bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the effectiveness of proline substitutions and glycine replacements in increasing the stability of phage T4 lysozyme
Authors: Nicholson H. , Tronrud D. E. , Becktel W. J. , Matthews B. W.
Published: December 2004

It was previously shown that the two replacements Gly 77 ↠ Ala (G77A) and Ala 82 ↠ Pro (A82P) increase the thermostability of phage T4 lysozyme at pH 6.5. Such replacements are presumed to restrict the degrees of freedom of the unfolded protein and so decrease the entropy of unfolding [B. W. Matthews, H. Nicholson, and W. J. Becktel (1987) Proceedings of the National Academy of Science USA Vol. 84, pp. 6663–6667].

To further test this approach, three additional replacements—G113A, K60P and A93P— have been constructed. On the basis of model building, each of these three replacements was judged to be less than optimal because it would tend to introduce unfavorable van der Waals contacts with neighboring parts of the protein. The presence of such contacts was verified for G113A and K60P by conformational adjustments seen in the crystal structures of these mutant proteins. In the case of G113A there are backbone conformational changes of 0.5–1.0 Å in the short α‐helix, 108–113, that includes the site of substitution. In the case of K60P the pyrrolidine ring shows evidence of strain. The thermal stability of each of the three variants at both pH 2.0 and pH 6.5 was found to be very close to that of wild‐type lysozyme. The results suggest that the procedure used to predict sites for both Xaa ↠ Pro and Gly ↠ Ala is, in principle, correct. At the same time, the increase in stability expected from substitutions of this type is modest, and can easily be offset by strain associated with introduction of the alanine or proline. This means that the criteria used to select substitutions that will increase thermostability have to be stringent at least. In the case of T4 lysozyme this severely limits the number of sites. The analysis reveals a significant discrepancy between the conformational energy surface predicted for the residue preceding a proline and the conformations observed in crystal structures. © 1992 John Wiley & Sons, Inc.
DNA torsional dynamics by multifrequency phase fluorometry
Authors: Collini Maddalena , Chirico Giuseppe , Baldini Giancarlo
Published: December 2004

The time decay of the fluorescence polarization anisotropy of calf thymus DNA‐ethidium complexes is obtained from measurements with sine‐modulated excitation employing the so‐called multifrequency phase fluorometry. A torsional dynamics model developed by J. M. Schurr [(1984) Chemical Physics, Vol. 84, pp. 71–96] and translated into the frequency domain is found here to describe accurately DNA‐ethidium fluorescence data collected under modulated excitation.

At a low dye/DNA ratio (1 : 400) the value of the DNA torsional constant (α = 4.63 ± 0.2 10−12 dyne cm) fitting the data agrees very well with the known values of α. When the measurements are extended to a higher ethidium/DNA ratio, energy transfer effects between intercalated dyes are observed. A theoretical prediction of the donor and acceptor dye contributions to the fluorescence polarization anisotropy is made here, taking into account also dye‐dye distance distributions. © 1992 John Wiley & Sons, Inc.
The shapes of backbones of chain molecules: Three‐dimensional characterization by spherical shape maps
Authors: Arteca Gustavo A. , Mezey Paul G.
Published: December 2004

In this work we present a new method to characterize the shape of flexible chain molecules. The procedure associates a sphere and a spherical shape map with each given molecular backbone. Each point on the sphere is classified according to the crossing pattern obtained when the backbone is looked at along a direction defined by the center of the sphere and the chosen point. The approach is simple to implement. It consists of the following steps: (a) enclosing the backbone by a sphere, whose center is the geometrical center of the backbone; (b) projecting the backbone onto a plane tangent to the sphere at the given point; and (c) characterizing the resulting planar curve by a vector specifying the number and handedness type of the crossings. When the procedure is repeated for all points on the spherical surface, the latter is divided into regions that are equivalence classes of points, corresponding to directions from where the backbone has the same overcrossing pattern. The computation of these equivalence classes is performed automatically by the computer, by determining the boundary of the regions characterized by different crossing vectors. The characterization provided is thus direction independent since it takes into accountallpossible directions from where a backbone can be analyzed. The procedure is illustrated for a number of supersecondary protein structures and small proteins. We find that a characterization of substructures can be obtained in terms of the arrangement of the equivalence region for the viewing directions from where the backbone showsno crossings. For instance, an α‐helix is represented by a spherical map with a small “band” region of no crossings perpendicular to the helical axis. Other supersecondary structural features are described in a similar fashion. A number of refinements of the method, based on the distances between crossings, are also given for the case of irregular backbones. © 1992 John Wiley & Sons, Inc.
A study of the quadrupolar NMR splittings of 7Li+, 23Na+, and 133Cs+ counterions in macroscopically oriented DNA fibers
Authors: Schultz Johan , Nordenskiöld Lars , Rupprecht Allan
Published: December 2004

The hydration and temperature dependencies of the 23Na+, and 133Cs+, and 7Li+ quadrupolar splitting have been determined in hydrated, macroscopically oriented DNA fibers. At low water contents the quadrupolar splitting is found to decrease as the water content increases, regardless of counterion, while at high water contents the hydration dependence is reversed. The 23Na+ and 133Cs+ quadrupolar splittings decrease as the temperature increases, while the 7Li+ splitting shows the opposite behavior. At high water contents the 23Na+ and 133Cs+ splittings decrease, and then, after passing zero splitting, increase as the temperature increases. The interpretation of the temperature dependence is discussed in terms of a two‐site model (free and bound ions) and a three‐site model (free ions and specifically or nonspecifically bound ions). It is suggested that a three‐site model is more consistent with the data for the present system. At high water contents, the temperature dependence of the 7Li+ splitting vanishes, indicating counterion condensation. The behavior of the 7Li+ splitting is confirmed by measurements on DNA fibers in equilibrium with a C2H5OD‐D2O‐LiCl solution. The salt dependence in this system is weak. The counterion quadrupolar splitting is seen to be very sensitive to structural transitions in double‐helical DNA. © 1992 John Wiley & Sons, Inc.
Helix propagation in trifluoroethanol solutions
Authors: Storrs Richard W. , Truckses Dagmar , Wemmer David E.
Published: December 2004

Helix propagation of the S‐peptide sequence (residues 1–19 of ribonuclease A) in 2,2,2‐trifluoroethanol (TFE) solutions has been investigated with CD and nmr Overhauser effect spectroscopies. In this study, the S‐peptide helix is covalently initiated at the N‐terminus through disulfide bonds to a helix scaffold derived from the N‐terminal sequence of the bee venom peptide apamin. The entire S‐peptide sequence of this hybrid sequence peptide becomes helical at high proportions of TFE. Residues 14–19 of the S‐peptide are not helical in the free peptide in TFE, nor are they helical in ribonuclease A. The “helix stop” signal encoded by the S‐peptide sequence near residue 13 does not persist at high TFE with this hybrid sequence peptide. The helix‐stabilizing effects of TFE are due at least in part to facilitated propagation of an extant helix. This stabilizing effect appears to be a general solvation effect and not due to specific interaction of the helical peptide with TFE. Specifically these data support the idea that TFE destabilizes the coil state by less effective hydrogen bonding of the peptide amide to the solvent. © 1992 John Wiley & Sons, Inc.
Solution structure of peptides containing two dehydro‐phenylalanine residues: A CD investigation
Authors: Pieroni Osvaldo , Fissi Adriano , Pratesi Claudio , Temussi Pier Andrea , Ciardelli Francesco
Published: December 2004

The peptides Ac‐ΔPhe‐Ala‐ΔPhe‐NHMe (1), Ac‐ΔPhe‐Val‐ΔPhe‐NHMe (2), Ac‐ΔPhe‐Gly‐ΔPhe‐Ala‐OMe (3), and Boc‐Ala‐ΔtPhe‐Gly‐ΔPhe‐Ala‐OMe (4), containing two dehydro‐phenylalanine (ΔPhe) residues, were synthesized and the solution structure investigated in various solvents. The nmr and CD measurements indicate that all the dehydropeptides examined adopt 310‐helical conformations in solution. The tripeptides 1 and 2 exibited an intense negative CD exciton couplet, which was assigned to the right‐handed screw sense, while the tetrapeptide 3 displayed a CD couplet having opposite sign, which was assigned to the left‐handed helical sense. In the pentapeptide 4 the sense of the helix was found to vary with solvent and temperature, as demonstrated by the sign reversal of the CD spectrum. The right‐handed sense dominates in hexafluoro‐2‐propanol, whereas a left‐handed helix prevails in chloroform, acetonitrile and methanol. A crucial role for this behavior is likely to be played by the two alanine residues positioned respectively at the head and tail of the sequence, which favor conformations having opposite screw senses. © 1993 John Wiley & Sons, Inc.
Physicochemical properties of (1 → 6)‐branched (1 → 3)‐β‐D‐glucans. 1. Physical dimensions estimated from hydrodynamic and electron microscopic data
Authors: Stokke Bjørn T. , Elgsaeter Arnljot , Hara Chihiro , Kitamura Shinichi , Takeo Kenichi
Published: December 2004

The physical dimensions of several (1 → 6) branched (1 → 3) ‐β‐D‐glucan samples obtained from different organisms and their derivatives have been studied by electron microscopy, light scattering measurements, viscometry, and gel permeation chromatography. The electron micrographs indicate that in most samples these biopolymers are adequately described as linear worm‐like coils. A sample reconstituted from alkaline media appeared as a blend of the linear, circular, and aggregated polymer morphologies. The average mass per unit length, ML = Mw/Lw for the macroscopically linear samples, was estimated to be 2100 ± 200 g mol−1 nm−1. The parameter mL was determined from the contour lengths obtained by electron microscopy and the molecular weight by light scattering measurements. The observed ML was consistent with the triple‐helical structure reported from x‐ray diffraction studies and observed degree of side‐chain substitution. From the molecular snapshots shown in the electron micrographs, the persistence lengths of these β‐D‐glucans were determined to be 140 ± 30 nm. The experimentally determined intrinsic viscosities were consistent with these estimates of ML and persistence length. Comparison of the molecular weight distributions obtained from gel permeation chromatography and those deduced from the electron micrographs indicates that number and weight average contour lengths are more reliable than z and z + 1 averages. © 1993 John Wiley & Sons, Inc.
Molecular dynamics study of iduronate ring conformation
Authors: Forster Mark J. , Mulloy Barbara
Published: December 2004

Molecular dynamics (MD) simulations of the conformation of the iduronate ring in a methyl glycoside and as the central residue in a trisaccharide have been carried out. Separate simulations were carried out with initial 1C4, 2S0, and 4C1 iduronate ring conformations. Simulations were followed by observing the time development of the Cremer–Pople ring puckering parameters θ,ϕ2. Starting with chair geometries gave trajectories showing only ring oscillations close to the initial geometry. Simulations were performed with a 2S0 starting geometry using explicit water and in vacuum with dielectric constants (ε) of 1 and 80, as well as with distance‐dependent dielectric functions of 2r and 4r. In both the explicit water simulation and the vacuum (ε = 80) simulations, extensive pseudorotational motion was observed in which boat and twist‐boat ring conformers interconvert. The overall range of ϕ22 variation in the trisaccharide was about half of that observed in the methyl glycoside. The Haasnoot procedure for calculating H‐H coupling constants in saccharides was applied to structures obtained from MD trajectories. Using MD time averaged couplings along with experimental data allowed the relative fractions of chair and boat/twist‐boat forms to be derived. © 1993 John Wiley & Sons, Inc.
Diagonalization in a mixed basis: A method to compute low‐frequency normal modes for large macromolecules
Authors: Mouawad Liliane , Perahia David
Published: December 2004

Low‐frequency collective motions in proteins are generally very important for their biological functions. To study such motions, harmonic dynamics proved most useful since it is a straightforward method; it consists of the diagonalization of the Hessian matrix of the potential energy, yielding the vibrational spectrum and the directions of internal motions. Unfortunately, the diagonalization of this matrix requires a large computer memory, which is a limiting factor when the protein contains several thousand atoms.

To circumvent this limitation we have developed three methods that enable us to diagonalize large matrices using much less computer memory than the usual harmonic dynamics. The first method is approximate; it consists of diagonalizing small blocks of the Hessian matrix, followed by the coupling of the low‐frequency modes obtained for each block. It yields the low‐frequency vibrational spectrum with a maximum error of 20%. The second method consists, after diagonalizing small blocks, of coupling the high‐ and low‐frequency modes using an iterative procedure. It yields the exact low‐frequency normal modes, but requires a long computational time with convergence problems. The third method, DIMB (Diagonalization in a Mixed Basis), which has the best performance, consists of coupling the approximate low‐frequency modes with the mass‐weighted cartesian coordinates, also using an iterative procedure. It reduces significantly the required computer memory and converges rapidly. The eigenvalues and eigenvectors obtained by this method are without significant error in the chosen frequency range. Moreover, it is a general method applicable to any problem of diagonalization of a large matrix.

We report the application of these methods to a deca‐alanine helix, trypsin inhibitor, a neurotoxin, and lysozyme. © 1993 John Wiley & Sons, Inc.
Self‐assembly of bioelastomeric structures from solutions: Mean‐field critical behavior and Flory–Huggins free energy of interactions
Authors: Sciortino F. , Prasad K. U. , Urry D. W. , Palma M. U.
Published: December 2004

Elastic and quasi‐elastic light scattering studies were performed on aqueous solutions of poly (Val‐Pro‐Gly‐Gly), a representative synthetic bioelastomer that differs from the previously studied poly (Val‐Pro‐Gly‐Val‐Gly) by the deletion of the hydrophobic Val in position four. When the spinodal line was approached from the region of thermodynamic stability, the intensity of light scattered by fluctuations, and the related lifetime and correlation length, were observed to diverge with mean‐field critical exponents for both systems. Fitting of the experimental data allowed determining the spinodal and binodal (coexistence) lines that characterize the phase diagrams of the two systems, and it also allowed a quantitative sorting out of the enthalpic and entropic contributions to the Flory–Huggins interaction parameters. The contribution of valine is derived by comparison of the two cases. This can be viewed as sorting out the effect of a modulation of the solute. The same approach may allow sorting out the entropic and enthalpic effect of modulations of the solvent by cosolutes (or by cosolvents). This could be of particular interest in the case of small osmolytes, affording important adaptive roles in nature, at the cost of very limited changes in genetic information.

Finally, the suggestion is further supported that statistical fluctuations of anomalous amplitude, such as those occurring in proximity of the spinodal line, have a role in promoting the process of self‐assembly of extended supramolecular structures. On the practical side, the present approach appears useful in the design of novel synthetic model systems for bioelastomers. © 1993 John Wiley & Sons, Inc.
A proton magnetic resonance study of two synthetic agonist–antagonist pairs of bradykinin analogues
Authors: Otter Albin , Bigler Peter , Stewart John M. , Kotovych George
Published: December 2004

The conformation of two agonist–antagonist pairs of bradykinin (Arg1‐Pro2‐Pro3‐Gly4‐Phe5‐Ser6‐Pro7‐Phe8‐Arg9) analogues were studied in CD3OH/H2O solution by 1H‐nmr techniques. The first agonist peptide studied, D‐Arg0‐Arg1‐Pro2‐Hyp3‐Gly4‐Thi5‐Ser6‐Pro7‐Thi8‐Arg9, differs from the bradykinin sequence by the addition of D‐Arg0, the replacement of the Phe moieties in positions 5 and 8 by Thi (Thi = β‐(2‐thienyl)‐L‐alanine), and Hyp3 (Hyp = L‐4‐hydroxy‐L‐proline) in position 3. In the corresponding antagonist sequence, Pro7 is replaced by D‐Phe7. The second agonist–antagonist pair studied does not contain the D‐Arg0 residue, which is present only to slow down the rate of metabolism. Based on complete resonance assignments from two‐dimensional total correlation spectroscopy and rotating frame nuclear Overhauser effect spectroscopy spectra at 500 MHz, the peptides were analyzed in terms of intraresidue, sequential, and medium‐range nuclear Overhauser effects, amide proton temperature coefficients, and vicinal coupling constants. Both agonist peptides show clear evidence for the existence of a type I β‐turn comprising the C‐terminal residues Ser6‐Pro7‐Thi8‐Arg9 in fast conformational equilibrium with extended structures throughout. Although the conformational space is dominated by extended structures, the presence of the β‐turn is spectroscopically clearly discernible. The two antagonist peptides, on the other hand, do not show evidence of turn formation but rather the presence of an extended conformation with some irregularities in the N‐terminal region of the peptide. While the existence of a turn at the C‐terminal end of bradykinin and its analogues with agonist activity has been predicted by empirical calculations and measurements in very apolar solvents, this study, for the first time, provides evidence based on physical data in a polar solvent environment that the turn is present, that it is type I and that it is essential for agonist activity. In the particular solvent used in these studies, the Pro7 to D‐Phe7 substitution precluded the formation of the turn for the C‐terminal residues of the antagonist. © 1993 John Wiley & Sons, Inc.
Differential binding of the enantiomers of chloroquine and quinacrine to polynucleotides: Implications for stereoselective metabolism
Authors: Scaria P. V. , Craig John C. , Shafer Richard H.
Published: December 2004

Interaction of the antimalarial drugs quinacrine and chloroquine with DNA has been studied extensively in order to understand the origin of their biological activity. These studies have shown that they bind to DNA through an intercalative mode and show little sequence specificity. All previous experiments were carried out using the racemic form of these drugs. We have investigated the binding of the enantiomeric forms of quinacrine and chloroquine to synthetic polynucleotides poly (dA‐dT) · poly(dA‐dT) and poly (dG‐dC) · poly(dG‐dC), and found interesting differences in their binding parameters.

Quinacrine enantiomers have a much higher binding affinity for the two polynucleotides compared to those of chloroquine. The negative enantiomers were found to have higher binding affinity than the positive ones. The binding constant for the binding of quinacrine (−) to poly(dG‐dC) · poly(dG‐dC) was found to be about 3 times that of quinacrine (+). The differences in these binding affinities were further confirmed by equilibrium dialysis of the complexes of the polynucleotides with the racemic form of the drugs, which resulted in the enrichment of the dialysate with the positive enantiomer. CD spectra of the enantiomers and their polynucleotide complexes are reported. Changes in the fluorescence properties of quinacrine in the presence of the two polynucleotides are also described. Biological implications of these findings are discussed. © 1993 John Wiley & Sons, Inc.
A reexamination of the conformational transitions of T4 thioredoxin
Authors: Shalongo William , Jagannadham Medicherla , Stellwagen Earle
Published: December 2004

The unfolding and refolding of T4 thioredoxin was observed by equilibrium and kinetic size exclusion chromatographic measurements in guanidine hydrochloride at 4°C and pH 7.0. All the observed chromatographic profiles can be simulated by a cubic mechanism using a consistent set of equilibrium and kinetic parameters describing each of the coupled transitions. The four components in the folded protein and in the unfolded protein are interrelated by configurational transitions having parameters characteristic for proline peptide isomerizations. Only two of the four folded conformations are significantly populated at equilibrium. Each of the four unfolded components can refold by a unique Conformational transition. No transiently populated folding intermediates are detected having hydrodynamic volumes intermediate between those characteristic for the folded and unfolded protein. © 1993 John Wiley & Sons, Inc.
Enhanced solubility of proteins and peptides in nonpolar solvents through hydrophobic ion pairing
Authors: Powers Michael E. , Matsuura James , Brassell James , Manning Mark C. , Shefter Eli
Published: December 2004

A new technique for enhancing the solubility of peptides and proteins in organic solvents is described. Complexation of polypeptides with stoichiometric amounts of an anionic detergent, such as sodium dodecyl sulfate (SDS), produces diminished aqueous solubility, but enhanced solubility in organic solvents. Consequently, the partitioning of a polypeptide into a nonpolar solvent can be increased by two to four orders of magnitude. In the case of an insulin–SDS complex, the solubility in 1‐octanol is more than tenfold greater than in water. In 1‐octanol, the native structure of insulin remains intact, as determined by CD spectroscopy, and the thermal denaturation temperature (Tm) is increased by approximately 50°C relative to unfolding in water. Finally, peptides and proteins can be extracted back into an aqueous phase provided the chloride concentration is sufficient to displace bound detergent molecules. © 1993 John Wiley & Sons, Inc.
A model of the rabies virus glycoprotein active site
Authors: Rustici Mauro , Bracci Luisa , Lozzi Luisa , Neri Paolo , Santucci Annalisa , Soldani Patrizia , Spreafico Adriano , Niccolai Neri
Published: December 2004

The glycoprotein from the neurotropic rabies virus shows a significant homology with the α neurotoxin that binds to the nicotinic acetylcholine receptor. The crystal structure of the α neurotoxins suggests that the Arg 37 guanidinium group and the Asp 31 side‐chain carboxylate of the erabutoxin have stereochemical features resembling those of acetylcholine. Conformational studies on the Asn194‐Ser195‐Arg196‐Gly197 tetrapeptide, an essential part of the binding site of the rabies virus glycoprotein, indicate that the side chains of Asn and Arg could also mimic the acetylcholine structure. This observation is consistent with the recently proposed mechanism of the viral infection. © 1993 John Wiley & Sons, Inc.
CD of proline‐rich polypeptides: Application to the study of the repetitive domain of maize glutelin‐2
Authors: Rabanal F. , Ludevid M. D. , Pons M. , Giralt E.
Published: December 2004

An overview of CD of proline‐rich peptides is reported. First, structural characteristics, theoretical CD studies, and the biological relevance of polyproline II structure in such peptides are discussed. Second, a CD study of peptides belonging to the repetitive domain of maize glutelin‐2, H‐(Val‐His‐Leu‐Pro‐Pro‐Pro)n‐OH (n = 3, 5, 8), is described. This series of peptides displayed the CD features of polyproline II structure in water (5°C, pH5). Moreover, it was shown that the addition of increasing amounts of the polyanionic molecule heparin forced a displacement of the conformational equilibrium of those peptides toward higher proportions of the polyproline II structure. In contrast, when the temperature is raised such a structure gradually disappears, leading to more disordered conformations. © 1993 John Wiley & Sons, Inc.
Bioactive peptides: Conformational study of a cystinyl cycloheptapeptide in its free and calcium complexed forms
Authors: Zanotti Giancarlo , Maione Annamaria , Rossi Filomena , Saviano Michele , Pedone Carlo , Tancredi Teodorico
Published: December 2004

The disulphide bridged heptapeptide magnified image has been synthesized by classical solution methods. An ion binding study showed the peptide's ability to complex calcium ions with definite stoichiometry. The solution conformation of the peptide in its free and calcium‐complexed form has been investigated by CD and nmr. The model structure derived from nmr data has been energy minimized and the resulting structure investigated by molecular dynamics simulation in water.

The structure of the equimolar peptide/Ca2− complex in acetonitrile at room temperature shows the presence of two transannular hydrogen bonds, with the formation of two ring structures of the C10 (type VIa) and C14 type. One peptide unit (Pro‐Pro) is cis, all others are trans. © 1993 John Wiley & Sons, Inc.
Modifications of the amide bond and conformational constraints in pseudopeptide analogues
Authors: Marraud Michel , Dupont Virginie , Grand Vincent , Zerkout Said , Lecoq Alain , Boussard Guy , Vidal Joelle , Collet Andre , Aubry Andre
Published: December 2004

We have investigated the conformational effects of modifying the amide group in model dipeptides. The N‐methyl amide ψ[CO‐NMe], N‐hydroxy amide ψ[CO‐N(OH)], N‐amino amide ψ[ CO‐N (NH2)], retro amide ψ[ NH‐CO], reduced amide in the neutral ψ[CH2‐NH] and protonated ψ[CH2‐N + H2] state, and hydrazide ψ[CO‐NH‐NH] have been introduced as surrogates of the amide link in pseudopeptide derivatives of the Pro‐Gly or Ala‐Gly model dipeptides protected on both termini by an amide group. These compounds have been studied in solution by proton nmr and ir spectroscopy, and in the solid state by x‐ray diffraction, giving an extended data set of experimental structural and conformational information on pseudopeptide sequences. The conformational effects depend both on the nature and the position of the modified amide link. Some modifications appear to have no intrinsic conformational induction (N‐amino and retro amide), but destabilize any local folded structure by hydrogen‐bond breaking. Because of the formation of strong intramolecular interactions, others are capable of stabilizing a β‐turn (for example protonated reduced amide), or of inducing a particular local conformation such as a β‐ or γ‐like turn (for example N‐hydroxy amide). The particular geometry of the cis N‐methyl amide and of the “hydrazino” proline favors the formation of a sharp turn of the main chain. All these structural data are of interest to the design of bioactive peptide mimics. © 1993 John Wiley & Sons, Inc.
Crystal structures of Boc‐D‐ and L‐Iva‐L‐Pro‐OBZl: Unturned conformation of Aib‐Pro sequence unaffected by replacement of Me with Et in Aib
Authors: Kawai Masao , Omori Yoshimasa , Yamamura Hatsuo , Butsugan Yasuo , Taga Tooru , Miwa Yoshihisa
Published: December 2004

The crystal structures of the isovaline (Iva) containing dipeptides, Boc‐D‐Iva‐L‐Pro‐OBzl and Boc‐L‐Iva‐L‐Pro‐OBzl, were determined by x‐ray diffraction. The diastereomeric peptides were shown to adopt unturned conformations closely similar to each other (ϕIva 52°, ψIva 46°, ϕPro–65°, and ψPro 143° for D‐Iva‐L‐Pro sequence and ϕIva 52°, ψIva 44°, ϕPro −63°, and ψpro 148° for L‐Iva‐L‐Pro sequence). The Pro ring of each peptide was in Cγ‐endo conformation. The unusually large ∠CIva‐NPro‐C values (131° in both peptides) were observed, that was due to steric repulsion between the δ‐methylene of Pro and the alkyl side chain of Iva residue. These conformations were essentially the same as that of the corresponding α‐aminoisobutyric acid (Aib)‐containing peptide Boc‐Aib‐L‐Pro‐OBzl. The result has demonstrated that replacement of either one of the two methyl groups of the Aib residue in Boc‐Aib‐L‐Pro‐OBzl with an ethyl group does not cause any significant change in the unturned conformation of the dipeptide. © 1993 John Wiley & Sons, Inc.
Statistics of RNA secondary structures
Authors: Fontana Walter , Konings Danielle A. M. , Stadler Peter F. , Schuster Peter
Published: December 2004

A statistical reference for RNA secondary structures with minimum free energies is computed by folding large ensembles of random RNA sequences. Four nucleotide alphabets are used: two binary alphabets, AU and GC, the biophysical AUGC and the synthetic GCXK alphabet. RNA secondary structures are made of structural elements, such as stacks, loops, joints, and free ends. Statistical properties of these elements are computed for small RNA molecules of chain lengths up to 100. The results of RNA structure statistics depend strongly on the particular alphabet chosen. The statistical reference is compared with the data derived from natural RNA molecules with similar base frequencies.

Secondary structures are represented as trees. Tree editing provides a quantitative measure for the distance dt, between two structures. We compute a structure density surface as the conditional probability of two structures having distance t given that their sequences have distance h. This surface indicates that the vast majority of possible minimum free energy secondary structures occur within a fairly small neighborhood of any typical (random) sequence.

Correlation lengths for secondary structures in their tree representations are computed from probability densities. They are appropriate measures for the complexity of the sequence‐structure relation. The correlation length also provides a quantitative estimate for the mean sensitivity of structures to point mutations. © 1993 John Wiley & Sons, Inc.
Molecular dynamics simulations of a winter flounder “antifreeze” polypeptide in aqueous solution
Authors: McDonald Shawn M. , Brady John W. , Clancy Paulette
Published: December 2004

A winter flounder antifreeze polypeptide (HPLC‐6) has been studied in vacuo and in aqueous solution using molecular dynamics computer simulation techniques. The helical conformation of this polypeptide was found to be stable both in vacuum and in solution. The major stabilizing interactions were found to be the main‐chain hydrogen bonds, a salt‐bridge interaction, and solute–solvent hydrogen bonds. A significant bending in the middle of the polypeptide chain was observed both in vacuo and in solvent at 300 K. Possible causes of the bending are discussed. From simulations of mutant polypeptide molecules in vacuo, it is concluded that the bend in the native polypeptide was caused by side chain to backbone hydrogen bond competition involving the Thr 24 side chain and facilitated by strains on the helix resulting from the Lys 18‐Glu 22 salt bridge. © 1993 John Wiley & Sons, Inc.
Structural versatility of peptides from Cα, α‐disubstituted glycines: Crystal‐state conformational analysis of homopeptides from Cα‐methyl, Cα‐benzylglycine [(αMe)Phe]n
Authors: Valle Giovanni , Pantano Monica , Formaggio Fernando , Crisma Marco , Toniolo Claudio , Précigoux Gilles , Sulzenbacher Gerlind , Boesten Wilhelmus H. J. , Broxterman Quirinus B. , Schoemaker Hans E. , Kamphuis Johan
Published: December 2004

The molecular and crystal structures of one derivative and three homopeptides (from the di‐to the tetrapeptide level) of the chiral, Cα, α‐disubstituted glycine Cα‐methyl, Cα‐benzylglycine [(αMe)Phe], have been determined by x‐ray diffraction. The derivative is mClAc‐D‐(αMe)Phe‐OH, and the peptides are pBrBz‐[D‐(αMe)Phe]2‐NHMe, pBrBz‐[D‐(αMe)Phe]3‐OH hemihydrate, and pBrBz‐[D‐(αMe)Phe]4‐OtBu sesquihydrate. All (αMe)Phe residues prefer ϕ,ψ torsion angles in the helical region of the conformational map. The dipeptide methylamide and the tripeptide carboxylic acid adopt a β‐turn conformation with a 1 ← 4 CO…︁HN intramolecular H bond. The structure of the tripeptide carboxylic acid is further stabilized by a 1 ← 4 CO…︁HO intramolecular H bond, forming an “oxy‐analogue” of a β‐turn. The tetrapeptide ester is folded in a regular (incipient) 310‐helix. In general, the relationship between (αMe)Phe chirality and helix screw sense is opposite to that exhibited by protein amino acids. A comparison is made with the conclusions extracted from published work on homopeptides from other Cα‐methylated α‐amino acids. © 1993 John Wiley & Sons, Inc.
Solid‐Phase synthesis and stability of triple‐helical peptides incorporating native collagen sequences
Authors: Fields Cynthia G. , Lovdahl Christine M. , Miles Andrew J. , Hageini Vickie L. Matthias , Fields Gregg B.
Published: December 2004

A generally applicable solid‐phase methodology has been developed for the synthesis of triple‐helical polypeptides incorporating native collagen sequences. Three nascent peptide chains are C‐terminal linked through one Nα‐amino and two Nε‐amino groups of Lys, while repeating Gly‐Pro‐Hyp triplets induce triple helicity. Different protecting group strategies, including several three‐dimensionally orthogonal schemes, have been utilized for the synthesis of four homotrimeric triple‐helical polypeptides (THPs) of 79–124 residues, three of which incorporate native type IV collagen sequences. Highly efficient assemblies were achieved by 9‐fluorenylmethoxycarbonyl (Fmoc) Nα‐amino group protection, in situ 2‐(1H‐benzotriazole‐l‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate mediated couplings, and 1,8‐diazabicyclo [5.4.0] undec‐7‐ene mediated Fmoc group removal. THPs were characterized by Edman degradation sequencing, size‐exclusion chromatography, mass spectrometry, reversed‐phase high performance liquid chromatography, and CD spectroscopy. THP thermal stabilities ranged from 35 to 59°C, with chain length and Hyp content being the influential factors. Melting temperatures and van't Hoff enthalpies for peptide triple‐helical denaturation could be correlated well to Hyp content. The THP synthetic protocol developed here will allow for the study of both structure and biological activity of specific collagen sequences in homotrimeric and heterotrimeric forms. © 1993 John Wiley & Sons, Inc.
127I‐NMR studies of anion binding to κ‐carrageenan
Authors: Zhang Wei , Furó István
Published: December 2004

A preliminary 127I‐NMR relaxation study of the binding of the I− ion to κ‐carrageenan is presented. Signal loss caused by motional correlation times out of extreme narrowing makes it difficult to analyze line widths. Longitudinal relaxation times are more suitable for a comprehensive relaxation study. From the observation of nonextreme motional narrowing, the lower limit of the residence time at the binding site is estimated to be on the order of 10 ns. © 1993 John Wiley & Sons, Inc.
The effects of Cu2+ and Pb2+ on the solution structure of calf thymus DNA: DNA condensation and denaturation studied by fourier transform IR difference spectroscopy
Authors: Tajmir‐Riahi Heidar‐Ali , Naoui Mancef , Ahmad Rohana
Published: December 2004

The interaction of calf thymus DNA with Cu2+and Pb2+ was studied in aqueous solution at pH 6.5 with metal/DNA (P) (P = phosphate) molar ratios (r) 1/80, 1/40, 1/20, 1/10, 1/4, 1/2, and 1, using Fourier Transform ir (FTIR) spectroscopy. Correlations between the ir spectral changes, metal ion binding mode, DNA condensation, and denaturation, as well as conformational features, were established.

Spectroscopic evidence has shown that at low metal/DNA (P) molar rations 1/80 and 1/40, copper and lead ions bind mainly to the PO of the backbone, resulting in increased base‐stacking interaction and duplex stability. The major copper ion base binding via G‐C base pairs begins at r > 1/40, while the lead ion base binding occurs at r > 1/20 with the A‐T base pairs. The denaturation of DNA begins at r = 1/10 and continues up to r = 1/2 in the presence of copper ions, whereas a partial destabilization of the helical structure was observed for the lead ion at high metal ion concentration (r = 1/2). Metal‐DNA binding also results in DNA condensation. No major departure from the B‐family structure was observed, upon DNA interaction with these metal ions. © 1993 John Wiley & Sons, Inc.
Quantitative two‐dimensional nmr study of dermenkephalin, a highly potent and selective δ‐opioid peptide
Authors: Naim Mohammed , Charpentier Stéphane , Nicolas Pierre , Baron Daniel
Published: December 2004

Dermenkephalin, H‐Tyr‐(D) Met‐Phe‐His‐Leu‐Met‐Asp‐NH2, a highly potent and selective δ‐opioid peptide isolated from frog skin, was studied in DMSO‐d6 solution by two‐dimensional nmr spectroscopy, including the determination of NH temperature coefficients, the evaluation of 3J coupling constants from phase‐sensitive correlated spectroscopy (COSY) and the volumes of nuclear Overhauser effect (NOE) correlations. The two‐dimensional NOE spectroscopy (NOESY) spectrum of dermenkephalin revealed sequential, medium‐, and long‐range effects. To put this information on a quantitative basis, special attention was devoted to J cross‐peak suppression, quantification of the NOE volumes and analysis of the overlaps, normalization of the NOEs against diagonal peaks and Hββ′ geminal interactions. Although most of the dihedral angles deduced from the 3JNα coupling constants together with several Niαi and αiNi + 1 NOEs pointed to a partially extended peptide backbone, several Ni Ni + 1 NOEs and βi Ni + 1 interactions argued in favor of a folded structure. Moreover, several long‐range correlations of strong intensities were found that supported a close spatial proximity between the side chains of D‐Met2 and Met6, Tyr1 and His4, Tyr1 and Asp7, and His4 and the C‐terminal amide group. In Phe, the g− rotamer in the side chain is deduced from the 3Jαβ coupling constants and αβ and Nβ NOE correlations. Whereas the amide proton dependency was not indicative of stable hydrogen bonds, the nonuniform values of the temperature coefficient may reflect an equilibrium mixture of folded and extended conformers. The overall data should provide realistic starting models for energy minimization and modelization studies. © 1993 John Wiley & Sons, Inc.
Solution conformation study of substance P methyl ester and [Nle10]‐neurokinin A (4–10) by nmr spectroscopy
Authors: Zhang Min , Wong Tuck C.
Published: December 2004

High‐resolution proton spectra at 500 MHz of two tachykinin peptides, substance P methyl ester (SPOMe) and [Nle10]‐neurokinin A (4–10), have been obtained in dimethylsulfoxide (DMSO), and for SPOMe, also in 2, 2, 2‐trifluoroethanol (TFE)/water mixtures. Complete chemical shift assignments for these peptides were made based on two‐dimensional (2D) nmr techniques, correlated spectroscopy and total COSY. J coupling measurement and nuclear Overhauser effect spectroscopy (NOESY) were then used to determine the conformation of these peptides in the various solvents. Based on the J coupling, NOE correlations, and temperature coefficients of the NH resonances, it is concluded that these two peptides exist in DMSO at room temperature as a mixture of conformers that are primarily extended. For SPOMe in TFE/water with high TFE content, however, helical structures are found to be present, and they become quite clear at temperatures between 270 and 280 K. The variation of the 13C chemical shifts of the Cα (the secondary shift) with TFE contents corroborates this conclusion. The NOE and Cα shifts show that the main helical region for SPOMe lies between 4P and 9G. The C‐terminus segment LMNH2 is found to be quite flexible, which appears to be quite common for neurokinin‐1 selective peptides. © 1993 John Wiley & Sons, Inc.
Synthesis and biological evaluation of novel NK‐1 tachykinin receptor antagonists: The use of cycloalkyl amino acids as a template
Authors: Sisto Alessandro , Bonelli Fabio , Centini Felice , Fincham Christopher I. , Potier Edoardo , Monteagudo Edith , Lombardi Paolo , Arcamone Federico , Goso Cristina , Manzini Stefano , Giolitti Alessandro , Maggi Carlo A. , Venanzi Mariano , Pispisa Basilio
Published: December 2004

In the course of a program aimed at synthesizing novel, potent NK‐1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the super imposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2‐aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo‐peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 Å distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well‐defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground‐state interactions between the chromophores. Implications of the π stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed. © 1995 John Wiley & Sons, Inc.
Design and operation of a pilot‐plant fermentor for the continuous propagation of filamentous microorganisms
Authors: Means C. W. , Savage G. M. , Reusser F. , Koepsell H. J.
Published: December 2004

A multi‐stage pilot fermentor was designed which consists of nine stainless steel cylindrical compartments 2 ft. in length and 8 in. in diameter, joined end to end, giving a tube with a total length of 18 ft. The whole apparatus was mounted with its axis in a horizontal plane. Each compartment was sealed off from its neighboring compartment by a separatory plate having a 1‐in. overflow hole in the upper half. Shafts 9½ feet long extended from each end of the fermentor through the compartments to the center. Agitation was provided by a multitude of stainless steel blades mounted at right angles to the shafts and spaced at regular intervals along the shafts. Stationary, stainless steel comb‐shaped baffle plates were installed vertically at the bottom of each compartment to increase mixing efficiency. Constant leaving and re‐entering of the fermentation liquor by the blades upon rotation imparts a shattering of the liquid surface thus preventing the accumulation of mycelial aggregates on the walls above the fermentation liquor. The mechanical performance of the described fermentor has proved excellent and several trial fermentations (antibiotic formation and steroid bioconversion) are described.
Microbial production of amino acids. I. The synthesis of lysine and threonine by ustilago species
Authors: Ericson L.‐E. , Kurz W. G.
Published: December 2004

The ability of a number of Ustilago species, especiallyUstilago maydis(DC.) Cda., to produce lysine and threonine was investigated. The organisms were grown in shake flasks or in 10‐l. fermentors. Lysine and threonine were found to be excreted into the medium both in the free and bound form. The bound amino acids could be released by acid hydrolysis or by enzymes from autolyzed cells. The optimal conditions for the release by autolysis were, in the case ofUstilago maydis(DC.) Cda., pH 4.3 and 45°C. An enzyme that could liberate lysine from the bound form(s) occurring in the broth was extracted from cells ofUstilago maydis(DC.) Cda. It exhibited an apparent pH optimum near 4.0. The effect of pH and temperature during the growth phase on the yield of lysine and threonine was studied in 10‐l. fermentations.
Antimicrobial agents as liquid chemosterilizers
Authors: Borick P. M.
Published: December 2004

Many antimicrobial agents are available for commercial use, but only a few are truly sporicidal and can be used as chemosterilizers. The action of ethylene oxide, propylene oxide, and β‐propiolactone is well documented. Strong acids, e.g., hydrochloric acid, or alkalis may be used as chemosterilizers but their use is extremely limited. The synthetic phenolics accompanied by boiling were recommended, but heat cannot always be employed. Chlorine and iodine have been used in higher concentrations. The use of 8% formaldehyde or 20% formalin was suggested by Spaulding.17 Other aldehydes possess antimicrobial potency. According to work performed in our laboratories, the sporicidal activity of certain alcoholic, saturated dialdehyde solutions equalled or surpassed that of formaldehyde. These included glyoxal, succinaldehyde, and glutaraldehyde. Aqueous alkaline glutaraldehyde solutions killed the most resistant bacterial spores, e.g., B. subtilis and Cl. tetani, within a few hours (Borick1,2). Glutaraldehyde, a 5‐carbon dialdehyde, was shown to be a chemosterilizer when tested by various methods, destroying bacteria, including M. tuberculosis, fungi, and viruses. Acid glutaraldehyde solutions stored at room temperature are highly stable, whereas alkaline solutions show a significant change in pH and a diminution of glutaraldehyde concentration on alkalinization. For this reason, fresh (not over 2 weeks old) alkaline glutaraldehyde solutions can be used as chemosterilizers.
The effect of ethanol on continuous culture stability
Authors: Zines D. O. , Rogers P. L.
Published: December 2004

The stability characteristics of a continuous culture system were studied following the addition of the natural product inhibitor, ethanol. For a steady state culture of Klebsiella (Aerobacter) aerogenes there was a linear dependence of growth rate on ethanol concentration. Following impulse and step addition of the inhibitor, response patterns of the growth rate (μ) and overall metabolism (Qo2, QCo2, QAC) were observed. A mathematical model of the transient behavior of a product‐limited system is proposed, and analog computer solutions fitted to the experimental data. The transient response of the growth rate could best be described by second or higher order equations, e.g.,

with values of the second order time constant (T2) = 5 min, and damping coefficient (ξ) = 0.4.
Kinetic model identification in mixed populations using continuous culture data
Authors: Chiu S. Y. , Erickson L. E. , Fan L. T. , Kao I. C.
Published: December 2004

The kinetic behavior of heterogeneous microbial populations of sewage origin was studied in a single‐stage, isothermal, continuous flow, completely mixed aeration tank. A series of experiments were carried out at various dilutions rates using glucose as the limiting substrate. The cell dry weight and substrate concentration in terms of chemical oxygen demand (COD) were continuously monitored. The results indicate that reproducible steady‐state conditions can generally be obtained; however, multiple steady states were observed at dilution rates near washout. At low dilution rates (below about 0.1 hr−1) the contribution of microorganism decay became appreciable. Using the multiresponse data of cell dry weight and COD, the parameter values in various existing growth models were estimated. The analyses of variance and residuals revealed that models proposed by Moser, Monod, and Contois, each with a decay term added, were significantly better than the other models which were tested.