibrils.     

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     

A model for the secondary structure of beta-lactamases

The effects of the quaternary agent meproadifen on ACh-activated channel currents were studied on myoballs cultured from hind limb muscles of neonatal rats. Meproadifen (0.02-0.1 microM) combined with ACh (0.1-0.3 microM) in the patch pipette caused an increase, followed by a decrease, in the frequency of channel openings. At concentrations greater than 0.2 microM the initial phase was not detected and a rapid and marked reduction in the opening frequency was observed. Meproadifen (up to 2.5 microM) produced no change in the duration or conductance of the open state of ACh-activated channels. In addition, this agent induced the appearance of events with a marked increase in the 'noise' during the opening phase. The lack of effect under inside-out patch conditions suggested that meproadifen binds to a site located at the external portion of the nicotinic macromolecule and has no access to it through the cell membrane. This study indicated that non-competitive antagonists such as meproadifen can facilitate receptor activation and desensitization.     

Complete 1H, 15N and 13C assignments, secondary structure, and topology of recombinant human interleukin-6

Summary Essentially complete backbone and side-chain ¹H, ¹⁵N and ¹³C resonance assignments for the 185-aminoacid cytokine interleukin-6 (IL-6) are presented. NMR experiments were performed on uniformly [¹⁵N]-and [¹⁵N, ¹³C]-labeled recombinant human IL-6 (rIL-6) using a variety of heteronuclear NMR experiments. A combination of ¹³C-chemical shift, amide hydrogen-bond exchange, and ¹⁵N-edited NOESY data allowed for analysis of the secondary structure of IL-6. The observed secondary structure of IL-6 is composed of loop regions connecting five -helices, four of which are consistent in their length and disposition with the four-helix bundle motif present in other related cytokines and previously postulated for IL-6. In addition, the topology of the overall fold was found to be consistent with a left-handed up-up-down-down four-helix bundle based on a number of long-range interhelical NOEs. The results presented here provide deeper insight into structure-function relationships among members of the four-helix bundle family of proteins.     

SFESA: a web server for pairwise alignment refinement by secondary structure shifts

Background

Protein sequence alignment is essential for a variety of tasks such as homology modeling and active site prediction. Alignment errors remain the main cause of low-quality structure models. A bioinformatics tool to refine alignments is needed to make protein alignments more accurate.

Results

We developed the SFESA web server to refine pairwise protein sequence alignments. Compared to the previous version of SFESA, which required a set of 3D coordinates for a protein, the new server will search a sequence database for the closest homolog with an available 3D structure to be used as a template. For each alignment block defined by secondary structure elements in the template, SFESA evaluates alignment variants generated by local shifts and selects the best-scoring alignment variant. A scoring function that combines the sequence score of profile-profile comparison and the structure score of template-derived contact energy is used for evaluation of alignments. PROMALS pairwise alignments refined by SFESA are more accurate than those produced by current advanced alignment methods such as HHpred and CNFpred. In addition, SFESA also improves alignments generated by other software.

Conclusions

SFESA is a web-based tool for alignment refinement, designed for researchers to compute, refine, and evaluate pairwise alignments with a combined sequence and structure scoring of alignment blocks. To our knowledge, the SFESA web server is the only tool that refines alignments by evaluating local shifts of secondary structure elements. The SFESA web server is available at http://prodata.swmed.edu/sfesa.     

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.     

Correlation between 15N NMR chemical shifts in proteins and secondary structure

Summary An empirical correlation between the peptide ¹⁵N chemical shift, ¹⁵N_i, and the backbone torsion angles _i, _i–1 is reported. By using two-dimensional shielding surfaces (_i1–1), it is possible in many cases to make reasonably accurate predictions of ¹⁵N chemical shifts for a given structure. On average, the rms error between experiment and prediction is about 3.5 ppm. Results for threonine, valine and isoleucine are worse (4.8 ppm), due presumably to ₁-distribution/-gauche effects. The rms errors for the other amino acids are 3 ppm, for a typical maximal chemical shift range of 15–20 ppm. Thus, there is a significant correlation between ¹⁵N chemical shift and secondary structure.     

Fluorescence study of secondary structure of DNA within bacteriophage lambda

Bromoacetaldehyde (BAA) was used to study the secondary structure of DNA in lambda-phage particles. It was determined that about 1% of the adenines in the intraphage lambda-DNA reacts readily with BAA, thus, they are placed in DNA sites with disturbed complementary interactions. These adenines are close to the tryptophan residues of the phage protein. Fluorescence emission of epsilon A in the intraphage DNA is dramatically quenched. This, apparently, indicates the interaction between epsilon A and Trp- and/or Tyr- and/or Met-residues of phage protein.     

Assignment and secondary structure of calcium-bound human S100B

The NMR assignments of backbone ¹H, ¹³C,and ¹⁵N resonances for calcium-bound human S100B werecompleted via heteronuclear multidimensional NMR spectroscopic techniques.NOE correlations, amide exchange, ³J_HN_Hcoupling constants, and CSI analysis were used to identify the secondarystructure for Ca-S100B. The protein is comprised of four helices (helix I,Glu²-;Arg²⁰; helix II,Glu³¹-;Asn³⁸; helix III,Gln⁵⁰-;Thr⁵⁹; helix IV,Phe⁷⁰-;Phe⁸⁷), three loops (loop I,Glu²¹-;His²⁵; loop II,Glu³⁹-;Glu⁴⁹; loop III,Leu⁶⁰-;Gly⁶⁶), and two -strands(strand I, Lys²⁶>-;Lys²⁸; strand II,Glu⁶⁷-;Asp⁶⁹) which form a shortantiparallel -sheet. Helix IV is extended by approximately one turnwhen compared to the secondary structures of apo-rat [Drohat et al. (1996)Biochemistry, 35, 11577-;11588] and bovine S100B [Kilby et al. (1996)Structure, 4, 1041-;1052]. In addition, several residues outside thecalcium-binding loops in S100B undergo significant backbone chemical shiftchanges upon binding calcium which are not observed in the related proteincalbindin D_9k. Together these observations support previoussite-directed mutagenesis, absorption spectroscopy, and cysteine chemicalreactivity experiments, suggesting that the C-terminus in Ca-S100B isimportant for interactions with other proteins.     

Predictions of the secondary structure and antigenicity of human and bovine tropoelastins

Secondary structure and antigenicity predictive methods have been applied to the sequences of human and bovine tropoelastins in order to have some insight into the molecular structure of its insoluble counterpart, i.e., elastin. For both tropoelastins, all the predictions yielded 11 major regions, in which the pleated conformation was predominant, separated by 10 strong helical segments of various lengths located within alanyl rich regions of the chains. The overall conformations of human and bovine tropoelastins were estimated to contain 18 ± 5% -helices, 63 ± 17% -sheets, 13 ± 13% -turns and 6 ± 6% random coil. For both tropoelastins, antigenicity predictions indicated the presence of seven synthetic decapeptides corresponding to continuous linear epitopes of the molecule. Some of the predicted epitopes are located in the same regions in both species while others are not. These predictions have allowed us to propose an / conformation for tropoelastin. Therefore this extracellular matrix macromolecule might be more structured (10 helical segments for about 18% of the overall structure) than previously suggested.Abbreviations HTPE human tropoelastin - BTPE bovine tropoelastin - AG antigenic index - CF Chou and Fasman algorithm - GOR method of Garnier Osguthorpe and Robson - DC decision constant - CD circular dichroism - NMR nuclear magnetic resonance     

Human but not rat amylin shares neurotoxic properties with Abeta42 in long-term hippocampal and cortical cultures

Type 2 diabetes mellitus (DM) and Alzheimer's disease (AD) share epidemiological and biochemical features. Both are characterized by insoluble protein aggregates with a fibrillar conformation--amylin in Type 2 DM pancreatic islets, and Abeta in AD brain. To determine whether amylin shares neurotoxic properties with Abeta, we incubated hippocampal and cortical neurons with Abeta42 and human amylin. Different from non-amyloidogenic rat amylin, both caused a dose-, time- and cell type-specific neurotoxicity supporting the notion of a similar toxic mechanism. Depending on the cell type, this finding is also supported by co-incubation of human amylin and Abeta.     

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     

Resonance assignment and secondary structure determination and stability of the recombinant human uteroglobin with heteronuclear multidimensional NMR

Human uteroglobin (h-UG) or Clara cell 10kDa (cc10kDa) is a steroid-dependent, 17 kDahomodimeric, secretory protein with potent anti-inflammatory/immunomodulatory properties.However, the exact physiological role still remains to be determined. It has been hypothesisedthat its activity is exerted through the binding of a specific target represented by a smallmolecule (still unknown), and that the binding is regulated by the formation/disruption of twocysteine bonds. The binding properties of the reduced UG have been proved in vitro forseveral different molecules, but no in vivo data are available to date. However, binding hasbeen observed between reduced rabbit UG and a protein of an apparent molecular mass of90 kDa and, more recently, we found an h-UG-binding protein (putative receptor), of anapparent molecular mass of 190 kDa, on the surface of several cell types. The recognitioninvolves oxidised h-UG. These findings pose the problem of the relevance of the oxidationstate in the recognition process. To determine the solution structure of the oxidised h-UG, weproduced wild-type as well as uniformly 15N- and 15N/13C-labelled samples of therecombinant protein. The assignments of the 1H, 15N and 13C resonances are presented,based on a series of homonuclear 2D and 3D and heteronuclear 2D and 3D double and tripleresonance NMR experiments. Our results indicate that h-UG is an extremely stable proteinunder a wide range of temperatures and pH conditions. The secondary structure in solutionis in general agreement with previously reported crystal structures of rabbit UG, suggestingthat cc10kDa and h-UG are indeed the same protein. Small local differences found in the N-and C-terminal helices seem to support the hypothesis that flexibility involves these residues;moreover, it possibly accounts for the residual binding properties observed when the proteinis in the oxidised state.     

Nucleotide sequence and presumed secondary structure of the 28S rRNA of pea aphid implication for diversification of insect rRNA

Determination of the entire nucleotide sequence of the aphid 28S ribosomal RNA gene (28S rDNA) revealed that it is 4,147 by in length with a G + C content of 60.3%. Based on the nucleotide sequence, we constructed a presumed secondary-structure model of the aphid 28S rRNA which indicated that the aphid 28S rRNA is characterized by the length and high G + C content of its variable regions. The G + C content of the aphid's variable regions was much higher than that of the entire sequence of the 28S rRNA, which formed a striking contrast to those ofDrosophila with the G + C content much lower than the entire 28S molecule. In this respect, the aphid 28S rRNA somewhat resembled those of vertebrates. This is the third report of a complete large-subunit rRNA sequence from an arthropod, and the first 28S rRNA sequence for a nondipterous insect. Correspondence to: H. Ishikawa     

Assignments, secondary structure and dynamics of the inhibitor-free catalytic fragment of human fibroblast collagenase

Fibroblast collagenase (MMP-1), a 169-residue protein with amolecular mass of 18.7 kDa, is a matrix metalloproteinase which has beenassociated with pathologies such as arthritis and cancer. The assignments ofthe ¹H, ¹⁵N, ¹³CO and¹³C resonances, determination of the secondary structure andanalysis of ¹⁵N relaxation data of the inhibitor-freecatalytic fragment of recombinant human fibroblast collagenase (MMP-1) arepresented. It is shown that MMP-1 is composed of a -sheet consistingof five -strands in a mixed parallel and antiparallel arrangement(residues 13–19, 48–53, 59–65, 82–85 and94–99) and three -helices (residues 27–43, 112–124and 150–160). This is nearly identical to the secondary structuredetermined from the refined X-ray crystal structures of inhibited MMP-1. Themajor difference observed between the NMR solution structure ofinhibitor-free MMP-1 and the X-ray structures of inhibited MMP-1 is thedynamics of the active site. The 2D ¹⁵N-¹H HSQCspectra, the lack of information in the ¹⁵N-edited NOESYspectra, and the generalized order parameters (S²) determinedfrom ¹⁵N T₁, T₂ and NOE datasuggest a slow conformational exchange for residues comprising the activesite (helix B, zinc ligated histidines and the nearby loop region) and ahigh mobility for residues Pro¹³⁸-Gly¹⁴⁴ in thevicinity of the active site for inhibitor-free collagenase. In contrast tothe X-ray structures, only the slow conformational exchange is lost in thepresence of an inhibitor.     

Primary and secondary plasmodesmata: structure, origin, and functioning

Summary In the multicellular organisms of higher plants, plasmodesmata provide pathways for intimate symplasmic communication between neighboring cells. The arguments summarized in the present review demonstrate that plasmodesmata are diverse and highly dynamic structures. Differences in the plasmodesmal origin and modifications of the plasmodesmal structure and functioning at the various cell interfaces are the basic means which give rise to a complicated and flexibile symplasmic network. This complex communication system is discussed to serve a significant role in the coordinated development and in the concerted physiological functioning of the cells within the plant tissues, organs, and organisms.     

Prediction and comparison of the secondary structure of legume lectins

Secondary structure prediction for the 4 legume lectins: Concanavalin A, soybean agglutinin, favabean lectin and lentil lectin, was done by the method of Chou and Fasman. This prediction shows that these four lectins fall into a structurally distinct class of proteins, containing high amounts of β-sheet and β-turns. There is a notable similarity in the gross structure of these proteins; all four of them contain about 40–50% of β-sheet, 35–45 % β-turn and 0–10% of α-helix. When the secondary structure of corresponding residues in each pair of these lectins was compared, there was a striking similarity in the Concanavalin A-soybean agglutinin and favabean lectin-lentil lectin pairs, and considerably less similarity in the other pairs, suggesting that these legume lectins have probably evolved in a divergent manner from a common ancestor. A comparison of the predicted potential β-turn sites also supports the hypothesis of divergent evolution in this class of lectins.