bluuebunny/crossref_metadata_2025 huggingface dataset:
Electric birefringence imaging of electrokinetic agarose orientation
Authors: Lanan Maureen , Shick Reed , Morris Michael D.
Published: December 2004

Time‐resolved and steady‐state electric birefringence imaging with a slow‐scan video camera is used to study orientation during DNA agarose gel electrophoresis. The hydrodynamically induced gel distortion is shown to be the major source of birefringence under electrophoresis running conditions and to generate a birefringence image that approximates the image of the DNA concentration gradient in the electric field direction. A fluid kinematic model is presented to describe the spatial distribution of steady‐state birefringence and is verified with fluorescence measurements of DNA distribution. The stress‐optic coefficient of 1% agarose gel is measured by mechanical compression and used to evaluate the magnitude of the induced strain on the gel during electrophoresis.
Structural versatility of peptides from Cα,α‐disubstituted glycines: Preferred conformation of the chiral isovaline residue
Authors: Nebel K. , Altmann E. , Mutter M. , Bardi R. , Piazzesi A. M. , Crisma M. , Bonora G. M. , Toniolo C.
Published: December 2004

The molecular structures of four protected isovaline‐ (Iva‐) containing peptides to the pentamer level have been determined by x‐ray diffraction. The peptides are t‐Boc‐Ala‐(S)‐Iva‐Ala‐OMe (t‐Boc : tert‐butyloxycarbonyl; OMe : methoxy) and its (R)‐Iva diastereomer, and t‐Boc‐[Ala‐(R)‐Iva]2‐Ala‐OH and its (S)‐Iva diastereomeric methyl ester analogue. The two tripeptides are folded in an open type II β‐bend conformation. The fully developed right‐handed 310‐helix formed by the (R)‐Iva pentapeptide, which includes an unusual intramolecular (acid) OH ⃛OC(peptide) H bond, is partially unfolded (near the C‐terminus) in the (S) ‐Iva pentapeptide. 1H‐nmr and Fourier transform ir absorption studies suggest that in CDCl3 solution (a) the two tripeptides maintain a type II β‐bend conformation of comparable stability and (b) both diastereomeric pentapeptide sequences adopt a fully developed 310‐helix. A comparison with the preferred conformation of other extensively investigated Cα,α‐disubstituted glycines is made and the implications for the use of the Iva residue in designing conformationally constrained analogues of bioactive peptides are briefly discussed.
Induced CD and interaction of some porphyrin derivatives with α‐helical polypeptides in aqueous solutions
Authors: Ikeda Shoichi , Nezu Takashi , Ebert Gotthold
Published: December 2004

Absorption spectra and induced CD have been measured on aqueous solutions of water‐soluble porphyrins with α‐helical poly(L‐glutamic acid) or α‐helical poly (L‐lysine) at different mixing ratios. For the former, porphyrin is porphine‐meso‐tetra (4‐N‐methylpyridinium) (TMpyP), and for the latter, it is porphine‐meso‐tetra (4‐benzenesulfonate) (TPPS) or porphine‐meso‐tetra(4‐benzoate) (TPPC). All the solutions of porphyrin‐polypeptide systems show hypochromism in the Soret band and induced CD in the Soret region. The CD is characterized by a positive band at a shorter wavelength and a stronger negative band at a longer wavelength. The hypochromicity and the magnitude of molar ellipticities are much larger for the TPPS– and TPPC–poly (L‐lysine) systems than for the TMpyP–poly (L‐glutamic acid) system. Porphyrin ions bind to the α‐helix electrostatically, and the two components of the Soret transition of porphyrin are subject to dissymmetric perturbation. TMpyP ions bind to the α‐helix at isolated sites, while TPPS ions and TPPC ions are in pairs on the α‐helix, that is, two ions bind consecutively and dissymmetrically. In the TMpyP–poly (L‐glutamic acid) system a single CD band is associated with each of the two components of the Soret transition, and these are of opposite sign. In the TPPS– and TPPC–poly (L‐lysine) systems, a pair of positive and negative CD bands is associated with each of the two components, thus giving apparently a single pair of CD bands with a shoulder, owing to partial overlapping.
The interaction of auramine O with calmodulin: Location of the binding site on the connecting strand
Authors: Steiner Robert F. , Albaugh Sharon , Nenortas Elizabeth , Norris Lynn
Published: December 2004

The cationic dye auramine O forms a fluorescent complex with Ca2+‐liganded calmodulin. One moderately strong binding site is present, as well as one or more weaker sites. The binding site for auramine O is different from those for toluidinyl‐naphthalene sulfonate. The dependence of binding upon electrolyte concentration suggests a substantial electrostatic component of the free energy of binding. The splitting of the bond between residues 77 and 78 by trypsin digestion abolishes auramine O binding; the N‐ and C‐terminal half‐molecules have virtually no binding capacity. This suggests that the primary binding site is located near the midpoint of the connecting strand and includes elements of both half‐molecules. Thrombin digestion, which splits calmodulin between residues 106 and 107, also substantially reduces auramine O binding; this may be interpreted in terms of the stabilization of the structure of the connecting strand by interaction with residues within binding domain IV. The binding affinity at pH 5.0, where the helical organization of the connecting strand may be intact, is greater than at neutral pH.
The interactions of neuropeptides with membrane model systems: A case study
Authors: Hicks Rickey P. , Beard Debbie J. , Young John K.
Published: December 2004

The interactions between the positively charged neuropeptides substance P (SP), bradykinin (BK), and zwitterionic Met‐enkephalin (ME) neuropeptides, and negatively charged SDS and zwitterionic lysophosphatidylcholine (LPC) membrane model systems, have been investigated using one‐ and two–dimensional nmr experiments. Proton longitudinal relaxation studies were used to characterize these interactions as intrinsic or extrinsic. An extrinsic interaction are similar to those observed for extrinsic membrane proteins. An intrinsic interaction are similar to those observed for intrinsic membrane proteins, and would require that the hydrophobic residues penetrate or insert into the hydrophobic core of the membrane. The interactions between both SP and BK and SDS, based on nmr results, may be characterized as intrinsic, and the interaction between ME and SDS may be characterized as extrinsic. Two–dimensional nuclear Overhauser enhancement spectroscopy experiments proved the insertion of the phenylalanine residues on both SP and BK into the hydrophobic core of SDS micelles. The interaction between SP and BK with LPC based on nmr results are characterized as extrinsic, with the interaction between ME and SDS characterized as weakly intrinsic.
Studies on the yeast α‐mating factor: A model for mammalian peptide hormones
Authors: Naider Fred , Gounarides John , Xue Chu‐Biao , Bargiota Effimia , Becker Jeffrey M.
Published: December 2004

Small peptides initiate sexual conjugation in the yeast Saccharomyces cereuisiae and this phenomenon is an ideal paradigm for studying the mode of action of mammalian peptide hormones, 1H‐nmr spectroscopy was used to examine the conformation of linear and cyclic analogues of the α‐factor (WHWLQLKPGQPMY) in aqueous solution. In all cases peptides that exhibit nmr parameters expected for a type II β‐turn have higher biological activities than those that do not appear to assume this conformation. Based on a simple model for the interaction of the pheromone with its receptor, we prepared fragments of the α‐factor. Several of these fragments either antagonize or potentiate the activity of the α‐factor. The latter represent the first example of peptide fragments that synergize the activity of the parent pheromone.
Peptide mechanics: A force field for peptides and proteins working with entire residues as smallest units
Authors: Gerber Paul R.
Published: December 2004

The central ingredient of any structure modeling tool is a molecular model or force field that accounts for proper geometry and energy calculation. For protein and peptide modeling based on entire amino acids as building blocks, we describe a peptide force field in which each amino acid is represented by a single point in space, taken at the position of the α‐carbon atom. Apart from the positional coordinates, these units carry the two torsional angles φ and Ψ as additional degrees of freedom to account for the orientations of the peptide links. While some of the energy terms are analogous to expressions in atomic force fields, the presence of the angular variables leads to fundamental differences with new features and additional terms with no atomic counterparts.

The force field reproduces secondary structure elements with very good accuracy. Globular parts of tertiary packing stay near the experimental structures with a rms deviation in C‐α positions of 0.1–0.3 nm and about 25° in φ and Ψ depending on the size of the structure. A tendency for larger discrepancies is observed in exposed loops or terminal segments the conformations of which may be strongly influenced by neighboring domains.

Finally, a scope of possible applications is presented. They range from modeling activities, such as model building by homology, to coarse scanning of conformation space in conformation analysis and structure determination. An extension to a dynamics model would offer the possibility to eliminate the less interesting high‐frequency modes that in all‐atom force‐field dynamics absorb most of the computational effort.
Conformations in solution of angiotensin II, and its 1–7 and 1–6 fragments
Authors: Cushman J. A. , Mishra P. K. , Bothner‐By A. A. , Khosla M. S.
Published: December 2004

High‐resolution proton spectra at 620 MHz of human angiotensin II (1–8), angiotensin II (1–7), and angiotensin II (1–6) have been obtained in aqueous solution at acidic pH, and in dimethylsulfoxide solution. Complete chemical shift assignments for all three angiotensin peptides were made based on two‐dimensional (2D) correlated spectroscopy and 2D‐CA‐MELSPIN spectra. Based on the measured values of 3J HNCH, the pattern of observed transverse Overhauser effects, and side‐chain coupling constants, it is concluded that all three analogues exist in H2O or DMSO‐d6 as a mixture of conformers that is largely extended, with negligible content of folded structures, such as β‐turns, γ‐turns, or helix content. The results fit well with those of Nikiforovich et al.1, 2. © 1992 John Wiley & Sons, Inc.
Shape distributions of protein topography
Authors: Connolly Michael L.
Published: December 2004

A method is presented for measuring protein surface shape. It is an improvement of an earlier method that intersects a sphere with the solvent‐excluded volume of a protein molecule. The new method, called a shape distribution, produces a more sophisticated description of the region of the sphere inside the protein than is provided by simply measuring the region's area or solid angle. This method is applied to the prediction of molecular complexes in three systems: the hemoglobin nonallosteric interface, trypsin and trypsin inhibitor, and heme and apomyoglobin. It does not uniquely predict the correct structure, even though the individual structures are taken from the experimentally determined complex structure. However, it does provide a list of several hundred predicted complexes, one of which is correct, and from which the correct complex might be extracted by a subsequent chemical filter. © 1992 John Wiley & Sons, Inc.
Variable ranges of interactions in polypeptide conformations with a method to complement molecular modeling
Authors: Jacchieri S. G. , Jernigan R. L
Published: December 2004

Formulations of conformational weights for helix‐coil transitions can be extended to substantially more complex situations than are usually pursued. General rules for matrix multiplication that depend parametrically on the interaction ranges and numbers of rotamers of residues are presented. The orders of the matrices of statistical weights can be increased with chain length, so that an individual matrix element can represent any specified single conformation, as needed. By the appropriate choice of interaction ranges and numbers of available conformers, approximations can be introduced in which: (1) an average of the conformations of any chain segment is obtained, (2) specific residue‐residue interactions are excluded, or (3) the conformation of a part of the chain is restricted or fixed. The method is appropriate for treating specific interactions in peptides and could be used together with available experimental information to develop models of conformational transitions. As such, the methods represent a class of calculations aimed at more rigorous calculations built around known features of a molecule. The aim is to facilitate calculations that bridge the gap between nonquantitative molecular model building and more rigorous but less directed molecular mechanics calculations. The method can directly include any desired longer range of interactions, if the interaction range is not too long to make impossible the manipulation of the requisite matrices. An outline is presented of an application to treat salt bridges in the C peptide of ribonuclease A. © 1992 John Wiley & Sons, Inc.
Nmr studies of an extrahelical cytosine in an A·T rich region of a deoxyribodecanucleotide
Authors: Morden Kathleen M. , Maskos Karol
Published: December 2004

One‐dimensional and two‐dimensional (2D) nmr experiments were carried out on an oligonucleotide duplex that contains an unpaired cytosine, d(GCGAACAAGCG)·d(CGCTTTTCGC), which will be referred to as the C‐bulge decamer. Evidence from one‐dimensional nuclear Overhauser effect (NOE) experiments on the exchangeable protons indicates that the unpaired cytosine is extrahelical. This conclusion is also supported by numerous cross‐peaks in the 2D NOE spectroscopy (NOESY) spectrum of the nonexchangeable protons. The assignments for all of the resonances, with the exception of the H5′ and H5″ resonances, have been made through the use of 2D NOESY, correlated spectroscopy (COSY), and relayed COSY experiments. The temperature dependence of the C(H6) resonance chemical shifts indicates that the unpaired cytosine shows unusual behavior compared to other cytosines in the duplex. A comparison of chemical shifts for all, the assigned resonances of the duplexes with and without the unpaired cytosine suggests that the majority of the structural perturbation is localized in the A·T tract surrounding the unpaired base. The behavior of the imino resonances as a function of temperature also indicates that the perturbation to the duplex is localized and destabilizes the A·T base pairs adjacent to the unpaired base. © 1993 John Wiley & Sons, Inc.
Degree of linear polarization method for determination of intrinsic optical anisotropy of southern bean mosaic virus
Authors: Sano Yoh
Published: December 2004

Southern bean mosaic virus (SBMV) is a spherical plant virus. It possesses an intrinsic (structural) anisotropy due to the orientation of valence bonds of the amino acid and/or nucleic acid residues. According to the light scattering theory of Rayleigh and Gans for optically anisotropic spheres, the degree of linear polarization of the scattered light depends on the intrinsic anisotropy, the relative refractive index of the sphere to the solvent, the scattering angle, and the inclination angle of linearly polarized incident light to the scattering plane. By using the theoretical expression of the degree of linear polarization, the intrinsic anisotropy parameter of southern bean mosaic virus in the compact spherical state was calculated with the appropriate experimental values. This novel method should be useful in elucidating the internal structure of spherical viruses and other spherical complexes of macromolecules. © 1993 John Wiley & Sons, Inc.
Application of distance geometry to the proton assignment problem
Authors: Oshiro C. M. , Kuntz I. D.
Published: December 2004

Assignment of the hydrogen spectrum is the first step in the conventional procedure for the determination of molecular structure by 1H‐nmr. In this paper, we explore the possibility of directly exploiting the distances derived from nuclear Overhauser effect experiments to generate a three‐dimensional structure that is then assigned based on knowledge of the connectivity or primary sequence. This effort is analogous to that of the protein crystallographers in tracing electron density of the peptide chain. In particular, we compare structures produced by distance geometry to known peptide secondary structures to see what level of information is required to “trace” the backbone α‐carbon and amide hydrogens and the β‐carbon hydrogens. We conclude that this approach is only useful with excellent quality stereo‐resolved data. © 1993 John Wiley & Sons, Inc.
Kinetic analysis of the hydrodynamic transition accompanying protein folding using size exclusion chromatography. 2. Comparison of spectral and chromatographic kinetic analyses
Authors: Shalongo William , Jagannadham Medicherla , Stellwagen Earle
Published: December 2004

The kinetics of the hydrodynamic volume change associated with the unfolding and refolding of a globular protein can be observed using high performance size exclusion chromatography. Chromatographic profiles that evidence such dynamics can be simulated using equations in which Chromatographic partitioning and the conformational transition are described in terms of a finite difference algorithm incorporating an apparent binding model to generate broad and asymmetric peaks. Application of these equations to the simple two‐state unfolding transition of ribonuclease A in guanidine hydrochloride indicates that reliable kinetic parameters can be obtained using these equations. © 1993 John Wiley & Sons, Inc.
Necessary conditions for avoiding incorrect polypeptide folds in conformational search by energy minimization
Authors: Vajda S. , Jafri M. S. , Sezerman O. U. , DeLisi C.
Published: December 2004

Low energy conformations have been generated for melittin, pancreatic polypeptide, and ribonuclease S‐peptide, both in the vicinity of x‐ray structures by energy refinement and by an unconstrained search over the entire conformational space. Since the structural polymorphism of these medium‐sized peptides in crystal and solution is moderate, comparing the calculated conformations to x‐ray and nmr data provides information on local and global behavior of potential functions. Local analysis includes standardization calculations, which show that models with standard geometry can approximate good resolution x‐ray data with less than 0.5 Å rms deviation (RMSD). However, the atomic coordinates are shifted up to 2 Å RMSD by local energy minimization, and thus 2 Å is generally the smallest RMSD value one can target in a conformational search using the same energy evaluation models.

The unconstrained search was performed by a buildup‐type method based on dynamic programming. To accelerate the generation of structures in the conformational search, we used the ECEPP potential, defined in terms of standard polypeptide geometry. A number of low energy conformations were further refined by relaxing the assumption of standard bond lengths and bond angles through the use of the CHARMM potential, and the hydrophobic folding energies of Eisenberg and McLachlan were calculated. Each conformation is described in terms of the RMSD from the native, hydrogen‐bonding structure, solvent‐acessible surface area, and the ratio of surfaces corresponding to nonpolar and polar residues.

The unconstrained search finds conformations that are different from the native, sometimes substantially, and in addition, have lower conformational energies than the native. The origin of deviations is different for each of the three peptides, but in all examples the refined x‐ray structures have lower energies than the calculated incorrect folds when (1) the assumption of standard bond lengths and bond angles is relaxed; (2) a small and constant effective dielectric permittivity (ε < 10) is used; and (3) the hydrophobic folding energy is incorporated into the potential. © 1993 John Wiley & Sons, Inc.
Peptide design 310‐helical conformation of a linear pentapeptide containing two dehydrophenylalanines, Boc‐Gly‐ΔZPhe‐Leu‐ΔZPhe‐Ala‐NHCH3
Authors: Bhandary Krishna K. , Chauhan V. S.
Published: December 2004

α, β‐Dehydroamino acids are expected to provide conformational constraint to the peptide backbone. A pentapeptide containing two dehydrophenylalanines (ΔZPhe) separated by one L‐amino acid has been synthesized and its solid state conformation determined. The pentapeptide, Boc‐Gly‐ΔZPhe‐Leu‐ΔZPhe‐Ala‐NHCH3, crystallizes from aqueous methanol in the orthorhombic space group P212121. There are four formula units, C35H46N6O7, in a unit cell of dimensions a = 10.155(3), b = 15.175(1), and c = 23.447(2) Å, at room temperature. The structure was solved by direct methods program, SIR88, and refined to a final R = 0.038 based on 3049 reflections with I > 2σ(I). All the peptide links are trans and the backbone conformation of the pentapeptide can be described as a 310‐helix, with mean ϕ, ψ values of −65.1° and −22.8° (the value is averaged over the first four residues). There are four intramolecular 4 → 1 type hydrogen bonds characteristic of 310‐type helices. In the crystal, the helices are held together by intermolecular NH…︁OC head‐to‐tail and lateral hydrogen bonding between symmetry related molecules. This mode of packing is similar to the packing motifs observed so often in other oligopeptides that adopt a 310‐helical structure. © 1993 John Wiley & Sons, Inc.
X‐ray studies on crystalline complexes involving amino acids and peptides. XXIV. Different ionization states and novel aggregation patterns in the complexes of succinic acid with DL‐and L‐histidine
Authors: Prasad G. Sridhar , Vijayan M.
Published: December 2004

Diffusion of acetonitrile into an aqueous solution of DL‐histidine and succinic acid in 1:3 molar proportions results in the crystals of DL‐histidine hemisuccinate dihydrate [triclinic, P1, a = 7.654(1), b = 8.723(1), c = 9.260(1) Å, α = 77.23(1), β = 72.37(1) and γ = 82.32 (1)°]. The replacement of DL‐histidine by L‐histidine in the crystallization experiment under identical conditions leads to crystals of L‐histidine semisuccinate trihydrate [orthorhombic, P212121, a = 7.030 (1), b = 8.773 (1), and c = 24.332 (3) Å]. The structures were solved using counter data and refined to R values of 0.056 and 0.054 for 2356 and 1778 observed reflections, respectively. Histidine molecules in both the complexes exist in open conformation I. Succinate and semisuccinate ions in them are planar, and exactly or nearly centrosymmetric. In the DL‐histidine complex, the amino acid molecules form double ribbons and the succinate ions occupy voids left behind when the double ribbons aggregate, as in inclusion compounds. In the L‐histidine complex, the amino acid molecules form columns; so do the semisuccinate ions and water molecules. The two columns interdigitate to form the complex crystal. There are similarities between the molecular aggregation in the complexes and that in the crystals of L‐ and DL‐histidine. However, the presence of succinic acid has the effect of disrupting, partially or totally, head‐to‐tail sequences involving amino acid molecules. © 1993 John Wiley & Sons, Inc.
Effects of molecular association on structure and dynamics of a collagenous peptide
Authors: Daragan Vladimir A. , Ilyina Elena , Mayo Kevin H.
Published: December 2004

Peptide GVKGDKGNPGWPGAPY from the triple‐helix domain of type IV collagen aggregates in solution at a critical aggregation concentration of 18 mM. This molecular self association process is investigated by 1H‐ and 13C‐nmr spectroscopy. As a function of increasing peptide concentration, selective 1H resonances are cooperatively chemically shifted by up to 0.04 ppm to apparently saturable values at high concentration. Pulsed field gradient nmr was used to derive translation diffusion constants that, as the peptide concentration is increased, also cooperatively and monotonically decrease to an apparent limiting value. An average number of 6 monomer units per aggregate have been estimated from diffusion constant and 13C relaxation data. Comparative 1H nuclear Overhauser effect spectroscopy (NOESY) spectra accumulated at high and low peptide concentrations suggest that average internuclear distances are decreased as a result of peptide association. 13C‐nmr multiplet spin‐lattice relaxation and 13C‐ {1H} NOE effects on 13C‐enriched glycine methylene positions in the peptide demonstrate that overall molecular tumbling and backbone internal motions are attenuated in the aggregate state. Lowering the solution pD from pD 6 to pD 2 disrupts the aggregate state, suggesting a role for electrostatic interactions in the association process. Based on thermodynamic considerations, hydrophobic interactions also probably act to stabilize the aggregate state. These data are discussed in terms of an nmr/NOE constrained computer‐modeled structure of the peptide. © 1993 John Wiley & Sons, Inc.
Stereochemical constraints in peptide design: Analysis of the influence of a disulfide bridge and an α‐aminoisobutyryl residue on the conformation of a hexapeptide
Authors: Uma K. , Kishore R. , Balaram P.
Published: December 2004

The competing effects of a disulfide bridge and an α‐aminoisobutyryl residue (Aib) in determining the conformation of a hexapeptide have been investigated, by comparing the cyclic disulfide
magnified image

and the acylic peptide Boc‐Cys(SBzl)‐Val‐Aib‐Ala‐Leu‐Cys(SBzl)‐NHMe (2). Previously published nmr and crystallographic studies [R. Kishore, S. Raghothama, and P. Balaram (1987) Biopolymers, Vol. 26, pp. 873–891; I. L. Karle, R. Kishore, S. Raghothama, & P. Balaram, (1988) Journal of the American Chemical Society Vol. 110, pp. 1958–1963] have established an antiparallel β‐hairpin structure for 1 with a central Aib‐Ala β‐turn. A comparison of nmr data for 1 and 2 in chloroform and dimethylsulfoxide reveals that the acyclic peptide is conformationally labile. Evidence for a 310‐helical conformation in CDCl3 is obtained from sensitivity of NH chemical shifts to temperature and solvent perturbation and low JHNCαH values. Studies in solvent mixtures establish a conformational transition on going from CDCl3 to (CD3)2SO. The changes in NH nmr parameters, together with the observation of several interresidue C H‐Ni + 1H nuclear Overhauser effects support a conformation having a central β‐turn with extended arms in (CD3)2SO. A single Aib residue appears to stabilize a helix in apolar solvents, for the acyclic hexapeptide, while the disulfide bridge serves to lock the β‐hairpin conformation. © 1993 John Wiley & Sons, Inc.
Conformational transitions using molecular dynamics with minimum biasing
Authors: Harvey Stephen C. , Gabb Henry A.
Published: December 2004

The molecular dynamics algorithm (MD), which simulates intramolecular motions on the subnanosecond timescale, has been modified to allow the investigation of slow conformational transitions that do not necessarily occur spontaneously in MD simulations. The method is designated CONTRA MD (CONformational TRAnsitions by Molecular Dynamics with minimum biasing). The method requires the prior definition of a single conformational variable that is required to vary monotonically from an initial conformation to a final target conformation. The simulation is broken up into a series of short free MD segments, and we determine, after each segment of MD, whether or not the system has evolved toward the final conformation. Those segments that do not move the system in that direction are deleted. Those that do move it toward the final conformation are patched together sequentially to generate a single representative trajectory along the transition pathway. The CONTRA MD method is demonstrated first by application to the simultaneous C2′‐endo to C3′‐endo repucker and anti to syn N‐glycosidic torsion transitions in 2′‐deoxyadenosine and then to the large‐scale bending in phenylalanine transfer RNA. © 1993 John Wiley & Sons, Inc.
The hydration of proteins in nearly anhydrous organic solvent suspensions
Authors: McMinn J. H. , Sowa M. J. , Charnick S. B. , Paulaitis M. E.
Published: December 2004

Water sorption isotherms at 27°C have been measured for lysozyme and chymotrypsin in suspensions of toluene, di(n‐butyl) ether, n‐propanol, and a solution of 1M n‐propanol in benzene. Sorption isotherms for the different suspensions are compared by converting solvent water content to the thermodynamic activity of water in each solvent. The sorption behavior is also compared to that for the two proteins hydrated from the vapor phase. At low water activities, all sorption isotherms are similar when compared on the basis of water activity. However, at higher activities, water sorption by the proteins in the organic suspensions is suppressed relative to the sorption of water vapor. The greatest suppression is observed for n ‐propanol, which suggests that the suppression may be due to a competition for water‐binding sites on the protein by the organic solvent. Sorption isotherms at low water activities have also been predicted using a thermodynamic model in which it is assumed that water binds selectively to the ionizable residues on the surface of the protein. A comparison of predicted and measured sorption isotherms shows that the model can provide reasonable estimates of water sorption in nonpolar or moderately polar organic solvent suspensions at low levels of hydration. © 1993 John Wiley & Sons, Inc.
Stretching and overstretching of DNA in pulsed field gel electrophoresis. II. Coupling of orientation and transport in initial response to the field
Authors: Mayer Pascal , Sturm Jean , Weill Gilbert
Published: December 2004

The rise of the birefringence following the application of an electric field to DNA in an agarose gel has been quantitatively analyzed to demonstrate the major role played by the chain overstretching. By analyzing the field free decay after short times, we demonstrate that the overshoot is completely due to overstretching. Its time of appearance τov varies with the field and DNA length in fair agreement with the E−lN0ln N0 prediction of Lim et al. [ (1990), Journal of Chemical Physics, Vol. 92, pp. 709–721]. The τov is also the time corresponding to one tube renewal after relaxation of the overstretching. © 1993 John Wiley & Sons, Inc.
Energetics of the disulfide bridge: An ab initio study
Authors: Qian Weili , Krimm Samuel
Published: December 2004

The energetics of the χχ3χ portion of the disulfide bridge have been obtained from an ab initio study of diethyl disulfide. Calculations at the 3‐21G* level were done on relaxed structures at every ∼ 30° in χ and χ, and the additional energies for small Δχ3 were obtained. Complete E(χ, χ) and χ(χ, χ) maps were computed from Fourier series expansions. These results have been used to calculate the energetics of 92 disulfide bridges in known protein structures, and to compare ab initio and molecular mechanics energies for some observed and predicted bridges. The differences found in relative energies and in χ values suggest that present energy functions give a limited description of the structural and energetic properties of the disulfide bridge. © 1993 John Wiley & Sons, Inc.
Conformationally readdressed CCK‐B/δ‐opioid pepitide ligands
Authors: Nikiforovich Gregory V. , Kolodziej Stephen A. , Nock Bruce , Bernad Nicole , Martinez Jean , Marshall Garland R.
Published: December 2004

The sequence of a cholecystokinin (CCK) related peptide was modified to obtain analogues, which intereact selectively either with CCK‐B, or with δ‐opioid receptors. Two kinds of peptides were designed, namely, the cyclic peptides of the H‐Tyr‐cyclo(D‐Pen‐Gly‐Trp‐L‐/D‐3‐transmecaptoproline)‐Asp‐Phe‐NH2 sequence (compounds 1a and 1b, respectively), and the linear peptides of the H‐Tyr‐D‐Val‐Gly‐Trp‐L/D‐3‐trans‐methylmercaptoproline‐Asp‐Phe‐NH2 sequence (compounds 2a and 2b, respectively). The only difference between the chemical structures of the linear analogues compared to the cyclic ones is that one covalent bond has been eliminated and a sulfur atom is replaced by a methyl group. Molecular modeling showed that, among low‐energy conformers of cyclic compounds 1, there are three‐dimensional structures compatible to the model for δ‐ receptor‐ bound conformer, suggested earlier[G. V. Nikiforovich. V. J. Hruby. O. Prakash, and C. A. Gehrig (1991) Biopolymers. vol. 31. pp. 941–955]. Results of binding assays fully supported the rationale for the design of compounds 1 and 2. The cyclic analogue 1a has Ki values of 4.5 and > 5000 n M at δ‐ and μ‐opioid receptors, respectively; IC50 values of 3000 n M for both CCK‐A and CCK‐B receptors, whereas its linear counterpart 2a has ki values of 462 and 229 nM at δ‐ and μ‐opioid receptors, respectively; and IC50 values of 1.6 and > 10.000 nM for CCK‐A and CCK‐B receptors, respectively. The results of this study demonstrate a possibility to redirect a peptide sequence that interacts with one type of receptors (CCK‐B receptors) toward interaction with another type (δ‐opioid receptors) belonging to a different physiological system. This redirection could be performed by changing the conformational properties of the peptide with very minimal changes in its chemical structure. © 1995 John Wiley & Sons, Inc.
Intramolecular electronic energy transfer in peptides carrying naphthalene and protoporphyrin molecules: A spectroscopic and conformational statistics investigation
Authors: Pispisa B. , Venanzi M. , Palleschi A. , Zanotti G.
Published: December 2004

Short linear peptides, carrying an AA spacer in the backbone chain (AA = Aib or Ala),and naphthalene(N) and protoporphyrin IX(P) covalently bound to ϵ‐amino groups of lysine side chains, were synthesized. The general formula is Boc‐Leu‐Leu‐Lys(P)‐(AA)n‐Leu‐Leu‐Lys(N)‐OtBu, with n = 0–2. The photophysical behavior of these compounds was investigated in water/methanol 75/25(v/v) solution by steady‐state and time‐resolved fluorescence experiments. Quenching of excited naphthyl chromophore lakes place by electronic energy transfer to the porphyrin ground state, and proceeds on a time scale of 3–8 ns, while a minor and slower(≈45 ns) fluorescence lifetime measures the decay of the exciplexes. The results were compared with those earlier obtained with the P(Ala)nN peptides(n = 0–4) in methanol solution, showing that addition of water does not significantly alter the dynamic relaxation behavior of the systems investigated, but affects the dissipation mechanism of the energy transferred to P. Quenching efficiencies from both fluorescence intensity and fluorescence lifetime measurements follow a different trend as the number of AA units increases, depending on whether AA = Aib or Ala, indicating that there are differences in the structural features of the two series of peptides. Consistently, CD spectral results suggest that the former compounds attain ordered conformations, possibly of the 310‐helical type, while the latter populate α‐helical structures to an extent depending on the chain length. The ir data in dilute CD3OD or CDCl3 solution confirm this conclusion in that there is an increased percentage of intramolecular H bonds in the P(Aib)nN as compared to the corresponding P(Ala)nN peptides. The photophysical results can be well described by a long‐range dipole–dipole interaction model, provided the separation distances distribution and mutual orientation of N and P groups are taken into account. The need of using the angular relationships between the probes implies that interconversion among conformational substates of chromophores linkages is slow on the time scale of the transfer process, very likely because of both the amide bond in the linkages and the bulkiness of the donor–acceptor pair. © 1995 John Wiley & Sons, Inc.
Gibberellic acid formation in continuous culture
Authors: Holme Tord , Zacharias Bengt
Published: December 2004

The formation of gibberellic acid in single‐stage continuous cultures was studied. Stirred fermenters of 3 and 20‐l. working volumes were used. Maximum output of gibberellic acid was obtained at a holding time of 200 hr. The rate of formation, calculated on the basis of mycelial nitrogen, was comparable with the highest values reported for batch cultures.
Kinetic studies of the lactic acid fermentation in batch and continuous cultures
Authors: Hanson T. P. , Tsao G. T.
Published: December 2004

The fermentation kinetics of the homofermentative organism Lactobacillus delbrueckii in a glucose‐yeast extract medium is studied in both batch and continuous culture under conditions of controlled pH. From a graphical analysis of the batch data, a mathematical model of the process is derived which relates bacterial growth, glucose utilization, and lactic acid formation. The parameters in the model represent the activity of the organism and are a function of pH, having a maximum value at about 5.90. In a continuous stirred tank fermentor (CSTF), the effect of pH, feed concentration, and residence time is observed. The feed medium is a constant ratio of two parts glucose to one part yeast extract plus added mineral salts. An approximate prediction of the steady‐state behavior of the CSTF can be made using a method based on the kinetic model derived for the batch case. In making step changes from one steady state to another, the transient response is observed. Using the kinetic model to simulate the transient period, the calculated behavior qualitatively predicts the observed response.
Effect of wall growth on scale‐up problems and dynamic operating characteristics of the biological reactor
Authors: Howell J. A. , Chi C. T. , Pawlowsky U.
Published: December 2004

If inhibitory substrates are being utilized in a well‐stirred biological reactor, microbiological growth on the walls of the reactor can create a scale‐up problem. A simple model is proposed which shows that without such growth, of the three existing steady states only one is stable and nontrivial, but with wall growth the trivial, stable, steady state (washout) is impossible. In addition, wall growth reduces the region over which three steady states are feasible and reduces the minimum residence time for which there is only one steady state that corresponds to a high conversion. Thus, a laboratory process with a high surface area to volume ratio can give an over optimistic prediction of both necessary residence; time and stability of the full scale process unless wall growth is accounted for.
Bacteria which attack petroleum hydrocarbons in a saline medium
Authors: Soli Giorgio , Bens E. M.
Published: December 2004

Bacterial strains were isolated from California coastal areas which showed the ability to oxidize normal paraffins, iso‐paraffins, and aromatic hydrocarbons in a synthetic seawater medium. The ability to utilize a particular hydrocarbon was established not only on the basis of visible bacterial growth but also through a chromatographic technique which was standardized and which could define the amount of each hydrocarbon consumed by the bacteria in a mixture. Some of the strains exhibited vigorous hydrocarbon oxidation when exposed to synthetic mixtures of hydrocarbons as well as crude oil. Under conditions of aeration and agitation, mixed cultures could destroy approximately 50% of a South Louisiana crude oil in a period of 48 hr.
Conversion of deteriorated metal cutting fluids into protein
Authors: Speidel Harold K. , Bennett E. O.
Published: December 2004

Six soil isolates were grown on a petroleum sulfonate medium and analyzed chromatographically to determine their amino acid composition. The organisms were also examined to determine their growth rate in the petroleum medium, their per cent conversion of hydrocarbon to dry cell mass, their protein content, and their possible use as a food or food supplement. It was found that the isolates varied extensively as to their amino acid composition. Several of the isolates proved to convert the hydrocarbons to cell mass with relative ease. The proteins of four of the isolates appear to be suitable as a good food source, and the protein of one of the remaining isolates appears to be a very good food supplement.
Sterilization by electrohydraulic discharges
Authors: Holland R. V. , Board P. W. , Richardson K. C.
Published: December 2004

High voltage electric discharges between two electrodes immersed in a liquid (“electrohydraulic discharges”) inactivate microorganisms suspended in the liquid. The intense pulse of UV radiation emitted from the plasma formed between the electrodes causes most of the bactericidal effects, rather than shock waves, or free radicals or other chemical species formed in the liquid medium. A method of sterilizing materials without contamination from electrode debris is described. Possible applications and limitations of the technique are outlined.
Purification of ten transfer riboiiucleic acids from E. coli K‐12 MO
Authors: Weeren H. O. , Ryon A. D. , Kelmers A. D.
Published: December 2004

A flowsheet has been developed which leads to the purification of 10 transfer ribonucleic acids (tRNAs) from E. coli K‐12 MO. Crude tRNAs were recovered by phenol extraction and two ethanol precipitation steps. The initial separation of the crude tRNA mixture was achieved by RPC‐3 reversed‐phase chromatography. Following rechromatography under other conditions, the following tRNAs were recovered at a purity of 70 to 100%: arginine, aspartic acid, glutamic acid, lysine, formylmethionine‐1, formylmethionine‐3, normal methionine, phcnylalanine‐1, phenylalaninc‐2, and valine. Recoveries for these tRNAs ranged from 15 to 60%. The flowsheet was demonstrated with chromatogaphic runs ranging from 1,300 to 250,000 A260 units per run. The chromatographic steps were simple and readily reproducible.
Lysozyme activity in the presence of nonionic detergent micelles
Authors: Bernath F. R. , Vieth W. R.
Published: December 2004

The effect of a nonionic surfactant, polyoxyethylenesorbitan monolaurate (Tween 20), on the hen egg‐white lysozyme catalyzed lysis of a dried cell suspension of Micrococcus lysodeikticus is analysed. A rate enhancement of up to 70% is observed in the presence of surfactant at concentrations above the critical micelle concentration. This activity increase may be explained by postulating the existence of a micelle‐enzyme complex in which enzyme molecules are bound to micelles with preferential orientation of their active sites.

The reaction is found to be second order with respect to substrate. A mechanism is postulated in which a substrate particle is assumed to be an energy‐furnishing collision partner to the enzyme‐substrate complex. This mechanism correlated data over a wide range of enzyme and substrate concentrations.

Data from kinetic, ultrafiltration, ultraviolet, and fluorescence studies provide convincing evidence for the existence of a micelle‐lysozyme complex. The results suggest that it is possible that immobilized enzymes mat in general be more reactive than corresponding free enzymes.
Large scale synthesis of dinucleoside monophosphates catalyzed by ribonuclease from Aspergillus clavatus
Authors: Bauer S. , Lamed R. , Lapidot Y.
Published: December 2004

A gram scale enzymatic synthesis of eight, dinucleoside monophosphates (ApC, ApU, CpC, CpU, GpC, GpU, UpC, and UpU) is described. The synthesis involves a reaction between the appropriate ribonucleoside‐2′,3′‐cyclie phosphates and cytidine or uridine in the presence of ribonuelease from Aspergillus clavatus at 30°C. The enzyme is removed from the reaction mixture by chromatography on Bio‐Gel P–4, and the dinucleoside monophosphate is further purified by chromatography on a DEAE‐Sephadex A–25, column.

A procedure for the large scale preparation of the ribonuclease from Aspergillus clavatus is also described.
Dynamics and control of continuous microbial propagators to subject substrate inhibition
Authors: Edwards Victor H. , Ko Raphael C. , Balogh Stephan A.
Published: December 2004

Inhibitory substrate levels are common in industrial fermentations and in biological waste‐water treatment of many industrial wastes. Continuous microbial cultures are unstable to certain disturbances, such as shock loading by inhibitory substrates. Two feedback proportional control strategies are analyzed and compared for a simple model culture assumed represent able by the culture concentrations of biomass and a single rate‐limiting and growth‐limiting nutrient (substrate). One control strategy, the well known turbidostat, consists of adjusting culture holding time (e.g., by flow rate adjustment) in response to deviations in turbidity or some other measure of culture biomass concentration. The other control strategy is to adjust holding time in response to deviations in limiting nutrient concentrations in the culture. This second control strategy, termed the nutristat, can be superior to the turbidostat in many applications. The sign and magnitude of the dimensionless group {(X/YD)[dμ/dS]s}, is shown to be an important determinant, in the behavior of the open loop and the two closed loop processes. This characteristic group is positive when the specific growth rate is increased by increases in the nutrient concentration, zero when the growth rate is unaffected by the nutrient concentration, and negative in the presence of nutrient or substrate inhibition.

The effects of process modifications and of modeling assumptions on the control of the process are discussed and more sophisticated control schemes are also proposed.
Utilization of organic nitrogen compounds by Hydrogenomonas eutropha
Authors: Wixom Robert L. , Sheng Yung‐Boo , Becker Richard S.
Published: December 2004

The chemolithotroph, Hydrogenomonas eutropha, was tested for its ability to utilize a variety of single nitrogen sources during growth in an atmosphere of H2O2CO2 The present data show that H. eutropha can utilize the nitrogen from many, but not all, amino acids, several sulfur‐containing amino acids, glucosamine, and two aliphatic amides. The nitrogen concentration that supported maximum growth for NH4Cl, L‐glutamate, L‐glutamine, urea, and glycine was in the 0.010–0.019M range. H. eutropha failed to remove the nitrogen from primary and secondary amines, eycloleucine, tert‐DL‐leucine, DL‐p‐fluorophenylalanine, DL‐5‐methyltryptophan, creatine, and creatine. This microorganism was able to partially degrade at least six substituted indoles and/or tryptophan catabolites and six substituted imidazoles and/or histidine catabolites. All of a series of 17 dipeptides were able to serve as a nitrogen source for growth in the absence of NH4Cl. Extracts of H. eutropha were able to catalyze the hydrolysis of 16 α‐dipeptides, 2 tripetides, a tetrapeptide, a polypeptide, a β‐aspartyl peptide, 2 γ‐glutamyl peptides, a N‐acetyl amino acid, and 4 amino acid amides. These results emphasize the effectiveness of H. eutropha in utilizing a wide diversity of organic nitrogenous compounds containing amino and amide groups, heterocyclic rings, and peptide bonds.
Kinetics of the xanthan fermentation
Authors: Moraine R. A. , Rogovin P.
Published: December 2004

Xanthan gum, a heteropolysaccharide with unusual and useful properties, is now produced commercially by fermentation with Xanthomonas compestris NRRL B–1459 in a medium containing glucose, minerals, and a complex nitrogen source—distillers' dried solubles (DDS). Understanding the kinetics of the fermentation should contribute to process improvements and increase the market potential for the gum. Earlier studies showed that although DDS determined initial growth rate, growth was stopped by some mechanism other than substrate exhaustion, probably an effect related to product formation.

Product formation did not require active growth, but its rate increased with cell concentration. Specific product formation rate declined at high viscosities. Varying glucose concentration from 0.5 to 5.0% and dissolved O2 tension between 20 and 90% air saturated had no effect on the rates, but pH had to be maintained near 7 and temperature near 28°C to permit continued product formation. Xanthan yield could be explained by the energy required for growth and polymerization, that energy coming from dissimilation of the part of the carbohydrate substrate not converted to polymer.