1H, 13C,and 15N assignments and secondary structure of the FK506 binding protein when bound to ascomycin

The ¹H, ¹³C, and ¹⁵N resonances of FKBP when bound to the immunosuppressant, ascomycin, were assigned using a computer-aided analysis of heteronuclear double and triple resonance three-dimensional nmr spectra of [U-¹⁵N] FKBP/ascomycin and [U-¹⁵N, ¹³C] FKBP/ascomycin. In addition, from a preliminary analysis of two heteronuclear four-dimensional data sets, ³J coupling constants, amide exchange data, and the differences between the C^α and C^β chemical shifts of FKBP to random coil values, the secondary structure of FKBP when bound to ascomycin was determined. The secondary structure of FKBP when bound to ascomycin in solution closely resembled the x-ray structure of the FKBP/FK506 complex but differed in some aspects from the structure of uncomplexed FKBP in solution. © 1993 John Wiley & Sons, Inc.     

Co-oligopeptides containing two aromatic residues spaced by glycyl residues. X. Proton magnetic resonance study of co-oligopeptides of tryptophan and glycine

The ¹H-nmr spectra of co-oligopeptides of tryptophan and glycine with structure H-Gly-Trp-(Gly)_n-Trp-Gly-OH (n = 0–2) and those of several di- and tripeptides have been recorded at 360 MHz with CD₃OD solutions containing 0.1N NaOD. The assignment of resonance signals was generally possible by comparing the spectra of structurally related peptides with each other. In order to solve the remaining ambiguities in the assignment, H-(αL,βS)(α,β-d₂)Trp-OH, H-Trp-(αL,βS)(α,β-d₂)Trp-OH, and H-Trp-(δ¹,ε²,ζ²,ζ³,η²-d₅)Trp-OH have been prepared and their spectra compared with those of the undeuterated compounds. The distribution of rotamers around the χ₁ and (in two cases) χ₂ torsion angles of the side chains has been obtained from the vicinal coupling constants ³J and from the long-range coupling constants ⁴J. These data and an analysis of the chemical shifts of the Gly-C^α protons suggest that the orientation of the aromatic side chain is influenced by the following order of decreasing interaction with the functional groups at N- and C-side: -NH₂ > –NHCO– > –CONH–> –COO^?. This rule does not hold for the second Trp residue of di- and tripeptides containing the -Trp-Trp- sequence, which has tentatively been attributed to steric effects.     

The solution conformation of malformin A

Solution conformations of the cyclic pentapeptide plant-hormone malformin A, whose conformational freedom is constrained by an intramolecular disulfide bridge, are derived and presented here. The nmr and CD data of Ptak are used to place restrictions on the search for possible malformin A solution conformers of low energy. Only two distinct conformers were found to be consistent with Ptak's data. Both structures are characterized by an internally buried (solvent-shielded) D -Cys² amide proton, a seven-membered (1–3)hydrogen bond between (N–H) and (O?C), and a disulfide bridge conformation with a P chirality as manifested in the nmr study by the temperature independence of the amide proton chemical shifts for the D -Cys² and D -Leu⁴ residues and the negative sign of the long wavelength maximum in the CD spectrum, respectively. Inspection of space-filling molecular models of both structures indicates severe steric barriers to their rapid interconversion. Thus, it appears that only one of the two conformers may be present in solution. The difference in their calculated dipole moments (4.6 and 6.9D) suggests an experimental method for distinguishing between the two proposed solution structures.     

NMR evidence for a type I β-turn in (Pro2)-tetrapeptides and interdependence of cis:Trans isomerism,ring flexibility,and backbone conformation

Tetrapeptides with proline in position 2, asparagine or leucine in position 3, and glycine in positions 1 and 4, with end groups free or blocked on the N-terminal side, were studied in their various ionic states in ²H₂O and in Me₂SO-d₆ by ¹H- and ¹³C-nmr. In order to clarify or refine some details, successive substitutions of the residues in these peptides with amino acids enriched to 85% in ¹³C, or to 85% ¹³C plus 97% ²H were carried out. The ¹H and ¹³C chemical shifts as well as the ¹H-¹H, ¹³C-¹³C, and ¹³C-¹H coupling constants and the signal intensities show strong similarity of behavior between the tetrapeptides of asparagine and leucine. The main conformational characteristics are (1) the almost total stabilization of the trans conformer in the type I β-turn structure when the peptide is in the zwitterion state dissolved in Me₂SO. This is deduced from the ³J and the ³J coupling constants, which both furnish a dihedral angle of ?³ = ?90°, and from the positive value of the temperature coefficient of the glycine-4 amide protons, which suggests a type 4 → 1 hydrogen bond; (2) the evolution of cis and trans isomer fractions which change with the ionic state of the peptides in Me₂SO, whereas they remain constant in aqueous solution; and (3) the conformation of the pyrrolidine ring as it follows the variations in cis:trans isomer populations together with the side-chain rotamer fractions of the residue in position 3. In the β-turn conformation the isomer cis is less abundant and the pyrrolidine ring is more flexible; this explains the perfect accommodation of the proline residue in position 2 of a bend. The interdependence of these phenomena where interactive forces play a predominant role underlines the importance of cooperative effects in the molecule. The results also suggest that the cis isomer of proline can adapt itself just as well as the trans isomer to position 2 of a type I β-turn.     

Structure of human salivary histatin 5 in aqueous and nonaqueous solutions

The solution structure of human salivary histatin 5 (D-S-H-A-K-R-H-H-G-Y-K-R-K-F-H-E-K-H-H-S-H-R-G-Y) was examined in water (pH 3.8) and dimethyl sulfoxide solutions using 500 MHz homo- and heteronuclear two-dimensional (2D) nmr. The resonance assignment of peptide backbone and side-chain protons was accomplished by 2D total correlated spectroscopy and nuclear Overhauser effect (NOE) spectroscopy. The high J values (≥7.4 Hz), absence of any characteristic NH-NH(i, i + 1) or C^αH-C^βH(i, i + 3) NOE connectivities, high dδ/dT values (≥0.004 ppm K⁻¹) and the fast ¹H/²H amide exchange suggest that histatin 5 molecules remain unstructured in aqueous solution at pH 3.8. In contrast, histatin 5 prefers largely α-helical conformation in dimethyl sulfoxide solution as evident from the J values (≤6.4 Hz), slow ¹H/²H exchange, low dδ/dT values (≤0.003 ppm K⁻¹) observed for amide resonances of residues 6–24, and the characteristic NH-NH(i, i + 1) and C^αH-C^βH(i, i +3) NOE connectivities. All backbone amide ¹⁵N-¹H connectivities fall within 6 ppm on the ¹⁵N scale in the 2D heteronuclear single quantum correlated spectrum, and the restrained structure calculations using DIANA suggest the prevalence of α-helical conformations stabilized by 19 (5 → 1) intramolecular backbone amide hydrogen bonds in polar aprotic medium such as dimethyl sulfoxide. The interside-chain hydrogen bonding and salt-bridge type interactions that normally stabilize the helical structure of linear peptides in aqueous solutions are not observed. Histatin 5, unlike other naturally occurring antimicrobial polypeptides such as magainins, defensins, and tachyplesins, does not adopt amphiphilic structure, precluding its insertion into microbial membranes and formation of ion channels across membranes. Electrostatic (ionic type) and hydrogen bonding interactions of the positively charged and polar residues with the head groups of microbial membranes or with a membrane-bound receptor could be the initial step involved in the mechanism of antimicrobial activity of histatins. © 1998 John Wiley & Sons, Inc. Biopoly 45: 51–67, 1998     

On the application of polyelectrolyte limiting laws to the helix-coil transition of DNA. IV. Depedence of helix stability on the concentration of divalent metal ions.

The molecular theory of the previous paper in this series is extended to determine the effect of divalent metal ions on helix stability relative to coil at fixed ionic strength and nucleotide phosphate concentration. Specification of the state of condensed counterions, as well as their concentration, is essential for the solution of this problem, and it is assumed that they translate freely within a thin cylindrical shell close to the polynucleotide. As a function of divalent counterion concentration m the relative stability of the helix is highly nonlinear. Although the overall trend is that the helix stability increases with addition of divalent metal ion, there is a narrow concentration range for which it decreases slightly. The behavior of the relative stability as a function of m is determined by the translational degrees of freedom of the counterions, both univalent and divalent, both condensed and uncondensed. Detailed comparison of the theory with data is not given here, but it is pointed out that the calculated values of the relative stability are consistent with the order of magnitude of the observed effect Mg²⁺ on the melting temperature.     

Conformational analysis of acetyl-L-phenylalanine p-acetyl and p-valeryl anilides

Empirical force-field calculations and ir and ¹H-nmr spectra indicate that five-membered (C₅) and seven-membered (C) hydrogen-bonded rings are the preferred conformations of acetyl-L -Phe p-acetyl and p-valeryl anilides in nonpolar media. The C₅/C ratio was found to be dependent on the dryness of the solute and the solvent. This fact and the results from conformational-energy calculations suggest that a molecule of water participates in the stabilization of the C conformation.     

Characterization of the transition state of Lysozyme unfolding. I. Effect of protein-solvent interactions on the transition state

The influence of proline cis-trans isomerization on the kinetics of lysozyme unfolding was examined carefully according to the theory of Hagerman and Baldwin [(1976) Biochemistry 15, 1462–1473]. As a result, the kinetics of lysozyme unfolding was found to follow the two-state transition model well. The temperature dependencies of k_uf and k_f over a wide temperature range showed that ΔC = 0 and ΔC = ?6.7 kJ K^?1 mol^?1 in solutions of different concentrations of GuHCl. The data observed in solutions containing other denaturants also supported the conclusion that ΔC is nearly equal to zero. The activation enthalpies of unfolding (ΔH) were observed at various concentrations of several kinds of denaturants. They were independent of species and concentrations of denaturants ΔH = 200 kJ mol^?1). These facts indicate that the aspect of interaction between protein and different kinds of solvent molecules varies only slightly during the unfolding to the transition state, that is, the transition state is at compact as the native one. Therefore, it is also suggested that ΔH of 200 kJ mol^?1 is primarily required for the disruption of long-range interactions among different structural domains through a subtle conformational change. We compared the effects of several kinds of denaturants on the unfolding rate. The addition of PrOH more remarkably increases the unfolding rate than do other hydrophilic denaturants. This is probably because PrOH molecules can penetrate into the hydrophobic core of lysozyme, but hydrophilic reagents cannot because of the compactness of the transition state.     

On the blue color of triiodide ions in starch and starch fractions. I. Characterization and assignment of absorption and circular dichroism spectra of triiodide ions in amylose

Four fundamental Raman lines were observed at 159, 111, 55 and 27 cm^-1 corresponding to the I bound (I) in amyloses with DP from 20 to 100, regardless of the degree of polymerization of I and the excitation wavelength. The spectral resolution was based on the molar extinction coefficient and molar ellipticity spectra of I. Eight bands, named, S₁, S₂, ?, S₈ from long to short wavelength, were isolated. These were found regardless of the DP. By a resonance excitation Raman study, the characteristics of S₃ and S₄, comprising the shoulder around 480 nm, were found to be different from those of S₁ and S₂, comprising the blue band. The assignment of the spectra was based on the electronic states of the monomeric I in the exciton-coupled dimeric unit. It was concluded that the blue band (S₁,S₂) belonged to the long-axis transitions and the shoulder band (S₃,S₄) to the short-axis ones on the monmeric coordinate system.     

Dielectrical study of thermal transformation in Na DNA solutions

A dielectric method is presented to measure the ejection of counterion during the thermal fusion of Na DNA solutions. The dielectric conductivity behavior at fixed frequency versus temperature is a measure of the molar concentration of counterfoils formed from the macromolecules. A linear dependence between Na⁺ cations released and DNA concentration was verified. We consider the variation of sodium activity coefficient Δ during thermal transconformation to be in good agreement with other experimental data.     

Levels of DNA strand breaks and superoxide in phorbol ester-treated human granulocytes

Phorbol ester treatment of granulocytes triggers release of superoxide (O) and a concomitant burst of DNA strand breaks. The relationship between the amount of O and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O generated over a 40-min period by 1 × 10⁶ granulocytes/mL, a discontinuous “10-min pulse” method employing cytochrome c was used; 140 nmol O per 1 × 10⁶ cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1–10 mM) and staurosporine (2–10 nM) both inhibited O production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O. Zinc chloride (50–200 μM) inhibited both O and DNA breaks, approximately equally. Dinophysistoxin-1 (okadaic acid) inhibited O production more effectively than it inhibited DNA breaks. O dismutes to H₂O₂, a reactive oxygen species known to cause DNA breaks. The addition of catalase to remove extracellular H₂O₂ had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected compared to the number detected by FADU, indicating that about 95% of breaks were single-stranded. The level of DNA breaks is not directly related to the amount of extracellular O or H₂O₂ in PMA-stimulated granulocytes. We conclude that either an intracellular pool of these reactive oxygen species is involved in breakage or that the metabolic inhibitors are affecting a novel strand break pathway. J. Cell. Biochem. 66:219–228, 1997. © 1997 Wiley-Liss Inc.     

Heat capacity changes and partial molal heat capacities of some di- and tripeptides in water

Integral enthalpies of solution of several dipeptides and tripeptides in water at low concentrations have been determined at 25 and 35°C. These data have been used to derive the changes in heat capacity on dissolution at infinite dilution ΔC at 30°C. Limiting partial molal heat capacities ΔC have been determined by combining ΔC with C_p2 (heat capacity of pure solid peptides). Using the data on ω-amino acids and these peptides, the partial molal heat capacity of a peptide group ? CONH? was semiquantitatively estimated.     

Conformational studies of N-acetyl-N′-methylamide derivatives of α-aminobutyric acid,norvaline, and valine. I. Preferred conformations in solution as studied by 1H-nmr spectroscopy

The ¹H-nmr studies were extensively carried out to elucidate preferred conformations of dipeptides CH₃C*O—X—NHCH₃, with X = Abu, nVal, and Val in various solvents. The vicinal ¹H—¹H coupling constants for the NH—C^αH moiety and those around the C^α—C^β bond in the articulated side chain provided the information regarding the average conformation of these molecules. The results indicate that transformation of skeletal conformations takes place in solution among conformers having similar dihedral angles, θ, in the Karplus expression.     

Compressibility studies of three proteins in CsCI solutions in the analytical ultracentrifuge

The compositional buoyant densities, ρ;, of human γ-immunoglobulin, bovine serum mercaptalbumin, and egg albumin have been measured in CsCl solutions in the analytical ultracentrifuge as a function or pressure. Standard pressure coefficients, ψ⁰, and standard partial specific volumes of the solvated proteins, υ ,0, have been computed from these data. The ψ⁰ values obtained are strikingly different from each other and from the only other pressure coefficients which have been measured, those values obtained for nucleic acids and nucleoproteins. The ψ value for γ-immunoglobulin is negative, the first nonpositive value obtained, and suggests an unusual internal structure for this protein. The pressure coefficient of mercaptalbumin is not constant. A second-order relation is derived and utilized to interpret these data. The slope of the ρ(P) plot for egg albumin was constant and negative and yielded values of ψ⁰ which are about 20% as large as those reported for DNA. Evaluation of published isopiestic data for egg albumin in CsCl solutions provided the dependence of preferential hydration on water activity. This quantity, (dΓ′/da) as well as α, were found to be negative. The values of ψ⁰ and α were used to compute the effective density gradient from which the correct molecular weight of egg albumin was obtained. The apparent specific volume of egg albumin in a buoyant CsCl solution was measured using the Mettler-Paar densimeter.     

Apparent molar volumes of some amino acids and peptides in aqueous urea solutions

Densities of solutions of several α-amino acids and peptides in 3 and 6m aqueous urea solvents have been determined at 298.15 K. These data have been used to evaluate the infinite-dilution apparent molar volumes of the solutes and the volume changes due to transfer (V ) of the α-amino acids and peptides at infinite dilution from water to aqueous urea solutions. The sign and magnitude of the V values have been rationalized in the framework of Friedman's cosphere-overlap model. The V values for the glycyl group (? CH₂CONH? ) and alkyl side chains have been estimated.     

Conformation and folding in histones H1 and H5

Denatured histones H1 and H5 can be readily refolded on salt addition. Their digestion by trypsin leads to limit peptides of about 80 residues having the same nmr and CD spectra as those of the intact parent histones. Scanning microcalorimetry shows that (1) the folded structures of H1 and H5 are located entirely in their limit peptides; (2) both have values of the specific denaturation enthalpy typical for small globular proteins; and that (3) both exhibit a classic “2-state” transition (ΔH = ΔH). The heat-denaturation profiles of H5 measured using intrinsic and extrinsic Cotton effect and side-chain nmr peaks do not coincide at all. Only the intrinsic Cotton effects give a T_m and ΔH close to that from microcalorimetry. We conclude that these proteins exhibit large-scale side-chain motions that precede the macroscopic cooperative transition.     

Estimation of Trichoderma QM 9414 biomass and growth rate by indirect means

Trichoderma QM 9414 was aerobically grown on glucose as the sole carbon and energy sources in a chemostat culture. The specific rates of glucose consumption (Q_G), oxygen consumption (Q), and carbon dioxide production (Q) at the steady state were measured to estimate the growth and maintenance requirements. From the results it was estimated that 2 mol adenosine triphosphate (ATP) were produced when1 mol NADH was oxidized through the respiratory chain of this microorganism. The true growth yield for ATP (Y_ATP) and specific ATP consumption rate for maintenance (Q) calculated with this value were 0.0106 g dry cell/mmol ATP and 5.2 mmol ATP/g dry cell/hr, respectively. Using the relationships between specific growth rate (μ) and (Q) and between μ and Q_G obtained from chemostat-culture data, cell and glucose concentration histories were estimated from the carbon dioxide production rate during the batch culture. The estimated cell concentrations agreed with the experimentally measured values. Glucose concentration were slightly overestimated.     

Kinetics of ethidium's intercalation in packaged bacteriophage T7 DNA: effects of DNA packing density

The kinetics of ethidium's intercalative binding to DNA packaged in bacteriophage T7 and two T7 deletion mutants have been determined, using enhancement of fluorescence to quantitate binding. At a constant ethidium concentration, the results can be described as first-order binding with two different rate constants, k (= k₁ + k_?1) and k (= k₂ + k_?2). The larger rate constant (k) was at least four orders of magnitude smaller than the comparable first-order forward rate constant for binding to DNA released from its capsid. At 25°C values of k decreased as the amount of DNA packaged per internal volume increased. This latter observation indicates that the rate of ethidium's binding to packaged T7 DNA is limited by an event that occurs inside of the DNA-containing region of T7, not by the crossing of T7 capsid's outer shell. Arrhenius plots of kM are biphasic, indicating a transition for packaged DNA at a temperature of 20°C. The data indicate that k s are limited by either sieving of ethidium during its passage through the packaged DNA or subsequent hindered intercalation.     

Order–Disorder conformation change of xanthan in 0.01M aqueous sodium chloride: Dimensional behavior

Weight-average molecular weights M_w, second virial coefficients, and z-average radii of gyration 〈S²〉 were determined by light scattering as a function of temperature T for four sodium salt samples of xanthan in 0.01M aqueous NaCl, in which the polysaccharide undergoes an order–disorder conformation change with increasing T. The data for 〈S²〉 and M_w at 25 and 80°C, the lowest and highest temperatures studied, confirmed the previous conclusion that the predominant conformation at the former T, i.e., in the ordered state, is a double helix, while that at the latter T, i.e., in the disordered state, is a dimerized coil expanded by electrostatic repulsions between charged groups of the polymer. As T was increased from 25 to 80°C, 〈S²〉 sigmoidally decreased or increased depending on the dimer's molecular weight. This temperature dependence of 〈S²〉 and that determined elsewhere for a high molecular weight sample were found to be described almost quantitatively by a simple dimer model in which the double helix melts from both ends, when the double-helical fraction in the dimer at a given T estimated previously from optical rotation data was used.