Main Chain Direction Research Articles

D-Peptide Ligands for the Co-chaperone DnaJ

The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, binds hydrophobic polypeptide segments in extended conformation. The co-chaperone DnaJ (Hsp40) has been reported to bind native and denatured proteins as well as peptides. We tested pseudo-peptides of D-amino acids as ligands for both chaperones. In comparison to the parent all-L peptide, these mimetics had either enantiomorphic side chain positions combined with retained main chain direction (normal all-D peptide) or unchanged side chain topology together with reverse direction of the peptide backbone (retro all-D peptide). The peptides were labeled with acrylodan (a), and their binding to DnaK and DnaJ was monitored by the accompanying increase in fluorescence intensity. The parent all-L peptide a-CALLLSAARR bound to both DnaK (Kd = 0.1 microM) and DnaJ (Kd = 9.2 microM). In contrast, the normal all-D and retro all-D peptides did not bind to DnaK; they bound, however, to DnaJ with Kd values of 6.8 microM and 0.9 microM, respectively. The emission spectra of the DnaJ-bound peptides suggests that DnaJ bound both D-peptides with the same main chain direction as L-peptides. Binding of the normal all-D and all-L peptides inhibited the DnaJ-induced stimulation of DnaK ATPase. However, binding of the retro all-D analog to DnaJ did not impair the stimulation, indicating the existence of separate binding sites for peptides and DnaK.

Structural comparison in solution of a native and retro peptide derived from the third helix ofStaphylococcus aureus protein A, domain B: retro peptides, a useful tool for the discrimination of helix stabilization factors dependent on the peptide chain orientation

A peptide fragment corresponding to the third helix of Staphylococcus Aureus protein A, domain B, was chosen to study the effect of the main‒chain direction upon secondary structure formation and stability, applying the retro‒enantio concept. For this purpose, two peptides consisting of the native (Ln) and reversed (Lr) sequences were synthesized and their conformational preferences analysed by CD and NMR spectroscopy. A combination of CD and NMR data, such as molar ellipcitity, NOE connectivities, Hα and NH chemical shifts, 3JαN coupling constants and amide temperature coefficients indicated the presence of nascent helices for both Ln and Lr in water, stabilized upon addition of the fluorinated solvents TFE and HFIP. Helix formation and stabilization appeared to be very similar in both normal and retro peptides, despite the unfavourable charge–macrodipole interactions and bad N-capping in the retro peptide. Thus, these helix stabilization factors are not a secondary structure as determined for this specific peptide. In general, the synthesis and confirmational analysis of peptide pairs with opposite main‒chain directions, normal and retro peptides, could be useful in the determination of secondary structure stabilization factors dependent on the direction. © 1997 European Peptide Society and John Wiley & Sons, Ltd.

Structural comparison in solution of a native and retro peptide derived from the third helix of Staphylococcus aureus protein A, domain B: retro peptides, a useful tool for the discrimination of helix stabilization factors dependent on the peptide chain orientation

A peptide fragment corresponding to the third helix of Staphylococcus Aureus protein A, domain B, was chosen to study the effect of the main‒chain direction upon secondary structure formation and stability, applying the retro‒enantio concept. For this purpose, two peptides consisting of the native (Ln) and reversed (Lr) sequences were synthesized and their conformational preferences analysed by CD and NMR spectroscopy. A combination of CD and NMR data, such as molar ellipcitity, NOE connectivities, Hα and NH chemical shifts, 3JαN coupling constants and amide temperature coefficients indicated the presence of nascent helices for both Ln and Lr in water, stabilized upon addition of the fluorinated solvents TFE and HFIP. Helix formation and stabilization appeared to be very similar in both normal and retro peptides, despite the unfavourable charge–macrodipole interactions and bad N-capping in the retro peptide. Thus, these helix stabilization factors are not a secondary structure as determined for this specific peptide. In general, the synthesis and confirmational analysis of peptide pairs with opposite main‒chain directions, normal and retro peptides, could be useful in the determination of secondary structure stabilization factors dependent on the direction. © 1997 European Peptide Society and John Wiley & Sons, Ltd.

Strain-induced birefringence and molecular structure of glassy polymers

The dynamic birefringence and viscoelasticity were measured for poly(ethylene naphthalene-2,6-dicarboxylate), and the contributions from the main chain orientation and the rotational orientation of repeating unit to the strain-induced birefringence were evaluated. The values, together with published ones for other glassy polymers, were compared with the anisotropy of polarizability tensor of the repeating units. The contribution from the main chain orientation for various polymers was in accord with that evaluated from the excess polarizability of the unit in the main chain direction assuming a quasi-affine orientation of the unit. The contribution from the rotation was well correlated with the anisotropy in the plane perpendicular to the chain axis. The degree of the rotational orientation was similar among the polymers. The rotational orientation was high for vinyl polymers with large side-groups.

Blends of a longitudinal polymer liquid crystal with polycarbonate: relation of the phase diagram to mechanical properties

Abstract This work is aimed at an evaluation of the capability to predict the mechanical behaviour of blends on the basis of the phase diagram. Connections between phase structures, the phase diagram and mechanical properties were studied for binary blends of polycarbonate (PC) based on bisphenol-a with PET/0.6PHB, where PET = poly(ethylene terephthalate), PHB = p -hydroxybenzoic acid and 0.6 = the mole fraction of PHB in the copolymer. PET/0.6PHB is a longitudinal polymer liquid crystal (PLC) with LC sequences in the backbone along the main-chain direction. The techniques used were differential scanning calorimetry (d.s.c.), thermomechanical analysis (t.m.a.) in the penetration and three-point-bending modes, thermally stimulated depolarization (t.s.d.) and two kinds of dynamic mechanical analysis (d.m.a.) as a function of frequency ω and temperature T . The resulting phase diagram is plotted as a quasi-binary one ( T versus concentration of PET/0.6PHB or PC) but provides information on miscibilities of all three pairs of constituents; it contains equilibrium as well as long-living non-equilibrium phases, including the quasi-liquid. D.s.c. and d.m.a. results show that glass transition temperatures for the PC-rich phase drop by ∼50°C after 1 h of annealing, with evident consequences for mechanical properties. Variations of storage modulus and strain as a function of time are reported for different blend compositions. Isothermal storage moduli and results of three-point bending as a function of composition are connected to the phase diagram. Prediction of mechanical behaviour is possible from miscibilities of constituents in specific regions of the diagram.

Molecular Interactions of Thrombin.

Thrombin possesses at least three independent binding sites for substrate, inhibitor, and co-factor molecules, four counting the Na+ ion binding site. The S1 subsite of the active site is specific for an arginine side group, while S2 is a more extended apolar site. The highly electropositive S' subsites of the fibrinogen exosite circumnavigate about a third of the thrombin surface, although evidence suggests molecular recognition of a tetra- or pentapeptide sequence is sufficient for binding. Another highly electropositive region of thrombin that binds the second kringle of prothrombin is the heparin binding site. All three of these sites display distinct binding modes with different molecules. These can arise from tolerance of imprecision of binding and/or from reversal of ligand main chain direction in active site and fibrinogen exosite binding. Preliminary indications suggest similar principles may apply to the heparin site. Such varied behavior easily accounts for the diversity of thrombin functions at the molecular level. The complexity of the behavior is compounded even more by a Na+ ion binding site that produces a procoagulant fast form of thrombin. The slow form (in the absence of Na+ ion) is anticoagulant.

Active site and exosite binding of alpha-thrombin.

alpha-thrombin possesses at least three independent binding sites for substrate, inhibitor and effector molecules. The S1 subsite of the active site is specific for an arginine side group while S2 is a more extended apolar site. The fibrinogen recognition exosite, which usually operates in concert with catalysis, appears to circumnavigate about a third of the surface, although evidence suggests that recognition of a tetra- or pentapeptide sequence is sufficient. Another highly electropositive region of thrombin, which binds the second kringle of prothrombin through salt bridges, is also most likely the heparin binding site. All three sites display distinct binding modes with different molecules. In the active site, these can arise from different optical enantiomorphs combined with reversal of main chain direction, while in the fibrinogen anion binding exosite, certain peptide side chains (hydrophobic) are tolerated of imprecision or are not relevant for binding or undergo a conformational change in substrate binding. Such apparently indiscriminant behaviour easily accounts for the diversity of thrombin functions at the molecular level.

Third-order nonlinear optical properties of polydiacetylene single crystal.

We report the sub-picosecond time-resolved nonlinear optical properties of poly-DCH single crystal from near resonance to on resonance. The magnitude of the third-order nonlinear susceptibilities of poly-DCH for polarization along the main chain direction were found to be of the order 10-6esu on resonance and 10-7esu near resonance. A photo- induced absorptive nonlinearity was found near resonance and an absorption saturation was found on resonance. The sign change in the imaginary susceptibility is due to the mixing of two phenomena, the free exciton level satutation and the reabsorption of the self-trapped exciton. The self-trapped exciton lifetime was observed to be 1.5-2.3ps near resonance.

Prediction of protein folding pathways

Recent 1H nuclear magnetic resonance (n.m.r.) hydrogen exchange experiments on five different proteins have delineated the secondary structures formed in trapped, partially folded intermediates. The early forming structural elements are identifiable through a technique described in this work to predict folding pathways. The method assumes that the sequential selection of structural fragments such as α-helices and β-strands involved in the folding process is founded upon the maximal burial of solvent accessible surface from both the formation of internal structure and substructure association. The substructural elements were defined objectively by major changes in main-chain direction. The predicted folding pathways are in complete correspondence with the n.m.r. results in that the formed structural fragments found in the folding intermediates are those predicted earliest in the pathways. The technique was also applied to proteins of known tertiary structure and with fold similar to one of the five proteins examined by 1H n.m.r. The pathways for these structures also showed general consistency with the n.m.r. observations, suggesting conservation of a secondary structural framework or molten globule about which folding nucleates and proceeds.

Hydrogen momentum distribution in anisotropic rigid-chain polymers

The authors have performed a trial deep inelastic neutron scattering (DINS) experiment on an aligned sample of a copolymer of poly(hydroxybenzoic acid) and poly(hydroxynaphthoic acid). The experiment has been carried out on the eVS inelastic spectrometer of the ISIS facility, at the Rutherford Appleton Laboratory (UK). The hydrogen atomic momentum distributions n/sub ///(p) and nperpendicular to (p), corresponding to momentum transfer Q parallel and perpendicular to the polymer main chain direction respectively, have been obtained as functions of the atomic momentum p. The experiment offers evidence of the insensitivity of the DINS technique to the degree of rigidity of the polymer main chain. Through the use of a quantum mechanical model they show that the hydrogen momentum distribution is defined by the C-H bond and that bonds connected to the carbon or other interactions have no significant influence.

Hydrogen momentum distribution in rigid chain polymers

We have performed a trial deep inelastic neutron scattering (DINS) experiment on an aligned sample of a copolymer of poly(hydroxybenzoic acid) and poly(hydroxynaphtoic acid), on the eVS inelastic spectrometer of the ISIS facility (UK). The hydrogen atomic momentum distribution n ∥( p) corresponding to momentum transfer Q parallel to the polymer main chain direction has been obtained as a function of the atomic momentum p. The experimental results have been interpreted on the basis of a quantum mechanical model, which indicates that the hydrogen momentum distribution is essentially determined by the C-H bond and by its characteristic motions.

One type of gamma-turn, rather than the other gives rise to chain-reversal in proteins

Gamma-turns may be defined by a hydrogen bond between the carbonyl group of one amino acid residue and the amino group of the acid two residues ahead in the sequence. They occur as two types, inverse γ-turns and classic γ-turns (classic γ-turns are usually called just γ-turns but we prefer to add the adjective classic to distinguish them from the word γ-turn, referring collectively to both). Of the two, classic γ-turns are less common and are considered by all authors to be extreme rarities in proteins. However, we find that a number do occur in a sample of proteins of known three-dimensional structure. One occurs at the edge of the second hypervariable region of the light chain in some immunoglobulins. All classic γ-turns except one are associated with a reversal in the main chain direction. In most cases, the turn lies at the loop end of a β-hairpin. By contrast, inverse γ-turns, although giving rise to a kink in the chain, rarely occur within β-hairpins and are seldom situated at a position of reversal, by 180 °, in chain direction.

Positron annihilation in (SN)x

A Doppler broadening study of the energy spectra of annihilation gamma rays from positron annihilation in (SN)x has been made. Using the Shape-parameter one set of the Doppler broadening data from rotation around the Kb axis shows a pair of peaks in the direction of the Ka axis. Two other sets of data from rotation around the Ka and Kc axis respectively, show peaks in the direction of Kb. This means that free (conduction) electrons in the Kb direction (main chain direction) are condensed. There is a difference between the Ka and the Kc Shape-parameter values, in that this parameter is higher for the Ka direction than for the Kc direction. It appears that (SN)x is quasi one-dimensional.

Formation of Anisotropic Phase of Low-molecular-weight Poly(γ-benzyl-l-glutamate)s in Solution

Abstract Low-molecular-weight poly(γ-benzyl-l-glutamate)s having degrees of polymerization (D. P.) 4 and 8 give an anisotropic phase in ethylene dichloride, if the concentration is appropriately chosen and the solution is incubated at a suitable temperature range. The optimum condition of concentration and temperature is more restricted for D. P. 8 than for D. P. 4. Ethylene dibromide solutions of the D. P. 4 specimen also give an anisotropic phase at some conditions. Infrared spectra and x-ray diffraction together with polarized microscopic observations suggest that the anisotropic phase consists of the β-structure of antiparallel arrangement of extended polypeptide chains; it has regular sequences of chains in the main chain direction and a stack of β-sheets in the side chain direction.

Kinetische und morphologische untersuchungen zur polymerisation von oxacyclischen monomeren im festen zustand, 2. Zur morphologie der bei der straheleninduzierten polymerisation von trioxan und tetroxan entstehenden poly(oxymethylene)

AbstractIn order to clarify the reaction mechanism of the solid‐state polymerisation of 1,3,5‐trioxane and 1,3,5,7‐tetroxane as induced by gamma‐radiation, the nascent textures and morphology of poly(oxymethylene) crystals were studied with light and transmission electron microscopy, X‐ray scattering and differential thermal analysis techniques. In both cases poly(oxymethylene) crystals with fibrous morphology and “twin‐structure” are obtained. The polymerization of 1,3,5,7‐tetroxane gives rise to the formation of crystals where the preferred orientation of the polymer chain is the monomer b‐axis; however, the fibrils showed ripples perpendicular to the main chain direction at a 70–240Å long period that depends extremely on the polymerization temperature. In the polymerization of 1,3,5‐trioxane, POM crystals with 60% of the polymer chains in the direction of the monomer c‐axis are obtained. The remaining chains are found in the “twin sub‐crystals”. Dark field electron micrographs showed the sub‐crystals to be an integral part of the fibrils. The material in the twin direction decreases, when the polymerization temperature or the yields increases; it never amounts to less than 30%. These results and the misfit between the lattices of the monomers and the nascent polymer indicate that the reactions which take place occur according to a simultaneous polymerization and crystallization mechanism through the gasphase. The polymerization process occurs easier at higher temperatures near the monomers' melting point due to the increase of monomer vapor pressure and diffusion processes in the monomer crystals. Molecular models of the chain growth are developed on the basis of the morphological observations.