Conformational studies of poly-L-alanine in water

The conformational properties of poly-L -alanine have been examined in aqueous solutions in order to investigate the influence of hydrophobic interactions on the helix–random coil transition. Since water is a poor solvent for poly-L -alanine, water-soluble copolymers of the type (D , L -lysine)_m–(L alanine)_n-(D , L -lysine)_m, having 10, 160, 450, and 1000 alanyl residues, respectively, in the central block, were synthezised. The optical rotatory dispersion of the samples was investigated in the range 190–500 mμ, and the rotation at 231 mμ was related to the α-helix content, θ_H, of the alanine section. In salt-free solutions, at neutral pH, the three large polymers show high θ_H values, which are greatly reduced when the temperature is increased from 5 to 80°C. No helicity was observed for the small (n = 10) polymer. By applying the Lifson-Roig theory, the following parameters were obtained for the transition of a residue from a coil to a helical state: ν = 0.012; ΔH = ?190 ± 40 cal./mole; ΔS = ?0.55 ± 0.12 e.u. Since ΔH and ΔS differ from the values expected for a process involving only the formation of a hydrogen bond, and in a manner predicted by theories for the influence of hydrophobic bonding on helix stability, it is concluded that a hydrophobic interaction is also involved. In the presence of salt (0.2M NaCl), or when the ε-amino groups of the lysyl residues are not protonated (pH = 12), the helical form of the two large polymers (n = 450 and n = 1000) is more stable than in water. Since the electrostatic repulsion between the lysine end blocks is greatly reduced under these conditions, the alanine helical sections fold back on themselves, and this conformation is stabilized by interchain hydrophobia bonds. This structure was predicted by the theory for the equilibrium between such interacting helices, non-interacting helices, and the random coil.     

Studies on poly(L-lysine50, L-tyrosine50)-DNA interaction

Interaction between poly(Lys⁵⁰, Tyr⁵⁰) and DNA has been studied by absorption, circular dichroism (CD), and fluorescence spectroscopy and thermal denaturation in 0.001M Tris, pH 6.8. The binding of this copolypeptide to DNA results in an absorbance enhancement and fluorescence quenching on tyrosine. There is also an increase in the tyrosine CD at 230 nm. The CD of DNA above 250 nm is slightly shifted to the longer wavelength which is qualitatively similar to, but quantitatively much smaller than, that induced by polylysine binding. At physiological pH the poly(Lys⁵⁰, Tyr⁵⁰)–DNA complex is soluble until there is one lysine and one tyrosine per nucleotide in the complex. The same ratio of amino acid residues to nucleotide has also been observed in copolypeptide-bound regions of the complex. The addition of more poly(Lys⁵⁰, Tyr⁵⁰) to DNA yields a constant melting temperature, T_m′, for bound base pairs at 90°C which is close to that of polylysine-bound DNA under the same condition. The melting temperature, T_m, of free base pairs at about 60°C on the other hand, is increased by 10°C as more copolypeptide is bound to DNA. As the temperature is raised, both absorption and CD spectra of the complexes with high coverage are changed, suggesting structural alteration, perhaps deprotonation, on bound tyrosine. The results in this report also suggest that intercalation of tyrosine in DNA is unlikely to be the mode of binding.     

Helix-coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes

Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10^?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises T_m, the melting temperature of free base pairs. The linear dependence of T_m on log Na⁺ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na⁺ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs T_m′ remains constant at various ionic strengths. On the other hand, the T_m′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of T_m′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na⁺ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers T_m′. This explanation is supported by circular dichroism (CD) results.     

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.     

Interaction of poly-L-lysine with nucleic acids. II. Poly(A+U), poly(A+2U), and rice dwarf virus RNA

The optical density–temperature profile of double-stranded poly(A + U), triple stranded poly(A + 2U), and double-stranded RNA from rice dwarf virus in solutions with and without poly-L -lysine has been examined. When poly-L -lysine is added, more than one melting temperature T_m is observed for poly(A + U) and poly(A + 2U). One of them is considered to correspond to the melting of the polynucleotide molecule free from poly-L -lysine, and another to the melting of a polynucleotide–poly-L -lysine complex. For rice dwarf virus RNA, the T_m assignable to the complex is not found to be lower than 99°C. In every case, however, the hyperchromicity observed at the T_m of the free poly-nucleotide molecule is lowered linearly as the amount of poly-L -lysine added to the solution increases. This fact is taken as indicating that there is a stoichiometric complex formed. The stoichiometric ratio lysine/nucleotide in each complex is determined by examining the relation between the amount of poly-L -lysine added to the solution and the percentage of hyperchromicity remaining at T_m of the free polynucleotide molecule. The ratio is found to be 2/3 for all of the three complexes. A discussion is given on the molecular conformations of four types of polynucleotide–polylysine complex hitherto found: (A) double-stranded DNA plus poly-L -lysine in which the lyslne/nucleotide ratio is 1, (B) three-stranded RNA [poly(A + 2U)] plus poly-L -lysine in which the ratio is 2/3, (C) double-stranded RNA [poly (A + U) or rice dwarf virus RNA] plus poly-L -lysine in which the ratio is 2/3, and (D) double-stranded RNA [poly(I + C)] plus poly-L -lysine in which the ratio is 1/2.     

Use of principal component analysis in interpretation of deoxyribonucleic acid relatedness

Principal component analysis (PCA) of published DNA-relatedness data showed the usefulness of this method in displaying relationships among closely related bacteria. Very similar ordinations were obtained when relative binding ratios (RBR) at 60°C or 75°C or ΔT _m values were used to form the data matrix. A curvilinear relationship and a (quasi) linear relationship were found, respectively, between 75°C and 60°C RBR and ΔT _m and 60°C RBR. These statistical relationships explain the similarity of PCA results using either measurement (60°C RBR, 75°C RBR, or ΔT _m). Use of PCA is suggested to delineate groups within a complex set of DNA-relatedness data. The level of ΔT _m within groups and between groups should help decide whether these groups are genospecies.     

Helix–coil transition and conformational studies of protamine–DNA complexes

Protamine–DNA complexes prepared by the method of direct and slow mixing in 2.5 × 10^?4M EDTA, pH 8.0, have been studied by thermal denaturation and circular dichroism. The complexes show biphasic melting with T_m at about 50 °C corresponding to the melting of free DNA regions and T_m′ at about 92 °C corresponding to the melting of protamine-bound regions. In protamine-bound regions there are 1.38 amino acid residues per nucleotide, indicating a nearly completely charge neutralization. T_m is increased but T_m′ is not when the ionic strength of the buffer is raised. This also supports a full charge neutralization in protamine-bound regions. The circular dichroism of the complexes can be decomposed into two components, Δ_ε0 of free DNA regions in B-form conformation and Δ_εb of protamine-bound regions in a characteristic conformation neither that of B- nor C-form but somewhere between them.     

Characterization of CaMKIIα holoenzyme stability

Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long‐term memory formation and other Ca²⁺‐dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X‐ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent T_m = 36°C), which is slightly stabilized by ATP/MgCl₂ binding (apparent T_m = 40°C) and significantly stabilized by regulatory segment binding (apparent T_m = 60°C). We crystallized the kinase domain of CaMKII bound to p‐coumaric acid in the active site. This structure reveals solvent‐exposed hydrophobic residues in the substrate‐binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent T_m ~ 90°C), and the holoenzyme structure has multiple unfolding transitions ranging from ~60°C to 100°C. Using MP, we compared a CaMKIIα holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact.     

Sex- and age-related changes in the biophysical properties of cuticular lipids of the housefly,Musca domestica

We examined the biophysical properties of cuticular lipids isolated from the housefly, Musca domestica. Melting temperatures (T_m) of surface lipids isolated from female houseflies decreased from 39.3 °C to 35.3 °C as the females attained sexual maturity and produced sex pheromone, whereas those prepared from males did not change with age. Lipids melted over a 10–25 °C temperature range, and their physical properties were a complex function of the properties of the component lipids. The T_m of total cuticular lipids was slightly below that of cuticular hydrocarbons (HC), the predominant lipid fraction. Hydrocarbons were further fractionated into saturated, unsaturated, and methyl-branched components. The order of decreasing T_m was total alkanes > total HCs > methyl-branched alkanes > alkenes. For 1-day-old flies, measured T_ms of hydrocarbons were 1.3–5.5 °C lower than T_ms calculated from a weighted average of T_ms for saturated and unsaturated components. For 4-day-old flies, calculated T_ms underestimated T_m by 11–14 °C. © 1995 Wiley-Liss, Inc.     

Polynucleotides. 13. Stoichiometric and thermodynamic studies of polynucleotide helices with non-complementary residues

Stoichiometry and thermodynamic properties of polyadenylate–polyuridylate helices containing varying proportions of near-randomly distributed non-complementary uridine residues were charactrized from an analysis of their mixing curves and melting profiles measured at 259 nm and at appropriate longer wavelength isochromic points. The noncomplementary residues in this homopolymer–copolymer system (in which the homopolymer has the capacity to readjust with respect to the residues with which it is in opposition) show absolute preference for an extrahelical conformation even when situated in … AAUAA … sequences and must occur therefore as single loops. As the frequency of extrahelical residues in creases, the electrostatic energy of these complexes becomes greater, and is particularly severe for the three-stranded helices. Thus, an adenyl-ate-uridylate copolymer containing 35.2 mole percent uridine residues does not form a three-stranded complex with polyuridylate even in 0.7M Na⁺at O°C. The imperfections introduced into the helix lattice by extrahelical residues decrease the cooperativity of thermal denaturation as well as T_m. However, for the helices with extrahelical residues in low frequency (～1 per helix turn) only small increases in concentration of charge-neutralizing ions are required to bring T_m to the level of their perfect analogs. Two-stranded helices with a higher density of extra helical residues (～5 per helix turn) show [Na⁺] dependence of T_m characteristic of perfect three-stranded helices. These findings together with the absence of an effect of these imperfections on the hypochromicity per base-pair suggest only minimal disruption of helix continuity or distortion of stacking interactions that normally in volve the base pairs or triplets.     

Unfolding properties of recombinant human serum albumin products are due to bioprocessing steps

We have used differential scanning calorimetry (DSC) to determine the unfolding properties of commercial products of human serum albumin (HSA) prepared from pooled human blood, transgenic yeast, and transgenic rice. The initial melting temperatures (T_m1) for the unfolding transitions of the HSA products varied from 62°C to 75°C. We characterized the samples for purity, fatty acid content, and molecular weight. The effects of adding fatty acids, heat pasteurization, and a low pH defatting technique on the transition temperatures were measured. Defatted HSA has a structure with the lowest stability (T_m of ～62°C). When fatty acids are bound to HSA, the structure is stabilized (T_m of ～64–72°C), and prolonged heating (pasteurization at 60°C) results in a heat‐stabilized structural form containing fatty acids (T_m of ～75–80°C). This process was shown to be reversible by a low pH defatting step. This study shows that the fatty acid composition and bioprocessing history of the HSA commercial products results in the large differences in the thermal stability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:62–69, 2015     

Germination and emergence of Sorghum bicolor. genotypic and environmentally induced variation in the response to temperature and depth of sowing

Abstract The germination of Sorghum bicolor seeds of 9 genotypes was tested at temperatures between 8°C and 48°C on a thermal gradient plate. Samples were tested from three regions of the panicle expected to differ in temperature during grain filling. Seeds of a tenth genotype, SPV 354, produced in controlled-environment glasshouses at different panicle temperatures, were tested similarly. In addition, the emergence of SPV 354 was measured from planting depths of 2 and 5 cm at mean soil temperatures of 15, 20 and 25°C. Four methods of calculating mean germination rate for the nine genotypes were compared. Germination characters like base, optimum and maximum temperature (T_b, T_o, T_m), thermal time (θ)and the germination rate at T_o(R_max showed only small differences between methods. There was a range of genotypic variation in all characters: T_b 8.5–11.9°C; T_o, 33.2–37.5°C; T_m, 46.8–49.2°C; θ, 23.4–38.0°Cd; R_max, 0.69–1.14-d_-1. In contrast, mean germinability (G) was between 90% and 100% over the temperature range 13–40°C. Panicle temperature had no effect on any germination character in SPV 354. However, deeper burial increased θ for emergence and decreased G, irrespective of soil temperature except at 5 cm. Increasing panicle temperature, by reducing seed size, reduced G and increased θ by about 10% only at 15°C and 5 cm depth.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

Solution conformations of B. subtilis ribosomal 5S RNA: a calorimetric study

The unfolding of B. subtilis 5S RNA is examined by direct calorimetric measurement in the presence of various concentrations of Na⁺ and Mg²⁺. The composite differential scanning calorimetry (DSC) curve is analyzed into 3–5 individual two-state melting transitions. In the absence of added Na⁺ or Mg²⁺, the 5S RNA segments melt together at T_m = 40°C. Addition of Na⁺ stabilizes the molecular structure (T_m = 56°C) and widens the melting temperature range, so that up to five component transitions are observed. Addition of Mg²⁺ alone produces a very stable structure (T_m = 75°C) with highly cooperative melting. Finally, addition of both Na⁺ and Mg²⁺ produces the highest stability (T_m = 76°C). The results are interpreted according to hypothetical secondary and tertiary base-pairing schemes. The conformational changes demonstrated here may facilitate the movement of the protein synthesis machinery during RNA translation.     

Chitosan-Soyprotein Interaction as Determined by Thermal Unfolding Experiments

Chitosan interaction with soybean β-conglycinin β₃ was investigated by thermal unfolding experiments using CD spectroscopy. The negative ellipticity of the protein was enhanced with rising solution temperature. The transition temperature of thermal unfolding of the protein (T _m) was 63.4 °C at pH 3.0 (0.15 M KCl). When chitosan was added to the protein solution, the T _m value was elevated by 7.7 °C, whereas the T _m elevation upon addition of chitosan hexamer (GlcN)₆ was 2.2 °C. These carbohydrates appear to interact with the protein stabilizing the protein structure, and the interaction ability could be evaluated from the T _m elevation. Similar experiments were conducted at various pHs from 2.0 to 3.5, and the T _m elevation was found to be enhanced in the higher pH region. We conclude that chitosan interacts with β-conglycinin through electrostatic interactions between the positive charges of the chitosan polysaccharide and the negative charges of the protein surface.     

Effect of temperature on development rate and intrinsic rate of increase ofAphis gossypii reared on greenhouse cucumbers

Thermal requirements for development and life table statistics ofAphis gossypii Glover (Homoptera, Aphididae) were determined over a range of constant temperatures from 10 to 30°C. The lower development threshold and the sum of effective temperatures were 6.9°C and 90.1°C, respectively, for preimaginal development, and 5.8°C and 113.6°C from birth to the onset of reproduction. Mean total fecundity ranged from 36 larvae per female at 10°C to 76 larvae at 30°C. On a time scale of days, net reproductive rate (R _o ) increased with increasing temperature while generation time (T) decreased causing the intrinsic rate of increase (r _m ) to increase linearly from 0.115 to 0.465. On a day-degree scaler _m only varied from 0.019 to 0.028 because the growth ofR _o was compensated by an increase inT with increase in temperature. The nearly constantr _m in terms of day-degrees, over a wide range of temperatures, greatly simplifies the prediction of future population numbers ofA. gossypii.     

Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 × 10⁵, and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T _g, T _m, and T _d (10%) were observed at −1.1°C, 158.8°C, and 252.7°C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.     

Toward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stability

Cutinases are powerful hydrolases that can cleave ester bonds of polyesters such as poly(ethylene terephthalate) (PET), opening up new options for enzymatic routes for polymer recycling and surface modification reactions. Cutinase from Aspergillus oryzae (AoC) is promising owing to the presence of an extended groove near the catalytic triad which is important for the orientation of polymeric chains. However, the catalytic efficiency of AoC on rigid polymers like PET is limited by its low thermostability; as it is essential to work at or over the glass transition temperature (T_g) of PET, that is, 70°C. Consequently, in this study we worked toward the thermostabilization of AoC. Use of Rosetta computational protein design software in conjunction with rational design led to a 6°C improvement in the thermal unfolding temperature (T_m) and a 10‐fold increase in the half‐life of the enzyme activity at 60°C. Surprisingly, thermostabilization did not improve the rate or temperature optimum of enzyme activity. Three notable findings are presented as steps toward designing more thermophilic cutinase: (a) surface salt bridge optimization produced enthalpic stabilization, (b) mutations to proline reduced the entropy loss upon folding, and (c) the lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the T_m of the global structure. Proteins 2016; 84:60–72. © 2015 Wiley Periodicals, Inc.     

Oligonucleotides containing a peptide internucleotide linkage. A novel class of modified oligonucleotides

Oligonucleotide analogues containing one or a few glycine, L-, and D-alanine residues instead of phosphodiester internucleotide linkages were synthesized (C3′-NH-C(O)-CH(X)-NH-C(O)-C4′, where X = H, (S)-CH₃, and (R)-CH₃. The stability of the duplexes of modified oligonucleotides with their wild-type complements was studied. The incorporation of glycine and L-alanine residues into internucleotide linkages was shown to noticeably decrease the stability of modified duplexes as compared to that of native ones (ΔT _m∼−2°C per modification), whereas analogues containing D-alanine linkers form duplexes with increased stability (ΔT _m∼+2°C per modification).