ALBERT

Notes: We have tested 21 different basis sets of synthetic DNA circular dichroism spectra and have slected one for use in spectral analyses of natural DNAs. This “standard” set consists of spectra of eight polymers: poly[d(A-A-T)·d(A-T-T)], poly[d(A-G-G)·d(C-C-T)], poly[d(A-T)·d(A-T)], poly[d(G-C)·d(G-C)], poly[d(A-G)·d(C-T)], poly[d(A-C)·d(G-T)], poly[d(A-T-C)·d(G-A-T)], and poly[d(A-G-C)·d(G-C-T)]. This basis set, applied according to the first-neighbor polymer procedure of Gray and Tinoco, allows a more uniformly accurate spectral analysis of six natural complex DNAs and eight (A+T)-rich satellite DNAs for base composition and first-neighbor frequencies than was previously possible. We find that spectra of poly[d(A)·d(T)] and/or poly[d(A-C-T-)·d(A-G-T)] are not generally required for good analysis results but we show in this and the following paper that these spectra are needed for the most accurate analyses of some satellite DNAs.

Notes: The interaction of counterions with a suitably long, charged oligomer appears susceptible to treatment in the context of polyelectrolyte theory by the introduction of an end-effect parameter that reflects the reduced association of counterions with the terminal regions of the oligo-ion. Use of a physically reasonable value for the end-effect parameter provides excellent agreement between theory and the experimental data of Elson, Scheffler, and Baldwin [J. Mol. Biol. 54, 401-415 (1970)] on the dependences of melting temperature on salt concentration and chain length for a series of hairpin helices formed by d(TA) oligomers. The differences in behavior expected for hairpin, dimer, and oligomer-polymer helices are discussed. The salt dependence of the end-joining equilibrium investigated for λ DNA by Wang and Davidson [Cold Spring Harbor Symp. Quant. Biol. 33, 409-415 (1968)] is treated as an oligomer-polymer interconversion. The dependence of equilibrium constant for this reaction on counterion concentration is in good agreement with that predicted by theory for an end-region totalling 24 nucleotides, the known length of the λ ends.

Notes: Solvent denaturation is developed along thermodynamic lines rather than from multiple-binding theory. Almost all the relations derivable from site-binding theory have their counterparts in the thermodynamic formulation showing that the details of binding models may be sufficient but are not necessary for the general description of solvent denaturation. Equations are derived for the effect of denaturant concentration on stability at constant temperature and on tm. It is recommended that the thermodynamic treatment be used instead of binding models unless stoichiometric interactions are demonstrable experimentally.

Notes: The optical properties of the 1,N6 - etheno derivative of ApA (εApεA) have been studied. Absorbance and CD measurements suggest that (1) neutral salts tend to unstack this molecule and (2) the stacking interaction is weaker than in ApA. εApεA is found to be quenched strongly with respect to the monomer. (εAMP); this quenching is solvent dependent (1M NaCl 〉 5M NaClo4〉40%glycerol) and increases with the ratio of temperature to viscosity (T/η) in each case. Fluorescence lifetime measurements also reveal a temperature- and solvent-dependent decay which is nonlinear on a semilog plot. In the presence of 95% glycerol, this decay return to linearity. These data have been considered from two points of view: (1) two-state pictures which are based on thermodynamic least-squares fit to quatum yield and CD curves, together with two exponential fits to the decay curves and (2) a dynamical model in which relatives fluorophore motion leads to deexcitation via intramolecular collision. A simple model of type (2) gives qualitative agreement with the observed behavior.

Notes: The native antibiotic polymyxin B, carrying five positive charges, displaces the monoacetylated on from its adsorbed state at the interface between negatively charged lipid layers ans water. A simulation model for asdorption, governed by electrostatic forces, is presented. The model adequently representes the competitive adsorption kinetics and equilibria.

Notes: In order to study the role of D-amino acid residues in keeping the stable β-sheet conformation and in the antimicrobial activity of gramicidin S (GS), the four analogs of GS containing D-Ala, L-Ala, Gly, and Aib (α-aminoisobutyric acid) in place of D-Phe were synthesized. D-Ala-and Gly-containing analogs showed antimicrobial activity, while those containing L-Ala and Aib showed no activity. Conformation of these analogs and their derivatives were studied by comparison of ORD and CD spectra and by slective methylation method. It is concluded that the biologically active analogs have β-sheet conformation while inactive analogs have a much different conformation from that of GS. This indicates that D-Ala-Pro and Gly-Pro sequences favor taking a β-bend form but L-Ala-Pro and Aib-Pro sequences do not because the presence of L-side methyl group on the α-carbon atom of LAla and Aib residues destabilizes the β-bend form. This would explain why the inactive analogs which take a different conformation from that of the active ones result in the loss of activity.

Notes: Earlier studies by the authors showed that the collagen fibrils in rat-tail tendon have a bi-modal distribution of fibril diameters from a time shortly after birth through to the onset of maturity at about 3-4 months. Present work has extended those observations for rats up to the age of 2 years. Histograms of the fibril diameter distributions for mature tail tendon and direct electron microscope observations show that the fibrils break down as the tendon ages. Further work on the constant diameter subfibrils of diameter 140 Å described previously, has confirmed that these are part of the elastic fibers present in tendon at all ages. It has been shown that there is relatively little variation in the collagen fibril diameter distribution as a function of the position of the specimen in the tail, and as the measured percentage of the area taken by the collagen fibrils present at any particular point. Estimation of the fibrillar collagen content of rat-tail tendon as a function of age indicates that it increases steadily from birth and reaches a maximum at the onset of maturity, beyond which the fibrillar collagen content appears to remain constant.

Notes: We have made quantitative estimates of some of the energetic factors to be considered in packaging of double-stranded DNA in virus particles. Numerical calculations were made using parameters appropriate for T4 bacteriophage. The unfavorable factors, and the Gibbs free energies per mole virus at 20°C associated with them, are bending, 1.5 × 103 kcal/mol; conformational restriction upon condensation, 5.1 × 102 kcal/mol; polyelectrolyte repulsion, 2.1 × 105kcal/mol; and melting or kinking, 6.9 × 103 kcal/mol. These must be counterbalanced in the assembled phage by noncovalent bonding interactions between protein subunits in the phage-head shell; by interactions between the DNA and polyvalent cations, especially putrescine and spermidine; nad perhaps by repulsive excluded volume and electrostatic interaction between the DNA and acidic polypeptides. Indeed, a rough estimate of the standard free energey of interaction between T4 DNA and the putrescine and spermidine contained in the head is --2.1 × 105 kcal/mol. In the absence of the other two sources of stabilization, each head protein subunit must contribute about 210 kcal/mol of binding energy.

Notes: A theoretical basis for thin-film dialysis involving binding between a ligand and nondialyzing species is presented. A general differential equation that applies to the case of equivalent, noninteracting sites is derived relating [A]F, [A]T, [P]T, and K. Numerical solutions to this equation are used to develop a series of escape curves corresponding to specific values of the parameters [P]T, [A]i, K, and k0. A general method for determining an equilibrium binding constant from thin-film dialysis data is given. A comparison of thin-film dialysis results predicted by this theory with literature data shows essential agreement.

Notes: The normal and vacuum uv spectra of cytosine, 5-azacytosine, and 6-azacytosine thin layers were determined. The spectra were resolved into a series of Gaussian bands and the experimental band positions and oscillator strengths were compared with the calculated values. The calculations were carried out with the PPP-SCF-CI method. The agreement of the calculations with those of others and with the experiment is discussed.

Notes: To obtain information on the solvent-solute interactions in the dinucleoside monophosphates pertaining to the dimeric subunits of RNA and DNA, we have computed the accessibility of a water molecule to the oxygen atoms of the subunits following the method of Lee and Richards [J. Mol. Biol. 55, 379-400 (1971)]. The solute molecules (dimeric unit) is represented by a set of interlocking spheres of appropriate van der Waals radii assigned to each atom, a solvent (water) molecule is rolled along the envelope of the van der Waals surface, and the total surface accessible to the solvent molecule - and hence the solvent accessibility of various atoms of the solute molecule for different conformations - are computed. From the calculated atomic accessibilities, solvation maps in the (ω′,ω) space have been constructed, keeping ψ at 60°, 180°, and -60°. The C(3′)-endo sugar system in the case of DNA subunit have been considered. The solvation maps describing the solvatability of single and groups of atoms give significant information on the backbone conformational domains that are preferred for solvent interaction, thus adding knowledge to the relative stability of the various possible conformations. The B-DNA-type conformer exposes three polar atoms - namely, PO1, O(3′), and O(1′) - to external solvent, whereas the A-DNA- and C-DNA-type conformers expose only one polar atom - O(3′) and O(1′), respectively - to the solvent. The O(2′) atom of the furanose ring system in the RNA subunit could give added stability via solvent association or interunit hydrogen bonding with or without a bridging water. The superposition solvation maps describing the accessibility of a group of polar atoms help to interpret a good number of phosphodiester conformations observed in a energetically less favored conformational domains in the tRNAPhe crystal. Another intresting fact that results from this study is the prediction that the trans oriented of ω is the most favorable conformations of random-coil polynucleotides in solution.

Notes: The Conformational properties of synthetic S-peptide analogs, in which the residues in the N-terminal sequence 1-6 were progressively deleted or replaced with amino acids of lower helical propensity, were studied by CD. Increasing the concentration of guanidine hydrochloride and decreasing the temperature were found to produce progressive destruction of ordered conformations, in the parallel with the increasing solubility of the peptide unit, while increasing the concentration of trifluoroethanol and decreasing the temperature produced the opposite effect. The maximum helicity determined in the these sets of experiments is found equal to or greater than that determined in the formation of the ribonuclease S′ complexes. With some peptides the maximum value of predicted helical conformation is reached, and the tendency of tertiary structure to reduce the maximum possible helicity is evident. We discuss the validity of the procedure by which conformational information, drawn from measurements in helicogenic solvents, is related to the state in native protein.

Notes: A thermal perturbation curve (TPC) is defined to be the derivative of the fractional degree of saturation, f, with respect to temperature, considered as a function of the natural logarithm of free ligand concentration, y. The theoretical framework for the use of such curves in the thermodynamic analysis of ligand binding to macromolecules is presented. The thermal perturbation curve either provides or complements the information obtained from the derivative binding isotherm ∂f/∂y. For a single set of identical and independent sites the TPC is identical to the derivative binding isotherm. Analysis of such a curve directly yield ΔH0 and ΔG0 for the binding reaction. In actual experimental work, however, the TPC can only be approximated because of “self-buffering” effects relations between the parameter of the approximate curve and the thermodynamic quantities have been developed. This technique is applied to the proflavin-α-chymotryspin system to demonstrate its usefulness. The general features of thermal perturbation curves for cases of multiple sets of independent sites and cooperatively interacting sites have also been developed. The analysis of thermal perturbation curves in combination with other methods should provide a more powerful approach to the characterization of ligand-macromolecule interactions.

Notes: The conformation of three sequential copolypeptides, poly(L-tyrosyl-L-lysine), poly(L-tyrosyl-L-lysyl-L-lysine), and poly[L-tyrosyl-(L-lysyl)2-L-lysine] have been studied by a variety of techniques, including CD, ir spectroscopy, analytical ultracentrifugation, and x-ray diffraction. Depending upon the pH and sovent composition, poly(L-tyrosyl-Llysyl-L-lysine) and poly [L-tyrosyl-(Llysyl)2-L-lysine] can adopt either the α-helical or random-coil conformation, while poly(L-tyrosyl-L-lysine) forms either inter- or intramolecular β-structures.

Notes: Molecular motion in solid poly(L-alanine), Poly(L-leucine), poly(L-valine), and polyglycine has been investigated through measurement of the portion spin-lattice relaxation time at 30 and 60 MHz between 110 and 350°K. Rapid random reoriention of sied-chain methyl groups provides the dominent source of relaxation in the first three; activation energies are 10.5 ± 1 1, 8.5 ± 1 kJ/mol, respectively, significantly lower than in the monomeric crystals. Relaxation times in poyglycine are two orders of magnitude longer than in the monomeric crystals. Relaxation times in polyglycine, significantly lower than in the monomeric crystals. Relaxation times in polyglycine are two orders of magnitude longer and are attributed mainly to segmental motions of the polymer chains. Evidence of nonexponential recovery of nuclear magnetization was encountered in the first three homopolyamino acids but not in polylycine, and was attributed to the correlated time to characterize these motions gave quite good agreement with the data; some improvement was obtained for two polymers using a Cole-Davidson distribution of correlation times. For biopolymers using a Cole-Davidson distribution of correlation times. For biopolymers generally it is concluded that rapid methyl group reorientation is a common dynamical feature and an important source of nuclear magnetic relaxation.

Notes: The karl Fischer reagent method has been used to study the drying of whale skeletal muscle myoglobin. Drying curves for metmyglobin were obtained by mechanical pumping (moderate vacuum) and diffusion pumping (high vacum) at 20, 10, 0, -10, and -20°C. Strongly held water is present in about the same amounts as were previously observed with lysozyme. Nearly all of this strongly held water is still present during the drying of acid denatured metmyoglobin. This suggest that a core structure containing strongly held water is still present at pH 4.0. Comparison of the drying of metmyoglobin, deoxymyoglobin, and oxymyoglobin indicates that the iron-coordinated water of methyoglobin is the second most strongly held water of the molocule.

Notes: Mixtures of bacteriophage T4D particles with up to six tail fibres attached were seprated and analyzed in a quasielastic light scattering-band electrophoresis apparatus. The electrophoretic mobilities and diffusion coefficients of the seprated bands were determined during the same experiment. Species differing in mobility by 0.05 × 10-4 cm2/V sec were resolved. Henry's electropheresis theory for spheres indicates that each tail fiber contributes about 140 effective positive charges to the phage structure. Estimate of the charge using the permanent dipole moment and the electrophorectic mobility (Bontje et. al. (1977) Biopolymers 16, 551-572) gives an effective charge of + 230 to -250 per tail fiber. The charge distribution on the fiberless particles was estimated to be -3380 on the head and -290 on the tail if the dipole moment was assumed positive and -1400 on the head and -1000 on the tail for a negative dipole. Tail-fiber attachement does not proceed to completion in our invitro system, as substantial distributions as a function of tail-fiber input indicates the reaction is a random, noncooperative process.

Notes: The contribution of the 1-6 N-terminal sequence to the conformational properties of the S-peptide (the 1-20 sequence of ribonuclease A) was assessed by determining in the ribonuclease S′ system the helical content and the binding capability of synthetic [Orn10]-S-peptide analogs, in which lysine1, glutamic2 and threonine3 were progressively deleted, alanine4 and alanine5were alternatively replaced by serine, and alanine6 was substituted by serine or proline. Both the deletion of the three N-terminal residues and the alanine6/proline replacement produces the loss of the helical structure up to lysine7. No or minor effects are found in all other cases. From the comparison of the binding data, the energy for the conformational stabilization of the N-terminal region was calculated to amount to 1.4 kcal/mol. The results are discussed in comparison with the known x-ray data of the enzyme, with some predictive rules of secondary structure which were applied to this region and with the known phylogenetic variance of the residues in this region.

Notes: In the Polymerization of phenylalanine N-carboxyanhydride (NCA) in No2Oh initiated by MeNHBzl, L-,D-, and DL-NCA As were polymerized at the same rate, and no stereoselectivity was observed. When the same experiment was carried out in HCONEt2, however, L- and D-NCA were both polymerized at a rate which was about twice as large as that of DL-NCA. In this case, the polymerization is stereoselective, ascribable to a preferable reaction between the optical enantiomorphs of the terminal residue of the growing chain and the NCA of the same chirality. On the other hand, the polymerization initiated by SarNMe2 and MeNH(CH2)2CONMe2 were stereoselective in NO2Ph and HCONEt2, but they were not stereoselective in m-(MeO)2Ph. These findings indicate that the polymerizations initiated by a strong base in highly dipolar solvents are stereoselective. Apparently, the reaction between a chiral, cyclic terminal of growing chain and a chiral, cyclic activated NCA in the activated-NCA mechanism is highly stereoselective. In addition, from a kinetic investigation on on the copolymerization between L- and D-NCAs, the penultimate chiral centers were also suggested to contribute to the stereoselection. Stereoselection by the α-helical conformation of the growing chain and by a chiral, linear terminal amine have been considered so far, and the contribution from the present type of stereoselection must have been overlooked.

Notes: Transient electric birefringence measurements of the bacteriophages T2L0 and T6 were performed under such conditions that the tail fibers are extended. The data obtained are compared to previously reported data for T4B. For all T-even phages the degree of extension of the tail fibers is a function of pH, ionic strength, and temperature. For T4B, much higher ionic strengths are needed than for T2L0 and T6 to accomplish complete tail-fiber extension. The rotational diffusion coefficients of the phages with fully extended fibers are equal to 120 ± 3 sec-1, 132 ± 5 sec-1, 157 ± 4 sec-1 for T2L0, T4B, and T6, respectively. The respective optical anistropies are - (2.66 ± 0.05) × 10-4, and - (3.07 ± 0.15) × 10-4. The differences in the rotational diffusion coefficient and optical anisotropy arise because the conformation of the fully extended tail fibers is different for the three phages. The tail fibers of T2L0 project further into the solution (away from the head) than do those of T4B and T6. The apparent permanent dipole moments of T2L0 and T6 decrease with increasing ionic strength. This decrease is caused by the screening of the surface charges on the phage body by the counter-ions in the solution. The biological relevance of this decrease is illustrated by the fact that the adsorption rate of T6 phages to E. coli B bacteria shows a similar dependence of ionic strength. Evidence is pressented that the tail fibers may move more or less independently of the phage body when an electric field is applied to the suspension.

Notes: A molecular theory of protein secondary structure is presented that takes account of both local interactions inside each chain region and long-range interactions between different regions, incorporating all these interactions in a single Ising-like model. Local interactions are evaluated from the stereochemical theory describing the relative stabilities of α- and β-structures for different residues in synthetic polypeptides, while long-range effects are approximated by the interaction of each chain region with the averaged hydrophobic template. Based on this theory, an algorithm of protein secondary structure prediction is proposed and examples are given of “blind” predictions made before the x-ray structural data became available.

Notes: To increase our understanding of peptide-water interactions, we are simulating the behavior of water molecules in the intermolecular channels of [Phe4Val6]antamanide dododecahydrate crystals. There is good overall agreement between the positions predicted using two alternative potential functions and those that have been observed by x-ray diffraction. Detailed differences between the predictions for the two potential functions are discussed.

Notes: Experimental measurements of disulfide bond stability at various stages of protein folding are considered in terms of the effective concentrations of the thiol groups relative to each other; values of up to 107M are observed, so that intramolecular interactions within the interior of a protein are much more stable, and provide greater stability to the folded conformation, than those on the surface or in a flexible segment. Intramolecular interactions can have substantially lower free energies than intermolecular, for solely entropic reasons; this implies that polar interactions, such as hydrogen bonds and salt bridges, can provide net stabilization to a folded conformation, in spite of the unfolded protein having intermolecular interactions with the solvent. These considerations can account for the lower free energy and enthalpy of the folded state and are useful for considering protein flexibility.

Notes: Near- and far-uv CD spectra of microtubule protein preparations have been examined to study the possible role of protein conformation in relation to the kinetics of the self-assembly of these proteins into microtubules in vitro. Although tubulin can form conformations with high helical content under apolar solution conditions, this transformation is apparently not involved in self-assembly. There is no major perturbation of tubulin near-uv CD by reagents and solution conditions favoring assembly. Thus, in these preparations, tubulin, as dimer and as oligomer with MAPs, is effectively in the conformation in which it undergoes self-assembly. This conclusion is consistent with a hybrid model of assembly of microtubule protein involving direct incorporation of oligomeric species as an alternative to the condensation polymerization of tubulin dimer as the exclusive assembly mechanism.

Notes: The ligand-modulated kinetics of the autoproteolysis of thermolysin and the high-molecular-weight products of the reaction provide evidence for the conclusion that separation of the two structural domains is most probably the first step on the unfolding pathway of the protein under native conditions.

Notes: The in vivo pathway of folding and subunit assembly of a trimeric bacteriophage protein has been studied by characterizing precursors to the native protein and by analyzing temperature-sensitive mutations that kinetically block the pathway. The native trimer is formed via an intermediate composed of three partially folded chains, the protrimer. At 39°C, temperature-sensitive mutations prevent the formation of both the native trimer and the protrimer, possibly by destabilizing earlier intermediates. However, the mutations do not affect the stability of the native protein, formed at 30°C. Thus, these mutations identify amino acid residues involved in interactions that determine the folding pathway.

Notes: We studied the kinetics of O2 release by oxyhemoglobin caused by sodium dithionite, in the presence and in the absence of organic cosolvents (monohydric alcohols and formamide) at 10°C. This study was performed by using standard stopped-flow techniques coupled with microprocessor-based data acquisition. We have fitted the experimental data to a mathematical expression obtained on the basis of a two-state model that takes into account the kinetic heterogeneity between α- and β-chains and the presence of αβ-dimers in oxyhemoglobin solutions. Results indicate that the cosolvents mainly affect the allosteric parameter L, i.e., the T ⇄ R conformational equilibrium of hemoglobin, leaving the intrinsic deoxygenation rates of both R and T states almost unaltered. The L values obtained in the present work are in excellent agreement with analogous values previously estimated from oxygen equilibrium measurements.

Notes: Porcine submaxillary mucin (PSM) is a glycoprotein composed of a protein core and frequent, short oligosaccharide side chains. We report static and dynamic light scattering experiments and intrinsic viscosities for PSM in aqueous solvent systems. In 0.1M NaCl solution, the data suggest PSM exists as large, internally branched, highly hydrated, polydisperse aggregates that slowly dissociate to give a stable species of weight-average molecular weight (Mw) 7.4 × 106. In 6M GdnHCl solution, the noncovalent bonds between PSM molecules are broken, giving a highly elongated molecule of Mw = 2.0 × 106. The irreversible nature of this dissociation suggests that the forces that stabilize the native aggregates of PSM in 0.1M NaCl are specific in nature. On reduction of PSM with mercaptoethanol, the polydispersity decreases and Mw also decreases to 9 × 105. A discrete change is observed in the solution properties of PSM in 0.1M NaCl at a concentration of 2mg/mL, manifested by a sudden decrease in the translational diffusion coefficient, an increase in viscosity number, and a decrease in slope of the osmotic compressibility. We tentatively propose that a weak and reversible secondary association process occurs at this concentration, although a purely hydrodynamic interaction cannot be ruled out.

Notes: The uv linear dichroism of calf thylus DNA has been studied at different degrees of orientation both in flow-oriented ethanol-water solutions and in a stretched aqueous host of poly(vinyl alcohol) (PVA). The reduced dichroism (LDR) curves in the region 250-290 nm for DNA in PVA films at 75 and 100% relative humidity (r.h.) are in fair agreement with the curves calculated for the A- and B-forms of DNA, based on the fiber structures and the π-π* transitions of the free bases. This suggests that DNA adopts its A and B conformations in PVA at 75 and 100% r.h. In ethanol, on the other hand, a deviation from the A-form spectrum shows that the conformation of DNA in the solution can differ from the fiber structure. At shorter wavelenghts, a positive contribution to LDR is explained in terms of an out-of-plane polarized n-π* transition.

Notes: A vibrational analysis has been performed for a double-helix-single-strand junction. A Green's function technique has been used in treating the junction as a defect on an otherwise perfect system of infinite chain homopolymers. We calculate that the hydrogen-bond stretching at the junction is amplified by a factor of two relative to the interior of the double helix, B poly(dG)-poly(dC). Breathing modes localized near the junction have also been predicted at 77 and 94 wave numbers. The calculated results are shown to be consistent with predictions from recent nmr studies.

Notes: Nmr studies of the protected and free tetrapeptide Gly-Pro-Gly-Gly were carried out in β-turn-supporting solvents, that is, in CDCl3 for Z-Gly-Pro-Gly-Gly-OMe and in Me2SO-d6 for H-Gly-Pro-Gly-Gly-OH. Comparisons with specifically α-deuterated analogs gave complete assignments of the NH and methylene regions. Analysis of chemical shifts, coupling constants, and the temperature dependence of chemical shifts show that the peptide adopts a type II β-turn conformation. This turn is stabilized for the protected tetrapeptide by two hydrogen bonds between (i) C=O (Gly1) and NH(Gly4), and (ii) urethane function NH and methyl ester C=O.

Notes: Ab initio minimal and split-valence basis set calculations have been performed on compounds that are involved in retro-inverso modifications, i.e., gem-diaminoalkyl and malonyl structures. These calculations are compared with empirical force field calculations and the minor differences discussed. All calculations agree that the preferred helical conformation of the isolated gem-diaminoalkyl and malonyl derivatives of residues found in the retro-inverso modified peptides is 5-8 kcal/mol lower than the Ceq7 conformation preferred by the isolated peptide residues. Population analysis and contour plots of the charge distribution are used to help explain the differences between the model compounds.

Notes: SCF-LCAO-MO ab initio calculations were carried out for the interaction between a charged amino acid and a water molecule. The results obtained were fitted by an analytical potential function of the atom-atom type, and the corresponding potential surfaces were examined by means of orientationally optimized isoenergy contour maps. Monte Carlo simulations were also carried out on a few selected solute-water clusters at T = 300 K, in order to obtain insight into the solvation structure.

Notes: An improved cube method has been developed for calculating the intensity of diffuse x-ray scattering of macromolecules in solution using a certain set of their atomic coordinates. The technique is based on the ideas of B. Lee and F. M. Richards [(1971) J. Mol. Biol. 55, 374-400] and Richards [(1977) Annu. Rev. Biophys. Bioeng. 6, 151-176] on the possibility of estimating the molecular and accessible surface of a particle by “rolling” a sphere, simulating a water molecule, on its molecular surface. It is shown that this technique is more advantageous than earlier versions of the cube methods. The improved technique for calculating scattering curves was utilized for several globular proteins, and for the first time, reliable scattering curves were obtained for protein-“bound” water complexes. In the case of globular proteins and tRNA, this technique has permitted a strict evaluation of their accessible surfaces, their volumes, and, apparently for the first time, their complete molecular surfaces.

Notes: An extended retro-inverso modification was introduced at the central six residues of the somatostatin molecule, the region of internal enzymatic degradations. The synthesis of the analog [Ala4,g-Phe6-r-D-Phe7-r-D-Trp8-r-D-Lys9-r-D-Thr10-m-R,S-Phe11]-somatostatin required a unique strategy accommodating the unusual structure. Side-chain protection based on the t-butyl group in combination with Fmoc and Nps α-amino protection was employed. The key component containing the gem-diaminoalkyl residue was generated by an iodobenzene bistrifluoroacetate-mediated reaction. The separation of diastereomers of the cyclic tetradecapeptide in highly pure form was accomplished by high-performance liguid chromatography on a semipreparative scale. The analogs exhibited very low potency in the growth hormone inhibition test in vitro. This is interpreted as the consequence of the complex structural changes created by the extended retro-inverso modification.

Notes: The nature of internal and overall motions in native (double-stranded) and denatured (single-stranded) DNA fragments 120-160 base pairs (bp) long is examined by molecular-dynamics modeling using 13C-nmr spin-relaxation data obtained over the frequency range of 37-125 MHz. The broad range of 13C frequencies is required to differentiate among various models. Relatively narrow linewidths, large nuclear Overhauser enhancements (NOEs), and short T1 values all vary significantly with frequency and indicate the presence of rapid, restricted internal motions on the nanosecond time scale. For double-stranded DNA monomer fragments (147 bp, 24 Å diam at 32°C), the overall motion is that of an axially symmetric cylinder (τx = ∼10-6 s;τZ = ∼1.8 × 10-8s), which is in good agreement with values calculated from hydrodynamic theory (τx = ∼1.8 × 10-6 s; τZ = ∼2.7 × 10-8 s). The DNA internal motion can be modeled as restricted amplitude internal diffusion of individual C—H vectors of deoxyribose methine carbons C1′, C3′, and C4′, either with conic boundary conditions (τw = ∼4 × 10-9 s, θcone = ∼21°) or as a bistable jump (τA = τB = ∼2 × 10-9 s, θ = ∼15°). We discuss the critical role in molecular-dynamics modeling played by the angle (β) that individual C—H vectors make with the long axis of the DNA helix. Heat denaturation brings about increases in both the rate and amplitude of the internal motion (described by the wobble model with τW = ∼0.2 × 10-9 s, θcone = ∼50°), and overall motion is affected by becoming essentially isotropic (τx = τZ = ∼5 × 10-8 s) for the single-stranded molecules. Since 13C-nmr data obtained at various DNA concentrations for C2′ of the deoxyribose ring is not described well by the above models, a new model incorporating an additional internal motion is proposed to take into account the rapid, extensive, and weakly coupled motion of C2′.

Notes: The interaction of methyl orange with poly(L-lysine) was studied kinetically by the stopped-flow technique with CD detection, as well as by static CD titration experiments. In the static experiments, the differences observed in the polymer-to-dye ratio dependences of the CD spectra and absorption spectra suggested at least two kinds of bound states of the methyl orange attached to the polymer. The kinetic experiments using the stopped-flow apparatus, however, revealed four distinct reaction processes. The reaction mechanism was elucidated from the concentration dependence of the time constant for each process as follows: the first process was attributed to the bimolecular binding step of methyl orange to the side chain of poly(L-lysine), the second and third process were ascribed to the intramolecular reaction of the polymer-dye complex, and the fourth process was found to be the intermolecular aggregation of the polymer-dye complex. The origin of the stacking of methyl orange on poly(L-lysine) is discussed on the basis of the characteristics of signal amplitudes obtained from the kinetic experiments for these processes.

Notes: Novel devices for the spectroscopic and chromatographic analysis of the denaturation curves of the protein are described. A multidimensional spectroscopic measuring system makes it possible to carry out simultaneous and continuous acquisition of a set of data of different spectroscopic dimensions at several wavelengths in the course of increasing or decreasing denaturational perturbation. GuHCl-gradient chromatography can provide information about the progressive change of the protein volume in the course of increasing GuHCl concentration. Thus, denaturation curves with a high data-point density can be obtained. The data-storing function by a magnetic disk memory provides enough precision for a rigorous investigation of the correlation among the curves that probe different aspects of denaturation. The GuHCl denaturation of RNase A, cytochrome c, and pepsinogen are studied to demonstrate the high performance of these devices. Three types of transitions are found in these three proteins and the multiphasic nature of the transitions is clearly detected in the last two proteins.

Notes: A sufficiently long semiflexible filamentous macromolecule is theroretically expected to exhibit three different domains of behavior of its apparent diffusion coefficient Dapp(K) as a function of scattering vector K: (1) the small wave vector limit, where Dapp(K) = D0 is the translational diffusion coefficient of the center-of-mass; (2) the universal K3 region, where Dapp(K) = (kBT/6πη)K is a universal function of K independent of any property of the molecule itself; (c) the plateau region at large K2, where Dapp(K) approaches either a plateau, or gradually sloping quasiplateau, characteristic of local (elastic) rigid-body motions of the filament. The existence of each of these different domains has now been established experimentally for at least some polymers. The boundaries of the universal K3 region and the plateau region are determined theoretically here using precise quantitative criteria for universal or plateau behavior of Dapp(K) for a Rouse-Zimm model containing N + 1 subchains with rms subchain extension b. Allowing a maximum of 13% nonuniversal behavior, the domain of the universal K3 region is given by K2R2G = K2Nb2/6 ≥ 7 and K2b2 ≤ 0.54. Allowing as much as 10% nonplateau behavior, the boundary for onset of plateau behavior is K2b2 = 18.3. Dapp(K) is at least 50% nonuniversal when K2b2/6 = 6 ln 3. Extension of these results to DNA is examined theoretically, and good agreement of the pertinent predictions with published experimental data is demonstrated.It is concluded that no truly universal K3 region exists for DNA with Mr ≤ 107 and persistence length a ≥ 450 Å, although marginally (≤17% nonuniversal) universal behavior, is exhibited in a very narrow domain 0.64 × 1010 ≤ K2 ≤ 0.84 × 1010 cm-2 for φ29 DNA (Mr = 11.5 × 106). More than 50% of Dapp(K) is governed by local (elastic) rigid-body motions when K2 = 5.23 × 1010 cm-2. The existence of a very wide region of nonuniversal apparent K3 behavior extending up to very large K2, far into the plateau region, is demonstrated in a plot of Dapp(K)/K vs K2 for the Rouse-Zimm model. This is shown to stem in part from visual artifacts of plotting Dapp(K)/K vs K2, even for rigid species.

Notes: The rotational relaxation tiem τ3 of DNA molecules (Mw ≃ 5 × 106) in solution has been determined by the transient electric birefringence method. The analysis of the birefringence decay makes it possible to study only the higher-molecular-weight fraction, the molecules being considered as rigid elongated particles in a short time scale. A marked concentration dependence of the relaxation time has been observed for DNA in low ionic strengths. Above a critical concentration c*, τ3 increases with the DNA concentration, c. The value of c* increases with the ionic strength. For 10-3 ionic strength (with NaCl), c* is about 10 μg/mL; then we observe the same strong concentration dependence of rotational relaxation times as recently reported for rodlike M-13 viruses [Maguire, J. F., McTague, J. P. & Rondelez, F. (1980) Phys. Rev. Lett. 45, 1891-1894]. These results may be discussed in terms of the Doi-Edwards theory for rotational relaxation time of rigid macromolecules [Doi, M. (1975) J. Phys. 36, 607-611; Doi, M. & Edwards, S. F. (1978) J. Chem. Soc. Faraday Trans. 74, 918-932] and the critical concentration above which the interactions between the molecules begin to appear allows determining the corresponding molecular length. We observe a very good agreement between the DNA lengths obtained from the c* values and by using the infinite dilution value of τ3 and Broersma's equation. Therefore, only highly diluted solutions can be used if intrinsic molecular properties based on the rotational diffusion of high-molecular-weight elongated molecules are studied.

Notes: A thermodynamic treatment of the helix-coil transition of synthetic polypeptides in binary organic solvent mixtures is extended to describe isobaric heat-capacity increments associated with the phenomenon. This development resolves such increments into three components: two associated respectively with intrinsic differences between the ordered and disordered states of the macromolecule and between the coil-solvent complex and its components, and a third term derived from the temperature dependence in the fraction of coil residues bound to active solvent. Insights derived from this analysis are also applied to the discussion of some heat capacity increments associated with the denaturation of globular proteins.

Notes: The temperature dependence of the humidity-sensitive spacing, d, related to the lateral packing of collagen molecules was measured for fully hydrated collagen. In the vicinity of 0°C, a sudden change in d was observed, which was reversible with temperature. In the diffraction profile, below 0°C, a set of diffraction peaks identified with the hexagonal crystalline form of ice was observed. With the reduction in water content, the intensity of the set of diffraction peaks decreased and was found to be zero at a water content of 0.38 g/g collagen. These results were considered to be caused by the frozen water in collagen fibril below 0°C. According to the water content dependence of d, it was considered that up to a certain water content water absorbed would be stowed in the intermolecular space of collagen and above that water content water molecules would aggregate to make pools, i. e., extrafibrillar spaces. The unfreezable bound water was considered to be located in the intermolecular space of collagen. Size of the extrafibrillar space, determined from the intensity analysis of a smallangle x-ray scattering pattern, corroborates the speculation that the water showed in the extrafibrillar space is freezable and free. The formation of the hexagonal crystalline form of ice in the extrafibrillar space was considered to cause the sudden change in d at 0°C.

Notes: Die Rheinanliegerstaaten haben im gemeinsamen Bemühen um die Qualität des Rheinwassers schon 1963 die „Internationale Kommission zum Schutze des Rheins gegen Verunreinigung“ (IKSR) ins Leben gerufen. Die IKSR überwacht die Wasserqualität und gibt einen jährlichen Bericht mit Analysendaten heraus. Außerdem erarbeitet sie Vorschläge für Maßnahmen zum Schutze des Stroms. In Ergänzung der jeweiligen nationalen Gewässerschutzmaßnahmen haben die Rheinanliegerstaaten 1976 das Übereinkommen zum Schutze des Rheins gegen chemische Verunreinigung (Chemieabkommen) abgeschlossen. Das Chemieakbkommen, durch das die Ableitung bestimmter gefährlicher Stoffe aus den Stoffgruppen und -familien: Queck-silber- und Cadmiumverbindungen, organische Halogen-, Phosphor- und Zinnverbin-dungen, cancerogene Stoffe so weit wie möglich verhindert und aus weiteren Stoffgruppen und -familien, darunter den meisten Metallen, streng überwacht und eingeschränkt werden soll, hat einen langfristigen präventiven Charakter. Das Chloridabkommen soll dagegen sofort zu Maßnahmen führen. Zu diesen Abkommen der Rheinanliegerstaaten treten eine Reihe weiterer internationaler Abkommen und Vereinbarungen mit Auswirkungen auf den Rhein, verschiedene Richtlinien der Europäischen Gemeinschaft, ein geplantes Abkommen der Länder im Europarat, ein Abkommen zum Schutze der Nordsee und des Nordatlantiks gegen die Verschmutzung vom Lande aus.

Notes: We have measured the ultraviolet CD spectra for covalently closed and linear forms of phage PM2 DNA in solution. We find that increased concentrations of salt or ethanol (up to 50% ethanol by weight) depress the long-wavelength positive CD bands in the spectra of both forms of DNA, although the spectrum of the native covalently closed DNA always has a slightly larger magnitude of these bands than does the spectrum of the linear DNA. In addition, both DNAs are equally capable of undergoing a transition to the A conformation in 70-80% ethanol at low Na+ concentrations. Thus, the constraint imposed by the covalent closure of a DNA molecule does not seem to hinder its conformational response to these changing solution conditions. Lang [(1973) J. Mol. Biol. 78, 247-254] has found by electron microscopy that T7 DNA has an inherent ability to condense into compact particles, suggested to be supercoils of multiple order. Both covalently closed and linear forms of PM2 DNA also become condensed when the DNA, in 0.2M ammonium acetate and 1 mM EDTA, is exposed to ethanol and subsequent drying on specimen grids [Lang, D., Taylor, T. N., Dobyan, D. C. & Gray, D. M. (1976) J. Mol. Biol. 106, 97-107]. Under similar conditions, in solutions of 0.2M ammonium acetate and 1 mM EDTA to which ethanol is added, we have measured the CD spectra of both covalently closed and linear forms of DNA. Below ethanol concentrations at which the DNA obviously precipitates, the CD spectra of both forms have reduced long-wavelength positive CD bands.

Notes: The polymeric cobalt(II) porphyrin complexes were prepared from cobalt(II) protoporphyrin IX dimethyl ester(Co(II)P) and copolymers of 4-vinylpyridine and styrene(PSP), and their binding ability of molecular oxygen was studied in toluene solution. The five- and six-coordinate structure of CoP-PSP complexes were confirmed by esr spectra. The esr parameters for the CoP-PSP complexes were not affected by the molecular weight and the vinylpyridine-unit content of PSP-ligand. The 1:1 dioxygen-Co complex was reversibly formed when the solution of CoP-PSP was exposed to oxygen atmosphere at low temperature. While the visible spectra and esr parameters for the dioxygen complexes of CoP-PSP were the same as those of the CoP-pyridine complex, the equilibrium constant for the oxygen binding increased with the vinylpyridine-unit content of the PSP-ligand. The larger entropy change was observed for the oxygenation in the CoP-PSP system especially, of which the vinylpyridine-unit content was large.

Notes: The low-temperature dynamic mechanical relaxation of ox Ligamentum nuchae elastin, swollen with water and ethylene glycol at various swelling ratios, has been investigated by means of the Rheovibron DDV II viscoelastometer, at frequencies from 3.5 to 110 Hz. The peak location on the temperature scale, its intensity and activation energy have been studied as a function of solvent content and it has been found that the two solvents influence the dynamic mechanical behavior in a quite different way. A similar interaction of the solvent molecules with the backbone peptide groups is assumed for both solvents, and the differences in the mechanical spectra, which are more marked the higher the solvation degree, are explained in terms of the different size of the protein-solvent relaxing units.

Notes: The relation between the chemical structure of a protein and the physical properties of a heat-set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress-strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross-links in the gel. The cross-links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross-links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory-Huggins lattice model, can also be used to calculate the number of cros-links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross-links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.

Notes: The single-strand helix-coil transition in various oligo- and polyadenylates is characterized by means of an improved cable temperature-jump technique. In all the polymers studied {poly(rA), poly(dA), poly[A(m2′)] and poly[A(e2′)]} helix-coil relaxation is observed in the time range from 30 to 1000 nsec. Relaxation-time constants observed at wavelengths λ〈280 nm (τα) are different from those found at λ 〉280 nm (τβ), indicating the presence of more than two conformational states. The time constants τα increase in the series poly(dA), poly[A(m2′)], constants τβ/τα is approximately 2.5, except in poly(dA) where τβ/τα ≈ 9. Relaxation measurements with r(A)n- oligomers show a decrease in conformational mobility with increasing chain length. The relaxation curves also demonstrate that “internal” residues have lower reaction rates than residues at the ends of the oligomer chain. Measurement in D2O reveal a solvent isotope effect for τα of +87% for poly(rA), and of +53% for poly(dA), whereas no isotope effect is found in τβ. The absence of “slow” relaxation processes in the model compound 9,9′ -trimethylenebisadenine shows that the relatively low rate of the single-strand helix-coil transitions is due to the coupling of base stacking with the folding of the sugar-phosphate chain. The absence of a seprate relaxation process (corresponding to τβ) in 9,9′-trimethylenebisadenine, as well as in the dinucleotides ApC and CpA, suggests that this relaxation process is dependent upon the presence of both the sugar-phosphate chain and of adjacent adenine bases. The experimental data provide evidence that there is more than one ordered conformation in various single-stranded oligo- and polyadenylates and that the transition between these conformations is influenced by the sugar conformation.

Notes: An algorithm has been developed that permits one to find all possible conformations of the sugar-phosphate backbone for any given disposition of DNA base pairs. For each of the conformations thus obtained, the energy of the helix was calculated by the method of atom-atom potentials. Several isolated regions in the space of the bases′ parameters (Arnott's parameters) have been found for energetically favorable helical structures. Two parameters, the distance of a base pair from the helix axis, D, and the windling angle, τ, allow one to subdivide possible conformations into the families of closely related forms. Two regions (ravines) on the (D, τ) map correspond to the know A and B families. In the B family a continuous transition has been obtained in which the double helix undergoes increasing winding, while the base pairs are moving toward the major (nonglycosidic) groove.Interrelationships between the variables, characterizing the spatial structure of the double helix, D, τ, TL and χ, when going along the bottom of the B ravine, were also obtained.Besides the Known A and B families, several new ones were found to be energetically possible. Among these the strongly underwound helices with the negative D values, as well as the forms with the C4-C5 angle in a trans position, should be mentioned.Biological roles of the different double-stranded conformations, in particular, in protein-nuclei acid interaction are discussed.

Notes: The theoretical basis for the statistical mechanical deconvolution of a thermally induced macromolecular melting profile is presented. It is demonstrated that all the thermodynamic quantities characterizing a multistate macromolecular transition can be obtained from the average excess enthalpy function, 〈ΔH〉, of the system, without any assumption of the particular model or mechanism of the reaction.Experimentally, 〈ΔH〉 is obtained from scanning calorimetric data by direct integration of the excess apparent molar heat capacity function, ΦCp. Once 〈ΔH〉 is known as a continuous function of the temperature, the partition function, Q, of the system can be calculated by means of the equation \documentclass{article}\pagestyle{empty}\begin{document}$$Q_{\left( T \right)} = \exp \smallint _{T_0}^T {\left( {\frac{{\left\langle {\Delta H} \right\rangle }} {{RT^2}}} \right)} dT $$\end{document} From the partition function all the thermodynamic quantities of the system can be obtained. It is shown that the number of discrete macroscopic energy states, the enthalpy and entropy changes between them, and the relative population of each state as a function of temperature can be calculated in a recursive form.

Notes: The high-resolution 1H-nmr study of the ferrichrome cyclohexapeptides, in d6-DMSO solutions, has been extended to the amide NH spectral region. A total of ten diamagnetic analogues of ferrichrome that differ in the coordinated metal ion (Al3+, Ga3+ or Co3+), the primary structure, the nature of the bidentate hydroxamate moiety, or the isotope compositions (14N, 15N) have been investigated. The 3JαNH values reflect regiorous conformational isomorphism throughout the complete suite of analogues, quite independent of the residue occupancy of each site. Totally resolved amide multiplets have been obtained in most cases and the four-line (doublet of doublets) appearances of glycyl NH resonances has been observed for the first times; these data enabled stereospecific assignment and accurate analysis of the NH-CαH proton spin systems. The high resolution was made possible by the use of a suitable spectral deconvolution shceme at 360 MHz. The fine structure, extraordinarily well displayed in the 15N-peptide spectra, provides a series of parameter values whose consistency has been checked by computer simulation. Since the crystallographic structure for two of the ferric peptides is known to 0.002-Å resolution, a 3J vs θ correspondence could be confidently established. A Karplus curve was derived from the combined x-ray and nmr data: \documentclass{article}\pagestyle{empty}\begin{document}$$ ^3 J_{\alpha {\rm NH}} = 5.4\cos ^2 \theta - 1.3\cos \theta + 2.2{\rm Hz} $$\end{document} It is suggested that seriously nonplanar amides can exhibit 3JαNH values higher than predicted by the ferrichrome curve.

Notes: The general formulas describing the low-angular dependence of polarized (Vv) and depolarized (Hv) light-scattering intensity by a chain macromolecule of arbitrary form with anisotropic polarizability are derived. It is shown that the value dHv/dtHvo (where t = [〈R2〉S/L]2, S being the scattering vector) is an indication of the chain-flexibility mechanism. This permits one to distinguish between the models of a wormlike chain, regular zigzag, or statistical zigzag. The results of numerical calculations useful for the interpretation of experimental data are presented.

Notes: The kinetics of the fluorescence decay of the energy donor in a homologous series of oligopeptides each containing at its ends a donor and an acceptor of electronic excitation energy was investigated in solvent mixtures of different viscosities. The repeating unit in the peptides was N5-(2-hydroxyethyl)-L-glutamine and the chromophores used as donor and acceptor were naphthalene and dansyl, respectively. The number of units in the peptides studied varied from four to nine. The solvents used were mixtures of glycerol and trifluoroethanol in various proportions. The decay rate of the donor fluorescence increases when the solvent viscosity decreases. This behavior is due to the disturbance of the equilibrium end-to-end distribution of distance of the excited molecules by the energy transfer process, which is more favorable foe short than for long distances. The subsequent rearrangement towards the equilibrium distribution by diffusion of the molecular ends relative to one another enhances the efficiency of the energy transfer. Assuming a modified Fick equation to describe this diffusion motion, the fluorescence decay data were analyzed in terms of a diffusion coefficient describing the Brownian motion of the molecular ends. The diffusion coefficients thus evaluated increase systematically upon decreasing the solvent viscosity. For example, for the oligopeptides studied it changes from unmeasurably small values in glycerol solution to values varying between 10-8 to 10-7 cm2/sec at room temperature in a glycerol trifluoroethanol solvent mixture of viscosity of 8 centipoise. The values obtained for the diffusion coefficient are smaller by about an order of magnitude than the values expected for the diffusion coefficients of the free chromophores in solvents of comparable viscosity. It is thus concluded that the backbone of the polymeric chains possesses appreciable internal friction which exerts resistance to the Brownian motion of the polymer chains. The diffusion coefficient of the end-to-end motion is systematically smaller for the shorter than for the longer chains. For example, at room temperature in a solvent mixture of 8 centipoise it is 3×10-8, 5×10-8, 7.6×10-8, and 8.5×10-8 cm2/sec for oligomers containing four, five, eight, and nine N5-(2-hydroxylethyl)-L-glutamine repeating units, respectively. The internal friction thus impedes the motion of the molecular ends more effectively in the shorter chains than in the longer ones.Analysis of the energy-transfer experiments in solvents spanning a wide range of viscosities shows that the orientational factor appearing in Förster's equation for energy transfer does not interfere perceptibly with the evaluation of the end-to-end distances from the fluorescence decay data when naphthalene and dansyl serve as donor and acceptor, respectively. This is due to the fact that both the donor emission and acceptor absorption in the range of spectral overlap are characterized by more than one transition dipole moment, as is evident from the corresponding polarization data.

Notes: Raman spectroscopic studies have been carried out on polymers of L-valine ranging in degree of polymerization (DP) from 2 to 930. The spectrum of the hexapeptide (DP = 6) is closely similar over the entire range 40-1750 cm-1 to those of polymers with much higher DP, and the structure is clearly shown to be that of the antiparallel pleated sheet (β-structure) by the amide I and III frequencies. The formation of a little α-helical structure occurs in polymers with DP above 500, although the amount does not appear to be a linear function of DP. The α-helical structure is unstable and readily destroyed in samples cast from trifluoroacetic acid solution. It is stabilized by the incorporation of L-alanine, a strong helix-former; polymers of the latter may in turn be forced into a α-structure in copolymers sufficiently rich in L-valine.

Notes: The fluorescence properties of several cooligopeptides of glycine, phenylalanine, and tryptophan, containing one or two aromatic residues, are investigated. In particular, a detailed analysis is made of the influence of pH upon the quantum yield and the position of the emission maximum (λmax) in H-Trp-Trp-OH, H-Trp-Gly-OH, H-Gly-Trp-OH, H-Gly-Trp-Gly-OH, H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, H-Gly-Trp-Trp-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, H-Gly-Phe-Trp-OH, and H-Gly-X-(Gly)n-Trp-Gly-OH, with X = Phe or Trp, and n = 0,1,2. It is shown that raising the pH from ca. 2 to 11 results in a red shift of λmax, and an increase in the quantum yield. These changes, mostly structure dependent, are in most cases attributable to electronic perturbations acting directly upon the λmax of the fluorophore(s) and upon the quenching efficiency of the free amino and carbonyl groups. For the compounds having two adjacent tryptophyl residues, it is shown that the two fluorophores do not appear to have the same emission properties and the quantum yield is lower than expected. The causes of this behavior are discussed in terms of conformational effects, stacking interactions, and radiationless energy transfer. Finally, an attempt is made to correlate fluorescence data with previous circular dichroism data which had indicated the occurrence of a conformationally rigid structure for some of the compounds having two adjacent aromatic residues.

Notes: Group-specific polysaccharides isolated by means of a cetavlon procedure are immunogenic in man and induce protective immunity against meningococcal meningitis. Minute quantities of the polymers in solution can act as vaccines. We now report the first characterization of a fractionated (C-1) group C polysaccharide in 0.4KM KCl and 0.05M sodium acetate by means of light-scattering spectroscopy. Independent measurements of refractive index increments, absolute scattered intensities, angular scattering intensities and line widths as a function of scattering angles and delay times at different concentrations using incident wavelengths of 632.8 nm from a He-Ne laser and of 488 nm from an argon-ion laser yield information on aggregation properties, molecular weight (Mr), radius of gyration 〈r0g〉1/2z, translational diffusion coefficient 〈D〉0z, and second virial coefficients A2 and B2 of C-1 polysaccharide.At relatively high ionic strength (0.04M KCl + 0.05M sodium acetate), we obtain for the C-1 polysaccharide in solution Mr = 5.15 × 105, 〈r2g〉1/2z = 345 Å, A2 = 1.25 × 10-4 ml/g, 〈D〉0z = 1.16 × 10-7 cm2/sec with a corresponding Stokes radius of 240 Å and B2 = 4.4 ml/g. A2 and B2 are the second virial coefficients from intensity- and diffusion-coefficient measurements. The C-1 polysaccharide aggregates in solution and behaves hydrodynamically like random coils. Viscosity and sedimentation studies further confirm our conclusions that the fractioned C-1 polysaccharide aggregates in solution and EDTA can partially break up those aggregates. However, the system remains polydisperse even after adding an excess amount of EDTA. The weight-average molecular weight of the C-1 polysaccharide in solution depends upon ionic strength and exhibits a minimum at ∼0.2M KCl. Finally, viscosity, light-scattering, and sedimentation results all show that the aggregated macromolecular system behaves like random-coiled polymers with no measurable shape factors.

Notes: We extend fluorescence correlation spectroscopy to systems that undergo translation or laminar flow in a sample cell. We include theoretical and experimental results; we consider uniform and nonuniform velocity profiles. Concentration correlation analysis extracts microscopic rate parameters from measurements of the spontaneous concentration fluctutations, which occur even at equilibrium. Fluorescence is one of the most sensitive means of monitoring these fluctuations. Analysis of flowing or translating systems (1) offers a method of measuring number concentrations of selected species, for example, of aggregates or polymers, (2) provides a nonperturbing velocity probe, (3) sometimes allows one to circumvent photolytic degradation, (4) has proved extremely helpful in testing and aliging apparatus for fluorescence correaltion measurement and in verifying theoretical analyses, and (5) may be required for interpretation of results obtained on systems in motion, even though that motion is undesired or initially unsuspected. We include both theoretical and experimental results for combined Poiseuille flow and diffusion in the geometry which is of most practical interest. Theoretical expressions for the much simpler cases of nondiffusive Poiseuille flow as well as uniform flow or translation with or without diffusion constitute limiting cases which are displayed explicitly.

Notes: In the preceding article1 we demonstrated that the partition function of a system is experimentally accessible from scanning calorimetric data. In this article the general results of the deconvolution theory are applied to the general case of cooperative transitions in macromolecules. It is demonstrated that, in the limit of very large systems, all the relevant molecular averages and molecular distribution functions can be directly obtained from the experiment. In doing this, the method of the grand partition function is used. It is shown that in the case of homopolymers, only one parameter, the stability constant, needs to be explicitly specified for a complete description of the system. Since the partition function is directly evaluated from the experiment, no special assumptions or artificial constraints directed to obtain a mathematically solvable model are required. This result offers the unique opportunity of having direct experimental access to statistical averages of systems in which the partition function cannot analytically be solved. Consequently, the theory can be extended to cooperative transitions occurring in two and three dimensions by introducing cluster distribution functions.

Notes: The pyridoxal Schiff bases of the polypeptides poly(L-lysine), poly(L-ornithine), and poly(L-α,γ-diaminobutyric acid) were prepared and investigated in water/methanol by CD spectra and equilibrium dialysis experiments. Only the poly(L-α,γ-diaminobutyric acid) derivative is characterized by a relevant optical activity similar to that found in pyridoxal enzymes. The stereospecific interactions between the pyridoxylideneimine group and the polypeptide chain prevent the hydrolysis reaction of the aldimine bond.

Notes: The protein turkey-heart cytochrome c is used as a model protein to study charge effects in sedimentation equilibrium experiments in three-component solutions. Data are given for the dependence of the apparent M (1-υρ) on ρ in solutions of KCl, RbCl, CsCl, and triethylamine hydrochloride. The results show the Donnan effect to have a significant influence on the apparent molecular weight, found by extrapolation of the data to a solution density of one. The apparent molecular weights are for protein at infinite dilution. A theoretical treatment is presented where the magnitude of this effect can be predicted accurately from the formal net charge of the protein as computed from the amino acid composition. The results are shown to be important in computing the preferential hydration of the protein in concentrated salt solutions. For such systems the Donnan effect should be subtracted from the total interaction coefficient for multicomponent system in order to obtain the preferential hydration.