D-pantolactone as a circular dichromism (CD) calibration.

A critical study of circular dichromism (CD) measurement at the wavelength region around 220 nm was made. Test sample methods gave deviations of abou 20%, at most, between instruments calibrated at 290 nm with D-10-camphorsulfonic acid. A calculation of the CD from optical rotatory dispersion (ORD) data was made for d-pantolactone which gave a CD maximum at 219 nm, [θ]₂₁₉²⁵ = ?17.3 × 10³ in water. A proposal is made to use this value for calibration of CD in the 220-nm region.     

Anticooperative Nearest-Neighbor Interactions between Residues in Unfolded Peptides and Proteins

Growing evidence suggests that the conformational distributions of amino acid residues in unfolded peptides and proteins depend on the nature of the nearest neighbors. To explore whether the underlying interactions would lead to a breakdown of the isolated pair hypothesis of the classical random coil model, we further analyzed the conformational propensities that were recently obtained for the two guest residues (x,y) of GxyG tetrapeptides. We constructed a statistical thermodynamics model that allows for cooperative as well as for anticooperative interactions between adjacent residues adopting either a polyproline II or a β-strand conformation. Our analysis reveals that the nearest-neighbor interactions between most of the central residues in the investigated GxyG peptides are anticooperative. Interaction Gibbs energies are rather large at high temperatures (350 K), at which point many proteins undergo thermal unfolding. At room temperature, these interaction energies are less pronounced. We used the obtained interaction parameter in a Zimm-Bragg/Ising-type approach to calculate the temperature dependence of the ultraviolet circular dichroism (CD) of the MAX3 peptide, which is predominantly built by KV repeats. The agreement between simulation and experimental data was found to be satisfactory. Finally, we analyzed the temperature dependence of the CD and ³J(H^NH^α) parameters of the amyloid β_1–9 fragment. The results of this analysis and a more qualitative consideration of the temperature dependence of denatured proteins probed by CD spectroscopy further corroborate the dominance of anticooperative nearest-neighbor interactions. Generally, our results show that unfolded peptides—and most likely also proteins—exhibit some similarity with antiferromagnetic systems.     

Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies

The temperature dependence of the composition of coacervate and equilibrium phases is examined for the polypentapeptide of elastin (L -Val¹-L -Pro²-Gly³-L -Val⁴-Gly⁵)_n in water. This provides for the development of a phase diagram. CD data is presented that provides information on associated polypeptide structure changes that, when added to previous CD, nmr, and dielectric relaxation data at lower water composition, allow construction of a phase-structure diagram of the polypentapeptide–water system. The molecular-weight dependence of phase change (coacervation) is included. The volume–composition studies as a function of temperature also provide temperature coefficients of expansion and of composition important in analyzing the mechanism of elasticity.     

Propagation Speed in Myelinated Nerve: I. Experimental Dependence on External Na+ and on Temperature

Conduction speed (θ) in single myelinated Rana pipiens sciatic nerve fibers has been precisely measured using intracellular recording and on-line digital computer techniques. The dependence of relative speed on external Na concentration at 15°C has been found to be ln(θ₁/θ₂) = 0.524 (±0.018) ln ([Na⁺]₁/[Na⁺]₂) + 0.003. Thus θ has very close to a square root dependence on [Na⁺]₀ for these fibers. This experimental finding is not in complete agreement with a theoretical prediction based on a solution of the Hodgkin-Huxley (H.H.) equations. The effect of small temperature variations around 15°C on θ has also been measured for Rana fibers in Ringer's solution. θ has close to an exponential dependence on T and a Q₁₀ of 2.95 has been estimated.     

Studies on groundnut proteins. V. Physico-chemical properties of arachin prepared by different methods.

ORD,¹ CD, and fluorescence spectra of arachin prepared by Tombs' (Biochem. J., 96, 119; 1965), Dawson's (Anal. Biochem., 41, 305; 1971) or Shetty and Rao's (Anal. Biochem., 62, 108; 1974) procedure were measured; the effect of denaturants such as SDS, GuHCl, and acid was also determined. ORD and CD spectra showed differences, whereas fluorescence spectra did not show any difference. The effect of the denaturants was the same on the three arachins. At low concentrations of GuHCl (<2 m), the denaturant was bound by the protein molecule without causing any conformational change. The binding affinity varied among the arachins.     

Phospholipid Reorientation at the Lipid/Water Interface Measured by High Resolution P Field Cycling NMR Spectroscopy

The magnetic field dependence of the ³¹P spin-lattice relaxation rate, R₁, of phospholipids can be used to differentiate motions for these molecules in a variety of unilamellar vesicles. In particular, internal motion with a 5- to 10-ns correlation time has been attributed to diffusion-in-a-cone of the phosphodiester region, analogous to motion of a cylinder in a liquid hydrocarbon. We use the temperature dependence of ³¹P R₁ at low field (0.03-0.08 T), which reflects this correlation time, to explore the energy barriers associated with this motion. Most phospholipids exhibit a similar energy barrier of 13.2 ± 1.9 kJ/mol at temperatures above that associated with their gel-to-liquid-crystalline transition (T_m); at temperatures below T_m, this barrier increases dramatically to 68.5 ± 7.3 kJ/mol. This temperature dependence is broadly interpreted as arising from diffusive motion of the lipid axis in a spatially rough potential energy landscape. The inclusion of cholesterol in these vesicles has only moderate effects for phospholipids at temperatures above their T_m, but significantly reduces the energy barrier (to 17 ± 4 kJ/mol) at temperatures below the T_m of the pure lipid. Very-low-field R₁ data indicate that cholesterol inclusion alters the averaged disposition of the phosphorus-to-glycerol-proton vector (both its average length and its average angle with respect to the membrane normal) that determines the ³¹P relaxation.     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.     

Temperature-dependent optical rotatory dispersion properties of helical muscle proteins and homopolymers

Thermally induced helix–coil transitions of myosin rod, light meromyosin, and tropomyosin were studied by optical rotatory dispersion (ORD). Fractional helicity was calculated from both the Moffitt-Yang parameter, b₀, and the corrected mean residue rotation [m′] at 231.4 nm. Between 3 and 30°C, [m′] increases linearly with a slope of 59/°C, whereas b₀ is virtually constant, indicating apparently different thermal melting behavior. Poly(L -lysine) and poly(L -glutamic acid) in their helical forms and myoglobin also show a nearly linear temperature dependence of [m′]_231.4. Muscle proteins in 6M guanidine hydrochloride and the random-coil forms of the homopolymers exhibit temperature-dependent values of [m′]_231.4 and b₀. We conclude from these observations that ORD properties of both α-helices and random-coil polypeptides have significant intrinsic temperature dependencies. A new method of estimating fractional helicity as a function of temperature is proposed.     

Assembly in vitro of the 50 S subunit from Escherichia coli ribosomes: proteins essential for the first heat-dependent conformational change.

An early intermediate during the assembly in vitro of the ribosomal 50 S subunit is the RI₅₀(1) particle, which undergoes a drastic change in s value to form the RI₅₀¹(1) particle. The formation of this RI₅₀¹(1) particle, which is essential for the assembly of a highly active 50 S particle, was analyzed. Total reconstitution experiments with purified proteins revealed that six ribosomal components (23 S RNA and the proteins L4, L13, L20, L22 and L24) are essential for the RI₅₀¹(1) formation. Protein L3 had a stimulatory effect, but was not essential. With these seven components, a particle with the RI₅₀¹ conformation could be formed.A comparison with assembly in vivo demonstrated that the pathways of protein assembly in vitro and in vivo are very similar at the beginning, but diverge towards the end of the process. Furthermore, the sequence in which the proteins can be split off the 50 S subunit by increasing concentrations of LiCl corresponds, to a first approximation, to the reverse of the order of assembly in vitro.     

Temperature shift effect on the Chlorobaculum tepidum chlorosomes

Chlorobaculum [Cba.] tepidum is known to grow optimally at 48–52 °C and can also be cultured at ambient temperatures. In this paper, we prepared constant temperature, temperature shift, and temperature shift followed by backshift cultures and investigated the intrinsic properties and spectral features of chlorosomes from those cultures using various approaches, including temperature-dependent measurements on circular dichroism (CD), UV–visible, and dynamic light scattering. Our studies indicate that (1) chlorosomes from constant temperature cultures at 50 and 30 °C exhibited more resistance to heat relative to temperature shift cultures; (2) as temperature increases bacteriochlorophyll c (BChl c) in chlorosomes is prone to demetalation, which forms bacteriopheophytin c, and degradation under aerobic conditions. Some BChl c aggregates inside reduced chlorosomes prepared in low-oxygen environments can reform after heat treatments; (3) temperature shift cultures synthesize and incorporate more BChl c homologs with a smaller substituent at C-8 on the chlorin ring and less BChl c homologs with a larger long-chain alcohol at C-17³ versus constant-temperature cultures. We hypothesize that the long-chain alcohol at C-17³ (and perhaps together with the substituent at C-8) may account for thermal stability of chlorosomes and the substituent at C-8 may assist self-assembling BChls; and (4) while almost identical absorption spectra are detected, chlorosomes from different growth conditions exhibited differences in the rotational length of the CD signal, and aerobic and reduced chlorosomes also display different Q_y CD intensities. Further, chlorosomes exhibited changes of CD features in response to temperature increases. Additionally, we compare temperature-dependent studies for the Cba. tepidum chlorosomes and previous studies for the Chloroflexus aurantiacus chlorosomes. Together, our work provides useful and novel insights on the properties and organization of chlorosomes.     

1H NMR studies of a two-iron: two-sulfur ferredoxin from halobacterium of the dead sea

Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in ²H₂O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C₂H and C₄H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pK_a for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T₁ relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.     

Optical rotatory dispersion and circular dichroism of carbanilyl polysaccharides

Measurements of optical rotatory dispersion (ORD) and circular dichroism (CD) have been made in the range of 600-210 mμ for the β-glycan carbanilates as for instance, 2,3,6-tricarbanilylcellulose (I), 2,3,6-tricarbanilylmannan (II), 2,3-dicarbanilylcellulose (III), and octacarbanilylcellobiose (IV) and also for the α-glycan carbanilates, such as 2,3,6-tricarbanilylamylose (V), tricarbanilylpullulan (VI), 2,3-dicarbanilylamylose (VII), and octacarbanilylmaltose (VIII). Furthermore, the 2,3,4,6-tetracarbanilyl-α-methyl-glucopyranoside (IX) and the 1,2,3,4,6-pentacarbanilylglucose (X) have been measured in dioxane at 20°C. For the β-glycans a small negative CD in the region of 238–240 mμ and nearly symmetrical ORD curve with a crossover point at 238–240 mμ are found; this indicates a simple negative Cotton effect. In the case of α-glycosides, a strong negative CD with a maximum at 240–242 mμ and a strong positive CD with a maximum at 223–225 mμ were found; the ORD curves are asymmetrical and cross the abscissa in two places, at 241–243 and 220–222 mμ. With 2,3,4,6-tetracarbanilyl-α-methylglucoside (IX) no CD and ORD in the ultraviolet region and with 1,2,3,4,6-pentacarbanilyl-glucopyranoside (X) the ORD, but not the CD, could be measured. The ORD curve is nearly symmetrical, like those of the β-glycans but is of opposite sign. It seems impossible to discuss the striking difference of the CD and ORD spectra between the α-and the β-glycans in terms of contributions of single independant chromophores influenced by their individual different steric arrangements and their spatial relation to the glycosidic bond in C₁. The exciton theory of Moffitt, which is suitable for explaining the ORD and CD spectra of helical polymers, has been applied to α- and β-glycans. A structure with helical parts is proposed for the α-glycans while a nearly planar arrangement is assumed for the β-glycans.     

The protein amide <Superscript>1</Superscript>H<Superscript>N</Superscript> chemical shift temperature coefficient reflects thermal expansion of the N–H···O=C hydrogen bond

The protein amide ¹H^N chemical shift temperature coefficient can be determined with high accuracy by recording spectra at different temperatures, but the physical mechanism responsible for this temperature dependence is not well understood. In this work, we find that this coefficient strongly correlates with the temperature coefficient of the through-hydrogen-bond coupling, ^3hJ_NC′, based on NMR measurements of protein GB3. Parallel tempering molecular dynamics simulation suggests that the hydrogen bond distance variation at different temperatures/replicas is largely responsible for the ¹H^N chemical shift temperature dependence, from which an empirical equation is proposed to predict the hydrogen bond thermal expansion coefficient, revealing responses of individual hydrogen bonds to temperature changes. Different expansion patterns have been observed for various networks formed by β strands.     

Hemoglobin-water interactions in normal and sickle erythrocytes by proton magnetic resonance T1ϱ measurements

The dependence of the water proton magnetic resonance spin-lattice relaxation rate (T_1?^?1) in the rotating frame on the strength of the spin-locking (H₁) field has been investigated for packed oxy and deoxy normal and sickle erythrocytes at temperatures from 9 to 40 °C. The T_1?^?1 of oxy or deoxy normal erythrocytes shows no dependence on H₁ up to ~7 G at any temperature studied. On the other hand, T_1?^?1 decreases from about 40 s^?1 to 15 s^?1 (H₁ from 0 to ~7 G) for deoxygenated packed sickle cells at 40 °C. The magnitude of this variation of T_1?^?1 with H₁ decreases with decreasing temperature. Oxy packed sickle cells also show a dependence of T_1?^?1 on H₁ but the magnitude is <10% of that of the deoxygenated samples. These results suggest that water proton T_1?^?1 measurements are a sensitive probe of hemoglobin S polymerization and provide a novel technique for the study of slow water motions in these systems. The T_1?^?1 results are compared with low frequency T₁^?1 results of other investigators on hemoglobin S solutions. Analysis of the data suggests that water proton motions with correlation times of the order of 10^?5 s are present in the deoxygenated sickle cell samples at temperatures above 10 °C.     

Direct measurement of the melting temperature of supported DNA by electrochemical method

The development of biosensors based on DNA hybridization requires a more precise knowledge of the thermodynamics of the hybridization at a solid interface. In particular, the selectivity of hybridization can be affected by a lot of parameters such as the single-strand (ss)DNA density, the pH, the ionic strength or the temperature. The melting temperature, T_m, is in part a function of the ionic strength and of the temperature and therefore provides a useful variable in the control of the selectivity and sensitivity of a DNA chip. The electrochemical technique has been used to determine the T_m values when the probe is tethered by a DNA self-assembled monolayer (SAM). We have built a special thin layer cell, which allows the recording of the cyclic voltammogram under controlled temperature conditions. T_m has been determined by recording the thermogram (current versus temperature) of a redox indicator on a double-stranded hybrid (dsDNA) modified electrode and comparison with the corresponding ssDNA response. T_m of supported DNA varies linearly with the ionic strength. The stability of the SAMs has been considered and comparison between T_m in solution and on a solid support has been discussed.     

CCR2+ Inflammatory Dendritic Cells and Translocation of Antigen by Type III Secretion Are Required for the Exceptionally Large CD8+ T Cell Response to the Protective YopE69-77 Epitope during Yersinia Infection

During Yersinia pseudotuberculosis infection of C57BL/6 mice, an exceptionally large CD8⁺ T cell response to a protective epitope in the type III secretion system effector YopE is produced. At the peak of the response, up to 50% of splenic CD8⁺ T cells recognize the epitope YopE_69-77. The features of the interaction between pathogen and host that result in this large CD8⁺ T cell response are unknown. Here, we used Y. pseudotuberculosis strains defective for production, secretion and/or translocation of YopE to infect wild-type or mutant mice deficient in specific dendritic cells (DCs). Bacterial colonization of organs and translocation of YopE into spleen cells was measured, and flow cytometry and tetramer staining were used to characterize the cellular immune response. We show that the splenic YopE_69-77-specific CD8⁺ T cells generated during the large response are polyclonal and are produced by a “translocation-dependent” pathway that requires injection of YopE into host cell cytosol. Additionally, a smaller YopE_69-77-specific CD8⁺ T cell response (~10% of the large expansion) can be generated in a “translocation-independent” pathway in which CD8α⁺ DCs cross present secreted YopE. CCR2-expressing inflammatory DCs were required for the large YopE_69-77-specific CD8⁺ T cell expansion because this response was significantly reduced in Ccr2^-/- mice, YopE was translocated into inflammatory DCs in vivo, inflammatory DCs purified from infected spleens activated YopE_69-77-specific CD8⁺ T cells ex vivo and promoted the expansion of YopE_69-77-specific CD8⁺ T cells in infected Ccr2^-/- mice after adoptive transfer. A requirement for inflammatory DCs in producing a protective CD8⁺ T cell response to a bacterial antigen has not previously been demonstrated. Therefore, the production of YopE_69-77-specific CD8⁺ T cells by inflammatory DCs that are injected with YopE during Y. pseudotuberculosis infection represents a novel mechanism for generating a massive and protective adaptive immune response.     

Promiscuity, stability and cold adaptation of a newly isolated acylaminoacyl peptidase

We report on the characterisation of a member of the acylaminoacyl peptidase family, the first isolated from bacteria. The enzyme was obtained from the psychrophilic bacterium Sporosarcina psychrophila and shows the typical features of cold adaptation (low T_m, optimal temperature of 40 °C, poor thermal stability). It was also tested for substrate specificity, effect of metals, temperature dependence and structure stability and revealed promiscuous catalytic activity on at least two chemically distinct substrates, with k_cat/K_m values for ester hydrolysis and acylamino acids cleavage of 1.7 × 10⁴ s⁻¹ M⁻¹ and 6.2 × 10³ s⁻¹ M⁻¹, respectively. Despite some properties cannot be explained with current models, results report on the relevance of structural and catalytic properties for the successful adaptation to cold temperatures.     

Secondary Structure of Nuclease P1

The molecular conformation of nuclease P₁ in aqueous solution was investigated by measuring the optical rotatory dispersion (ORD) and circular dichroism (CD). The optical rotatory dispersion constant, λ was 281 nm. The Moffit-Yang parameters, a₀ and b₀, were ?2 and ?195, respectively. The ORD spectrum showed a minimum at 234 nm and the reduced mean residue rotation at 233 nm, [m]233, was ?5880. The CD spectrum showed a double minimum at 213 and 226 nm and the molecular ellipticity at 222 nm, [θ]22, was -11,900. From these data, the α-helix content was calculated to be 29 to 31 %. The computer fit of CD suggests that the α-structure is about 6% and the random coil is about 63%. The helical structure was found to be quite stable to denaturing reagents such as urea and guanidine hydrochloride. However, removal of zinc atoms from the enzyme resulted in disruption of the helical structure with inactivation.     

Molecular mobilities in chromaffin granules magnetic field dependence of proton T1 relaxation times

The magnetic field dependence of the NMR spin-lattice relaxation time of water protons in intact bovine chromaffin vesicles has been studied over the range 1.00–23.49 kG. The T₁ relaxation time shows a dispersion a t field values near 20 kG. The observed proton resonance arises mainly from solvent protons (¹H₂O), but the relaxation rate, which is a weighted average over all sites with which the solvent protons rapidly exchange (i.e., NH and OH protons), is dominated by exchangeable protons in the most slowly moving soluble component. The field dependence of the T₁ dispersion demonstrates the existence of a site of exchangeable protons for which τ_r = 1.9±0.5 ns at 3°C. This site is assigned to ATP and cationic groups to which its phosphate esters are complexed, since previously measured correlation times of epinephrine and the chromogranin backbone are nearly an order of magnitude too short to explain the T₁ dispersion. Quantitative estimates of the relative numbers of exchangeable protons on the different soluble components support this interpretation. The temperature dependence of T₁ of the peak due to exchangeable protons has also been measured over a temperature range ?3 to 25°C. T₁ lengthens by about 30% over this range and exhibits no discontinuous behavior, as would be expected if a gel transition or structural alterations in the storage complex occurred. T₁ lengthens by less than 10% in chromaffin granule pastes that have been maintained at 25°C for 24 h, indicating considerable thermal stability in the storage complex. Possible effects on the solvent T₁ due to paramagnetic ions have been considered with the conclusion that they are probably negligible or of minor significance.     

Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5 °C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔH_binding) crosses zero (T_H) and becomes favorable (negative). This T_H/activity upshift temperature is 15 °C for Klenow and 30 °C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG^‡, ΔH^‡, and TΔS^‡ that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp^‡ of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH^‡) switches from positive to negative.