bluuebunny/crossref_metadata_2025 huggingface dataset:
Diffusion coefficients of segmentally flexible macromolecules with two subunits: A study of broken rods
Authors: Mellado P. , Iniesta A. , Diaz F. G. , De La Torre J. García
Published: December 2004

A general treatment for the solution dynamics of segmentally flexible macromolecules having two subunits is presented. Bead modeling allows for a complete inclusion of hydrodynamic interactions in this treatment. The finite size of the beads is also considered, so that it is therefore possible to account properly for torsional motions of the subunits. Expressions for the components of the resistance matrix are derived. From them, the translational and rotational diffusion coefficients can be calculated. Distinction is made between hinged macromolecules, whose only internal motion is bending, and swivel‐jointed macromolecules, for which torsions of the subunits are also allowed. Numerical results are presented for broken rods with the two types of flexibility. The effects of hydrodynamic interaction between arms of broken rods are about 25% for translation and under 10% for rotation. These findings give support to the treatments of Harvey, Wegener, and co‐workers in which interactions were neglected. The rotational dynamics of hinged and swivel‐jointed rods are compared. Although there are differences in the short‐time behavior, the longest relaxation time is the same for the two cases. Finally, the validity of Wegener's rotational diffusion constants is discussed.
Structural and dynamic properties of the heme pocket in myoglobin probed by optical spectroscopy
Authors: Cupane Antonio , Leone Maurizio , Vitrano Eugenio , Cordone Lorenzo
Published: December 2004

We report the optical absorption spectra of sperm whale deoxy‐, oxy‐, and carbonmonoxymyoglobin in the temperature range 300–20 K and in 65% glycerol or ethylene glycol–water mixtures. By lowering the temperature, all bands exhibit half‐width narrowing and peak frequency shift; moreover, the near‐ir bands of deoxymyoglobin show a marked increase of the integrated intensities. Opposed to what has already been reported for human hemoglobin, the temperature dependence of the first moment of the investigated bands does not follow the behavior predicted by the harmonic Franck–Condon approximation and is sizably affected by the solvent composition; this solvent effect is larger in liganded than in nonliganded myoglobin. However, for all the observed bands the behavior of the second moment can be quite well rationalized in terms of the harmonic Franck–Condon approximation and is not dependent on solvent composition. On the basis of these data we put forward some suggestions concerning the structural and dynamic properties of the heme pocket in myoglobin and their dependence upon solvent composition. We also discuss the different behaviors of myoglobin and hemoglobin in terms of the different heme pocket structures and deformabilities of the two proteins.
Conformational characteristics of peptides and unanticipated results from crystal structure analyses
Authors: Karle Isabella L.
Published: December 2004

Preferred conformation and types of molecular folding are some of the topics that can be addressed by structure analysis using x‐ray diffraction of single crystals. The conformations of small linear peptide molecules with 2–6 residues are affected by polarity of solvent, presence of water molecules, hydrogen bonding with neighboring molecules, and other packing forces. Larger peptides, both cyclic and linear, have many intramolecular hydrogen bonds, the effect of which outweighs any intermolecular attractions. Numerous polymorphs of decapeptides grown from a variety of solvents, with different cocrystallized solvents, show a constant conformation for each peptide.

Large conformational changes occur, however, upon complexation with metal ions. A new form of free valinomycin grown from DMSO exhibits near three‐fold symmetry with only three intramolecular hydrogen bonds. The peptide is in the form of a shallow bowl with a hydrophobic exterior. Near the bottom of the interior of the bowl are three carbonyl oxygens, spaced and directed so that they are in position to form three ligands to a K+, e.g., complexation can be completed by the three lobes containing the β‐bends closing over and encapsulating the K+ ion. In another example, free antamanide and the biologically inactive perhydro analogue, in which four phenyl groups become cyclic hexyl groups, have essentially the same folding of backbone and side chains. The conformation changes drastically upon complexation with Li+ or Na+. However, the metal ion complex of natural antamanide has a hydrophobic globlar form whereas the metal ion complex of the inactive perhydro analogue has a polar band around the middle. The structure results indicate that the antamanide molecule is in a complexed form during its biological activity.

Single crystal x‐ray diffraction structure analyses have identified the manner in which water molecules are essential to creating minipolar areas on apolar helices. Completely apolar peptides, such as membrane‐active peptides, can acquire amphiphilic character by insertion of a water molecule into the helical backbone of Boc‐Aib‐Ala‐Leu‐Aib‐Ala‐Leu‐Aib‐Ala‐Leu‐Aib‐OMe, for example. The C‐terminal half assumes an α‐helix conformation, whereas the N‐terminal half is distorted by an insertion of a water molecule W(1) between N(Ala5) and O(Ala2), forming and hydrogen bonds N(5)H ⃛ W(1) and W(1) ⃛ O(2). The distortion of the helix exposes CO (Aib1) and CO (Aib4) to the outside environment with the consequence of attracting additional water molecules. The leucyl side chains are on the other side of the molecule. Thus a helix with an apolar sequence can mimic an amphiphilic helix.
Cyclo(D‐Pro‐L‐Pro‐D‐Pro‐L‐Pro): Structural properties and cis/trans isomerization of the cyclotetrapeptide backbone
Authors: Mästle Werner , Weber Thomas , Thewalt Ulf , Rothe Manfred
Published: December 2004

Combinations of L‐ and D‐proline residues are useful compounds for finding new structures and properties of cyclic peptides. This is demonstrated with one striking example, the cyclic tetrapeptide c(D‐Pro‐L‐Pro‐D‐Pro‐L‐Pro). For this molecule composed of strictly alternating D‐ and L‐configurated residues, a highly symmetrical structure is expected, which should be an optically inactive meso‐form. Cyclization of the enantiomeric pure linear precursor D‐Pro‐L‐Pro‐D‐Pro‐L‐Pro, however, yields a racemic mixture of two enantiomeric cyclotetrapeptides, both with twofold symmetry and a cis–trans–cis–trans sequence of the peptide bonds. Remarkably, this formation of a racemate was not caused by racemization, but by cis/trans isomerization of all peptide bonds in the ring. This process may occur in the linear precursor during the ring formation (cyclization of conformers with trans–cis–trans or cis–trans–cis arrangement of the amide bonds) as well as in the enantiomeric pure cyclic tetrapeptide at higher temperature. In the latter case, an all‐cis structure should exist as the intermediate, which can form a cis–trans–cis–trans sequence in two equivalent ways, leading finally to two enantiomeric cyclotetrapeptides. In the first one, the cis peptide bonds are attributed to the L‐residues and the trans peptide bonds to the D‐residues; in the second one, the cis bonds belong to the D and the trans bonds to the L‐residues. The mixture of these two enantiomers does not crystallize in the racemic form, but in enantiomeric pure separate crystals. The structural properties could be proved by 1H‐ and 13C‐nmr spectroscopy and x‐ray analysis. The cis/trans isomerization process was confirmed by optical rotation measurements and CD spectroscopy, as well as DREIDING model studies. Calorimetric measurements in the solid state suggest the existence of the expected all‐cis intermediate. The backbone conformation of the 12‐membered medium‐sized ring shows only slight deviations—up to 6° —from the planarity of the peptide bonds. On the other hand, the four pyrrolidine rings show different types of puckering of the Cγ or the Cβ atoms.
Regularly alternating L,D‐peptides. III. Hexacyclic peptides from valine or phenylalanine
Authors: Pavone Vincenzo , Benedetti Ettore , Di Blasio Benedetto , Lombardi Angela , Pedone Carlo , Tomasich Lera , Lorenzi Gian Paolo
Published: December 2004

In the present paper we describe the single‐crystal x‐ray analyses of two cyclic hexapeptides containing an equal number of alternating L,D‐residues as putative analogues of the metal binding compounds, enniatin and beauvericine. Both the molecules of c(L‐Val‐D‐Val)3 and c(L‐Phe‐D‐Phe)3 retain in the solid state the center of symmetry and crystallize with six and eight trifluoroacetic acid molecules, respectively. The peptides are strongly hydrogen bonded to the solvent molecules. We estimate, on the basis of the molecular geometry and spatial arrangement of the peptide carbonyl groups and in comparison with other metal binding cyclic peptides, the ability of these molecules to interact with metal ions as 1:1 complexes.
Structure of conformationally constrained peptides: From model compounds to bioactive peptides
Authors: Toniolo C.
Published: December 2004

The use of backbone conformational constraints has acquired increasing importance in the design and synthesis of structurally restricted agonists and antagonists of bioactive peptides. Here I discuss the preferred conformations of four among the most popular types of such peptide surrogates: (a) Peptides from Cα,α‐dialkylated residues, (b) tetrazolyl peptides, (c) (γ‐ and δ‐) lactam‐containing peptides, and (d) thiated peptides. Emphasis is given to conformational energy computations and x‐ray diffraction analyses of selected model compounds and analogues of small bioactive peptides such as the formylmethionyl tripeptide chemoattractant and MIF.
Structure and stability of the Boc(D‐Pro‐L‐Pro)2OCH3 Na+ complex: A model of poly(D,L‐proline), an alkali ion channel across membranes
Authors: De Santis P. , Scipioni A. , Palleschi A. , Savino M.
Published: December 2004

In order to elucidate the molecular mechanisms of alkali ions conduction of poly(D,L‐proline), which shows the typical behavior of an ion channel across membranes, conformations of the tetrameric derivative Boc(D‐Pro‐L‐Pro)2OCH3 in solution, in the presence of the Na+ ion, were investigated by CD and nmr spectroscopy. Both theoretical and experimental data indicate the formation in solution of a monomeric complex between the tetramer, in the all‐trans conformation, and the cation. The results allow to confirm the channel model, previously proposed on the phenomenological behavior of poly(D,L‐proline) in membranes.
Tooth enamel protein, amelogenin, has a probable β‐spiral internal channel, Gln112–Leu138, within a single polypeptide chain: Preliminary molecular mechanics and dynamics studies
Authors: Renugopalakrishnan V. , Pattabiraman N. , Prabhakaran M. , Strawich E. , Glimcher M. J.
Published: December 2004

Molecular dynamics simulation, with backbone constraints for 20 ps of equilibration and simulation, of a repeating polypeptide segment, Gln‐Pro‐His‐Gln‐Pro‐Leu‐Gln‐Pro‐His‐Gln‐Pro‐Leu‐Gln‐Pro‐Met‐(Gln‐Pro‐Leu)4, constituting residues 112–138 of bovine amelolgenin, a 19.35 kD hydrophobic protein, are discussed. It is generally believed that the above polypeptide segment is important for the interaction of amelogenin with Ca++ ions, which occurs in the early phases of enamel mineralization. An energetically stable structure of the above polypeptide with recurrent β‐turns is observed and contains a pore of ∼ 1 Å radius along the helical that can accommodate an unhydrated Ca++ ion. The length of the polypeptide possesses correct dimensions to span a bilayer. The proposed structure is unique among known polypeptide and protein structures.
Conformation of bombesin in buffer and in the presence of lysolecithin micelles: Nmr, CD, and fluorescence studies
Authors: Cavatorta P. , Spisni A. , Szabo A. G. , Farruggia G. , Franzoni L. , Masotti L.
Published: December 2004

The conformation of the tetradecapeptide hormone bombesin has been studied in buffer and in the presence of lysolecithin micelles, using static and dynamic fluorescence, CD, and one‐ and two‐dimensional nmr. The results obtained show that in buffer bombesin is present in an extended flexible chain, with no evidence for any ordered secondary structure. A marked change in the CD spectrum is observed changing from buffer to the lipid suspension. Concomitantly, the 1H‐nmr spectrum of bombesin, in a D2O lipid dispersion, shows the persistence of resonances due to exchangeable protons and in similar conditions the fluorescence intensity increases. We think therefore that these results strongly support the hypothesis that bombesin interacts with the lipid phase, assuming ordered secondary structure. Finally, the marked dependence of tryptophan fluorescence quantum efficiency and order parameter from the hormone concentration in the presence of lysolecithin but not in buffer leads to the conclusion that bombesin can associate into the lipid matrix.
Molecular dynamics of the α‐helical epitope of a novel synthetic lipopeptide foot‐and‐mouth disease virus vaccine
Authors: Krug Michael , Folkers Gerd , Haas Bernd , Hess Günter , Wiesmüller Karl‐Heinz , Freund Stefan , Jung Günther
Published: December 2004

A novel synthetic foot‐and‐mouth disease virus (FMDV) peptide vaccine consisting of a synthetic B‐cell and macrophage activator covalently linked to an amphiphilic α‐helical T‐cell epitope was developed. The low molecular weight vaccine of 3400 daltons is composed of virus VP1 antigenic determinant and the immunologically active lipotripeptide tripalmitoyl‐S‐glyceryl‐cysteinyl‐seryl‐serine (P3CSS) as built‐in adjuvant. The vaccine, tripalmitoyl‐S‐glyceryl‐cysteinyl‐seryl‐seryl‐FMDV‐VP1 (VP1 = serotype O1K 135–154) induces protection against homologous challenge and serotype‐specific virus neutralizing antibodies in guinea pigs after single administration without further adjuvants or carriers. A P3CSS conjugate with the FMDV‐VP1 segment 135–154 of strain O Wuppertal produced only poor cross‐protection against challenge with O1K virus.

The antigenic determinant VP1(135–154) is an amphiphilic α‐helix, as shown by CD. Molecular dynamics simulations (MDS) carried out using the highly homologous α‐helical alcohol dehydrogenase (ADH) segment H3 as starting conformation for VP1(138–149) suggest that the FMDV segment 138–149 may adopt α‐helical conformation during binding to its T‐cell receptor, and that the development of the system during MDS may be considered as the dissociation step of the complex.
Comparison of the conformations of cyclolinopeptide a in the solid state and in solution
Authors: Tancredi T. , Zanotti G. , Rossi F. , Benedetti E. , Pedone C. , Temussi P. A.
Published: December 2004

Cyclolinopeptide A, a cyclic nonapeptide isolated from linseed, has lately attracted large interest for its cytoprotective activity. The recent elucidation of its solid state structure has prompted us to undertake a detailed conformational analysis in solution. Room‐temperature 1H‐nmr spectra in several solvents (DMSO‐d6, DMSO‐d6/D2O/H2O, CD3OH, (CD3)2CDOH, CDCl3) all show very broad lines, indicating the presence of chemical exchange among several conformers. It proved possible to freeze a single conformational state in CDCl3 at 214 K. Unusual chemical shifts and nuclear Overhauser enhancements are consistent with the main features of the solid state structure.
CH ⃛ O hydrogen bonding in solutions of methylated nucleic acid base analogs as revealed by nmr
Authors: Bruskov V. I. , Bushuev V. N. , Okon M. S. , Shulyupina N. V. , Poltev V. I.
Published: December 2004

Formation and thermodynamic characteristics of CH ⃛ O hydrogen bonding of methylated uracils and caffeine have been studied by nmr along two lines.

The concentration and temperature dependencies of the PMR spectra of 1,3‐dimethyluracil (m2 1,3Ura), 1,3‐dimethylthymine (m2 1,3Thy), and 1,3,6‐trimethyluracil (m3 1,3,6Ura) in chloroform at high concentrations of base analogs indicated the self‐association of m2 1,3Ura and m2 1,3Thy via C(6)H ⃛ O hydrogen bonding and the competitive formation of CH ⃛ O bonds between carbonyl oxygens and chloroform. The intermolecular interaction energy and the arrangement of molecules in the local minima of various m2 1,3Ura dimers were calculated by the method of atom‐atom potentials. The deepest minimum for the m2 1,3Ura coplanar dimer corresponds to a C(6)H ⃛ O hydrogen‐bond formation.

At low concentration of m2 1,3Ura and caffeine in CCl4, C(6)H ⃛ O bonding for m2 1,3Ura and C(8)‐H ⃛ O bonding for caffeine with oxygens of dimethyl sulfoxide (DMSO) and acetone were observed. The association constants of these complexes were obtained at different temperatures. The enthalpies ΔH, of the m2 1,3Ura–DMSO, m2 1,3Ura–accetone, caffeine–DMSO, and caffeine–acetone complexes were −2 ⃛ 0.1 kcal/mol. The calculations showed that the deepest minimum of the caffeine‐acetone coplanar complex corresponds to C(8)H ⃛ O bonding with energy of −3.5 kcal/mol and that of the m2 1,3Ura‐acetone complexes corresponds to C(6)H ⃛ O bonding with energy of −3.4 kcal/mol. The approximate correction for the solvent effect provides good agreement of the experimental data with the calculations.
Proton nmr relaxation study of the dynamics of anthopleurin‐A in solution
Authors: Torda Andrew E. , Norton Raymond S.
Published: December 2004

Spin–spin and spin–lattice 1H‐nmr relaxation times of the sea anemone polypeptide anthopleurin‐A were measured at frequencies of 200, 300, 400, and 500 MHz. Relaxation times were fitted iteratively by least squares regression to the isotropic tumbling model, Woessner's model for anisotropic motion, and Lipari and Szabo's “model‐independent” model. Data for aromatic and aliphatic methine protons could not be fitted satisfactority using the isotropic model. Good fits were obtained, however, using the model‐independent approach,‐indicating that high‐frequency internal motions of the polypeptide backbone were significant. In addition, a range of τC values from 2.2 to 3.2 ns was obtained for various methine protons, suggesting that overall rotational reorientation of the molecule was anisotropic. Methyl group relaxation data were fitted satisfactorily by Woessner's model. Some assessment has been made of the effect of experimental errors on the quality of fit to the data, as well as of the contribution of experimental values at certain frequencies to definition of the spectral density function.
Two‐dimensional proton NMR studies on poly(VPGVG) and its cyclic conformational correlate, cyclo(VPGVG)3
Authors: Urry D. W. , Chang D. K. , Krishna N. R. , Huang D. H. , Trapane T. L. , Prasad K. U.
Published: December 2004

Two‐dimensional nuclear Overhauser enhancement (2D NOESY) data are reported for the polypentapeptide of elastin, poly(VPGVG), and the cyclopentadecapeptide, cyclo(VPGVG)3. In both, the repeating type II Pro2‐Gly3 β‐turn can be derived from the NOE data, providing confirmation of many previous studies. In addition, other through‐space connectivities are detailed that also compare favorably with previously determined crystal and solution structures for cyclo(VPGVG)3. Also, near identical data for the cyclopentadecapeptide and the polypentapeptide demonstrate the cyclic conformation‐linear (helical) conformational correlate relationship between the two molecules. The 2D NOESY experiment is seen to be an effective means of establishing the presence or absence of a conformational relationship between a cyclic repeating sequence and its higher molecular weight linear counterpart. This is an approach of substantial practical value when developing the conformation of sequential polypeptides and when attempting to identify the presence of the conformation of a repeating peptide sequence within a more complex primary structure.

Having established the basic conformational relationship between a cyclic conformation and its linear helical counterpart, cross peaks present in the linear helical structure that are not present in the cyclic conformational correlate can provide information on the interactions between adjacent turns of the helix. In this connection, a ValγCH3 ⟷ ProβCH2 interaction is reported that can be the basis for determining the number of pentamers per turn of helix once it is determined whether it is dominantly the Val1 or Val4γCH3 that is interacting with the Pro2βCH2.
Prediction of DNA structure from sequence: A build‐up technique
Authors: Hingerty Brian E. , Figueroa Samuel , Hayden Thomas L. , Broyde Suse
Published: December 2004

A build‐up technique has been devised that permits prediction of DNA structure form sequence. No experimental information is employed other than the force field parameters. This strategy for dealing with the multiple minimum problem requires a supercomputer to make the necessary global searches. The number of energy minimization trials that were made for each of the 16 deoxydinucleoside monophosphate conformational building blocks of DNA was 1944. As a test case, the minimum energy conformations of d(GpC) and d(CpG) to 5.5 kcal/mole were then combined to generate energy‐minimized structures for d(CpGpC). The number of trials that were made for d(CpGpC) was 3752. Minima for this single‐stranded trimer to 15 kcal/mole were then employed to search for minimum energy conformations of the duplex d(CpGpC) · d(GpCpG). The number of starting conformations that were utilized at this stage was 1514. The lowest energy duplex had a Z‐II‐DNA conformation, followed by a B‐DNA form at 1.2 kcal/mole. The A‐ and Z‐I‐forms as well as many novel Watson–Crick base‐paired structures were found at higher energy. Finally, energy‐minimized structures of d(CG)6 in Z‐II and B‐DNA conformations were computed using torsion angles from the analogous duplex trimer minima.
Orientational dynamics of T2 DNA during agarose gel electrophoresis: Influence of gel concentration and electric field strength
Authors: Åkerman Björn , Jonsson Mats , Nordén Bengt , Lalande Marc
Published: December 2004

The understanding, on a molecular level, of the mechanisms responsible for the improved separation in DNA gel electrophoresis when using modulated electric fields requires detailed information about conformational distribution and dynamics in the DNA/gel system. The orientational order due to electrophoretic migration (“electrophoretic orientation”) is an interesting piece of information in this context that can be obtained through linear dichroism spectroscopy [M. Jonsson, B. Åkerman, and B. Nordén, (1988) Biopolymers 27, 381–414]. The technique permits measurement of the orientation factor S of DNA (S = 1 corresponds to perfect orientation) within an electrophoretic zone in the gel during the electrophoresis. It is reported that the degree of orientation of T2 DNA [170 kilo base pairs (kpb)] is considerable (S = 0.17 in 1% agarose at 10 V/cm) compared to relatively modest orientations of short fragments found earlier (for 23‐kbp DNA, S = 0.03 in 1% agarose at 10 V/cm), showing that large DNA coils are substantially deformed during the migration. Growth and relaxation dynamics of the orientational order of the T2 DNA are also reported, as functions of gel concentration (0.3–2%), electric field strength (0–40 V/cm), and pulse characteristics.

The rise profile of the DNA orientation, when applying a constant field, is a nonmonotonic function that displays a pronounced overshoot, followed by a minor undershoot, before it reaches steady‐state orientation (after 12 s in 1% agarose, 9 V/cm). The orientational relaxation in absence of field shows a multiexponential decay in a time region of some 10 s, when most of the DNA anisotropy has disappeared. A surprising phenomenon is a memory over minutes of the DNA/gel system to previous pulses: with two consecutive rectangular pulses (of the same polarity), the orientational overshoot and undershoot as a response to the second pulse are significantly reduced compared to the first pulse. The time required to recover 90% of their amplitudes is typically 1200 s (1% agarose, 9 V/cm), which may be compared to the time required to relax 90% of the DNA orientation, which is only 6 s. The major part of the over‐ and undershoot recovery is thus a reorganization of a system in which DNA is already randomly oriented.

The different response amplitudes and relaxation times, including the amplitude and recovery time of the overshoot, of the orientational order of DNA in the electrophoretic gel have been studied as functions of gel concentration and field strength. The results are discussed against relevant theories of polymer dynamics.
On the cooperative binding of large ligands to a one‐dimensional homogeneous lattice: The generalized three‐state lattice model
Authors: Bujalowski Wlodzimierz , Lohman Timothy M. , Anderson Charles F.
Published: December 2004

We present the general secular equation for three‐state lattice models for the cooperative binding of large ligands to a one‐dimensional lattice. In addition, a closed‐form expression for the isotherm is also obtained, that can be used with all values of the cooperativity parameter ω(0 < ω < ∞) thus eliminating the need for multiple equations.
Protein complexation with DNA phosphates as a cause for DNA duplex destabilization: A thermodynamic model
Authors: Van Genderen Marcel H. P. , Buck Henk M.
Published: December 2004

Complexation of positively charged sites in a protein with the negative DNA phosphate groups shields the phosphate charges. This diminishes interstrand electrostatic repulsions, which stabilizes the duplex. When phosphate shidlding is present in one DNA strand only, the conformation of this strand changes due to a decrease of intrastrand phosphate–phosphate repulsions. This destabilizes the duplex since then the strands differ in conformation. A thermodynamic model is formulated to describe this stabilization/destabilization effect in terms of changed enthalpies and entropies of hybridization. It is found that protein complexation with one DNA strand can indeed lower the TM value of a duplex. The model is applied to the action of helicases (replication), RNA polymerases (transcription), and restriction endonucleases. Mechanisms with unilateral charge shielding are proposed for their duplex‐destabilizing properties.
Theory of cooperative transitions in protein molecules. II. Phase diagram for a protein molecule in solution
Authors: Finkelstein Alexey V. , Shakhnovich Evgeny I.
Published: December 2004

The thermodynamically stable states of denatured protein in solution are investigated. These states are distinguished from the native state by the absence of tight packing of side chains while the compactness of denatured protein may vary within a wide region. The following regimes are outlined:

the “wet” molten globule, i.e., the compact state with pores occupied by solvent;

the swollen globule (“wet,” of course); and

the coil.

The “dry” molten globule, when solvent does not penetrate inside the protein, is excluded for all experimental conditions. All the transitions within the denatured globule state are gradual while the denatured globule–coil phase transition is a second order one. The conditions of protein denaturation as well as conditions of transitions and crossovers within the denatured state are outlined.
Allosteric formulation of thermal transitions in macromolecules, including effects of ligand binding and oligomerization
Authors: Robert Charles H. , Colosimo Alfredo , Gill Stanley J.
Published: December 2004

We examine the effects of concentration (aggregation), buffers, and ligation, under conditions of either constant ligand activity or limited total amount of ligand, upon thermal denaturation of macromolecules as measured by scanning calorimetry. In doing so we utilize and extend an earlier generalized allosteric treatment [S. J. Gill, B. Richey, G. Bishop, and J. Wyman (1985) Biophys. Chem. 21, 1–14], applicable to ligand binding, enthalpy changes, and volume changes in a macromolecular system. The approach is contrasted with formulations based on the idea of structural domains. We show how information from the full scanning calorimetric curves can be utilized in arriving at and testing appropriate models for observed behavior in selected examples.
IR vibrational CD in alanyl tripeptide: Indication of a stable solution conformer
Authors: Lee O. , Roberts G. M. , Diem M.
Published: December 2004

Infrared vibrational CD (VCD) of a small peptide, L‐alanyl‐L‐alanyl‐L‐alanine (Ala3), and a peptide model, N‐acetyl‐L‐alanine‐N′‐methyl‐amide (AAMA), in the 1550–1750‐cm−1 region has been observed. The “coupled oscillator” VCD feature observed for Ala3 in the amide I region is interpreted in terms of a solution structure stabilized by the electrostatic interaction of the zwitterionic groups. No such interactions are possible in basic aqueous solution of Ala3 nor in AAMA in neutral solution. Thus, the coupled oscillator features are lost in the latter two cases, indicating the absence of a simple stabilized conformation.
A critical examination of the reaction of pyridoxal 5‐phosphate with human hemoglobin Ao
Authors: Marini Mario A. , Moore Gerald L. , Fishman Ronald M. , Jessee Robert , Medina Frank , Snell Shawn M. , Zegna Angelo I.
Published: December 2004

Pyridoxylated normal adult human hemoglobin (HbAo) has been prepared using both oxygenated and deoxygenated HbAo at pH 6.8 and room temperature without the addition of Tris to produce a mixture with P50 of 30 ± 2 torr and a Hill coefficient of 2.3 ± 0.1 similar to that of the isolated adult human hemoglobin from the red blood cell. Reduction of the pyridoxylated HbAo in the oxygen‐ligated form by sodium borohydride gives unacceptable levels of methemoglobin (i.e., > 10%). Exessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with < 5% methemoglobin. Both reducing agents give products with multiple components as shown by analytical chromatography. Radioautography on the isoelectric focusing gels of HbAo treated with 14C pyridoxal 5‐phosphate (PLP) shows three major bands for the cyanoborohydride‐reduced derivatives and a much more complex mixture of labeled molecules after the sodium borohydride reduction. When pyridoxylated hemoglobin is prepared without reduction, the preparation, after passage through a mixed‐bed resin, contains 0.4 equivalents of PLP per heme, and has a P50 of 30 ± 2 torr and an n value of 2.3 similar to the values found after reduction. Upon anion exchange resin chromatography, the PLP is removed, indicating that the reaction forms a reversible Schiff base. On standing at 4°C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P50. Methemoglobin increased to 3% during this incubation. After four months in the cold, the yield of a single chromatographic species is 70% with 20% methemoglobin. This fraction appears to be stable and can be passed through an anion exchange column without release of the PLP. Separation of the individual chains by reverse‐phase chromatography indicates that the addition of PLP to HbAo is directed solely to the β‐chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH4 is used for the reduction, radioactively labeled PLP is found on both the α‐ and β‐chains.
Differential scanning calorimetric study of the thermal unfolding of myosin rod, light meromyosin, and subfragment 2
Authors: Lopez‐Lacomba Jose L. , Guzman M. , Cortijo Manuel , Mateo Pedro L. , Aguirre Raul , Harvey Stephen C. , Cheung Herbert C.
Published: December 2004

The thermal unfolding of myosin rod, light meromyosin (LMM), and myosin subfragment 2 (S‐2) was studied by differential scanning calorimetry (DSC) over the pH range of 6.5–9.0 in 0.5M KCl and either 0.20M sodium phosphate or 0.15M sodium pyrophosphate. Two rod samples were examined: one was purified by Sephadex G‐200 without prior denaturation (native rod), and the other was purified by a cycle of denaturation‐renaturation followed by Sephacryl S‐200 chromatography (renatured rod). There were clearly distinguishable differences in the calorimetric behavior of these two samples. At pH 7.0 in phosphate the DSC curves of native rod were deconvoluted into six endothermic two‐state transitions with melting temperatures in the range of 46–67°C and a total enthalpy of 4346 kJ/mol. Under identical conditions the melting profile of LMM was resolved into five endothermic peaks with transition temperatures in the range of 45–66°C, and the thermal profile of long S‐2 was resolved into two endotherms, 46 and 57°C. Transition 4 observed with native rod was present in the deconvoluted DSC curve for long S‐2, but absent in the DSC curve for LMM. This transition was identified with the high‐temperature transition detected with long S‐2 and attributed to the melting of the coiled‐coil α‐helical segment of subfragment 2 (short S‐2). The low‐temperature transition of long S‐2 was attributed to the unfolding of the hinge region. The smallest transition temperatures observed for all three fragments were 45–46°C. It is suggested that the most unstable domain in rod (domain 1) responsible for the 46°C transition includes both the hinge region, which is the C‐terminal segment of long S‐2, and a short N‐terminal segment of LMM. This domain, accounting for 21% of the rod structure, contains the S‐2/LMM junction, and upon proteolytic cleavage yields the C‐terminal and N‐terminal ends of long S‐2 and LMM, respectively. Over the pH range of 6.5–7.5, the observed specific heat of denaturation of rod was approximately equal to the sum of the specific heats of LMM and S‐2. This finding provides an additional argument for the existence of independent domains in myosin rod.
Autoxidation of ascorbic acid catalyzed by a semisynthetic enzyme
Authors: Germanas Juris P. , Kaiser Emil T.
Published: December 2004

The semisyntehtic enzyme 6 was prepared by alkylation of the cysteine‐25 sulfhydryl group of papain with the bipyridine 5 and was shown to stoichiometrically bind copper ion; 7 catalyzed the autoxidation of ascorbic acid derivatives with saturation kinetics approximately 20‐fold faster than a model system using 3‐Cu(II).
Structure and conformation of the nitroxyl spin‐label ethyl 3‐(2,2,5,5‐tetramethylpyrrolinyl‐1‐oxyl)‐propen‐2‐oate determined by electron nuclear double resonance: Comparison with the structure of a spin‐label substrate of carboxypeptidase A
Authors: Mustafi Devkumar , Boisvert William E. , Makinen Marvin W.
Published: December 2004

The conformation of the nitroxyl spin‐label ethyl 3‐(2,2,5,5‐tetramethylpyrrolinyl‐1‐oxyl)‐propen‐2‐oate has been determined by electron nuclear double resonance (ENDOR) spectroscopy and computer‐based molecular modeling. From ENDOR spectra of the compound in frozen solution, we have assigned resonance absorption features for each class of protons, and we have identified their principal hyperfine coupling (hfc) components from analysis of the dependence of ENDOR spectra on the static laboratory magnetic field. The dipolar hfc components yielded estimates of the electron‐proton separations for each class of protons of the ethyl propenoyl moeity. Torsion angle search calculations were carried out to determine the conformational space compatible with hard‐sphere nonbonded constraints and with the ENDOR‐determined distance constraints. Molecular graphics analysis revealed that the propenoyl side chain of the spin‐label exhibits an extended trans conformation and that the ethyl moiety of the ester group deviates significantly from coplanarity with the carboxylate COO atoms. The conformation of this molecule is compared with that of an analogous compound O‐[3‐(2,2,5,5‐tetramethylpyrrolinyl‐1‐oxyl)‐propen‐2‐oyl]‐L‐β‐phenyllactate, which has been employed as a spectroscopic substrate probe of carboxypeptidase A (L. C. Kuo, J. M. Fukuyama, and M. W. Makinen (1983) Journal of Molecular Biology 163, 63–105). The rotamer conformation of the free spin‐label ester in solution, as determined in this study, and that of the enzyme‐bound spin‐labeled phenyllactate are compared. Differences in rotamer structure are discussed in terms of stereoelectronic principles that govern the pathway of substrate hydrolysis catalyzed by carboxypeptidase A.
Inactivation of the catalytic subunit of bovine cAMP‐dependent protein kinase by a peptide‐based affinity inactivator
Authors: Mobashery Shahriar , Doughty Michael , Kaiser E. T.
Published: December 2004

A peptide affinity inactivator, Ac‐Leu‐Arg‐Arg‐Ala‐(BrAc)Orn‐Leu‐Gly, was used as a tool to probe for active site residues in the catalytic subunit of bovine cAMP‐dependent protein kinase. The peptide inactivated the catalytic subunit in an active site‐directed and monophasic manner with a first‐order rate constant of 0.03 min−1 and a dissociation constant of 675 μM. Studies with radioactive peptide indicated that approximately one equivalent of peptide was incorporated into each protein molecule. Protein sequencing identified the modified residue as Cys‐199. A possible location for Cys‐199 within the active site is suggested.
Construction, expression, and preliminary characterization of chimeric class μ glutathione S‐transferases with altered catalytic properties
Authors: Zhang Pinghui , Armstrong Richard N.
Published: December 2004

An expression plasmid for isoenzyme 3–3 of rat liver glutathione S‐transferase has been constructed from the cDNA clone pGTA/C44 and the pAS expression vector pMG27NS, and used for the efficient production of the enzyme in the Escherichia coli strain M5219. The plasmid has also been manipulated, through theuse of synthetic linkers, to encode chimeric polypeptides in which short sequnces of the closely related isoenzyme 4–4 have been substituted into the N‐terminal and C‐terminal variable domains of isoenzyme 3–3. The chimeric polypeptides designated 493208, 320948, and 49320048 are expressed with varying degrees of efficiency in E. coli. The active dimeric holoenzymes 3‐3, (493208)2, (320948)2, and (49320048)2 can be isolated. The spectroscopic and kinetic properties of the chimeric enzymes are significantly different than the native enzyme.
Light scattering studies of the effect of Ca2+ on the structure of porcine submaxillary mucin
Authors: Varma B. K. , Demers A. , Jamieson A. M. , Blackwell J. , Jentoft Neil
Published: December 2004

The effects of calcium ions on the solution properties of porcine submaxillary mucin (PSM) have been investigated by static and dynamic light scattering. The weight average molecular weights of PSM fractions are unaffected by the addition of up to 0.5M CaCl2: these data are within experimental error of those for solutions in 0.1M NaCl. The distribution of relaxation frequencies derived from the dynamic data shows the existence of two distinct relaxation modes. The average relaxation times have been interpreted to yield the z‐average translational diffusion coefficient and the longest intramolecular relaxation time τ1. A plot of τ1 vs 〈1/Rh〉 is linear, and consistent with plots of such data recorded for PSM in 0.1M NaCl and 6M GdnHCl solutions. However, the τ1 values and the associated results for 〈R〉 in 0.5M CaCl2 are smaller than those determined in 0.1M NaCl. This suggests that the conformation of PSM in CaCl2 solution is more contracted than those in the other two solvents. These results are consistent with the compact packaging of mucin in the secretary granules that have elevated Ca2+ levels.
Temperature dependence of enthalpy changes for ethidium and propidium binding to DNA: Effect of alkylamine chains
Authors: Hopkins Harry P. , Fumero Juan , Wilson W. David
Published: December 2004

Calorimetric titrations have been performed on the binding of ethidium and propidium to calf thymus DNA at temperatures in the 15–60°C range. Enthalpy changes (ΔHB) derived from these experiments performed with the new Omega reaction calorimeter have a precision of ±0.10 kcal/mol or less at all temperatures. For ethidium (a monocation), ΔHB varies little with temperature, and the heat capacity change (ΔCP) for the binding reaction derived from these parameters is 10 cal/deg/mol. In contrast, ΔHB changes from −6.5 to −8.1 kcal/mol for DNA binding of propidium (a dication due to a charged amine group at the end of an alkyl chain attached to the phenanthridine ring nitrogen), and ΔCP is −57 cal/deg/mol. At 21°C a plot of ΔHB vs mole ratio is curved downward for propidium in the 0.08–0.25 range, whereas the same plot at 45°C is a straight line from 0.05 to 0.15 and sharply downward thereafter. Similar plots for ethidium follow the latter pattern between 25 and 50°C. These observations and our analyses of ΔHB and ΔSB are consistent with the hypothesis that the location in the DNA complex and the rotational motion of the alkylamine chain change substantially over the temperature range in this study. Only near 50°C is ΔHB equal for the binding of these two cations to DNA, and caution must be used in analyses of enthalpic effects when the temperature dependence for ΔHB is not available.
A unique binding cavity for divalent cations in the DNA–metal–chromomycin A3 complex
Authors: Itzhaki Laura , Weinberger Sarah , Livnah Nurit , Berman Elisha
Published: December 2004

Binding of chromomycin A3 (CRA) to calf thymas DNA was investigated in the presence of divalent cations using visible absorption and 1H‐nmr spectroscopies. An apparent equilibrium binding constant (∼ 1011M−1) was obtained from metal competition experiments using EDTA to remove the metal cation from the DNA–M–CRA (M: metal) complex. The large binding constant of the drug to DNA enabled us to obtain essentially complete complexation of CRA to the short homogeneous d(ATGCAT)2 duplex using stoichiometric amounts of the metal cation. Large induced chemical shifts were observed in the 1H‐nmr spectrum of the above complex using the paramagnetic Co2+ cation indicating that the metal occupies a unique binding site. Since no induced 1H‐nmr chemical shifts were observed for the drug–Co2+ mixture, it was concluded that no metal–drug complex is formed. In addition, it was found that bound CRA is negatively charged at physiological pH and binding to the DNA could be affected only by using metal cations whose ionic radius size (< 0.85 Å) and charge (2+) were simultaneously satisfied. Stringent metal cation selectivity for the DNA–M–CRA complex may be intimately connected with the antitumor selectivity of CRA, since different types of cells generally possess widely differing molar concentrations of metal cations.
Electrophoresis and movements of fluorescence pattern after photobleaching of large DNA fragments in agarose gels
Authors: Wu Chi , Wang Zhulun , Chu Benjamin
Published: December 2004

By combining electrophoresis with movements of fluorescence pattern after photobleaching (MOFPAP), which is abbreviated as EMOFPAP, we are able to measure electrophoretic mobilities of large DNA fragments in an agarose gel within a fairly short time scale (about 10 min or even down to 1 min). The new method represents a significant improvement in experiment time when compared with the time (typically on the order of hours) required to determine the average electrophoretic mobility of large DNA fragments in agarose gels by means of either conventional gel electrophoresis or pulsed‐field gel electrophoresis. In this article, we present the EMOFPAP experimental setup and consider optical conditions, including beam profile geometry and fluorescence pattern formation. A realistic formula that can explain the parameters governing the EMOFPAP method using our present optical setup has been derived. A comparison of results between experimental and computer simulation data is made, and an optimization of the EMOFPAP method is proposed.
Simulations of the B‐DNA molecular dynamics of d(CGCGAATTCGCG)2 and d(GCGCGCGCGC)2: An analysis of the role of initial geometry and a comparison of united and all‐atom models
Authors: Rao Shashidhar N. , Kollman Peter
Published: December 2004

Molecular dynamics simulations on the sequence d(CGCGAATTCGCG)2 have been carried out using both united atom and all‐atom representations, and starting the simulations both from a regular repeating B‐DNA structure and from the x‐ray single crystal B‐DNA structure. An all‐atom B‐DNA simulation on the sequence d(GCGCGCGCGC)2 has also been carried out, in order to compare it with a previous united atom simulation. The helix repeats, H‐bonding, sugar pucker profiles, and average torsional angles are all in the range observed in crystallographic and nmr studies for B‐DNA helices. In some of the sequences, there is a significant bend in the DNA helices. The individual helix repeats, with focus on 3′CpG5′ and 3′GpC5′ units, show the opposite helix repeat to that suggested by Calladine's rules.
X‐Ray studies on crystalline complexes involving amino acids and peptides. XVII. Chirality and molecular aggregation: The crystal structures of DL‐arginine DL‐glutamate monohydrate and DL‐arginine DL‐aspartate
Authors: Soman Jayashree , Vijayan M. , Ramakrishnan B. , Row T. N. Guru
Published: December 2004

DL‐Arginine DL‐glutamate monohydrate and DL‐arginine DL‐aspartate, the first DL‐DL amino acid–amino acid complexes to be prepared and x‐ray analyzed, crystallize in the space group P1 with a = 5.139(2), b = 10.620(1), c = 14.473(2) Å, α = 101.34(1)°, β = 94.08(2)°, γ = 91.38(2)° and a = 5.402(3), b = 9.933(3), c = 13.881(2) Å, α = 99.24(2)°, β = 99.73(3)°, γ = 97.28(3)°, respectively. The structures were solved using counter data and refined to R values of 0.050 and 0.077 for 1827 and 1739 observed reflections, respectively. The basic element of aggregation in both structures is an infinite chain made up of pairs of molecules. Each pair, consisting of a L‐ and a D‐isomer, is stabilized by two centrosymmetrically or nearly centrosymmetrically related hydrogen bonds involving the α‐amino and the α‐carboxylate groups. Adjacent pairs in the chain are then connected by specific guanidyl–carboxylate interactions. The infinite chains are interconnected through hydrogen bonds to form molecular sheets. The sheets are then stacked along the shortest cell translation. The interactions between sheets involve two head‐to‐tail sequences in the glutamate complex and one such sequence in the aspartate complex. However, unlike in the corresponding LL and DL complexes, head‐to‐tail sequences are not the central feature of molecular aggregation in the DL‐DL complexes. Indeed, fundamental differences exist among the aggregation patterns in the LL, the LD, and the DL‐DL complexes.
Melanin‐concentrating hormone: A structural and conformational study based on synthesis, biological activity, high‐field nmr, and molecular modeling techniques
Authors: Brown D. W. , Campbell M. M. , Kinsman R. G. , White P. D. , Moss C. A. , Osguthorpe D. J. , Paul P. K. C. , Baker B. I.
Published: December 2004

A series of Melanin‐concentrating hormone (MCH) fragments have been synthesized and their biological activities compared with the parent peptide. The substructural units, 5‐14 linear and 5‐14 cyclic, have been used as models for MCH—
magnified image
in 1H‐nmr conformational studies. Conformational features predicted by molecular dynamics analyses find support in the nmr experiments.