Monomolecular Transition Research Articles

A convenient tool for studying the stability of proteins and nucleic acids

The aim of this work is to discuss the thermodynamic properties, obtained by differential scanning calorimetry (DSC), of the thermal transition of proteins and nucleic acids and to analyze these data using statistical thermodynamic relations. The denaturation of the ordered, specific structures of biological macromolecules is a cooperative process and in many cases the macromolecules undergo a two-state transition. Differential scanning calorimetry, giving direct thermodynamic information, has proved to be very useful in clarifying the energetics of macromolecule transitions and in characterizing their thermal stability. Here, various examples are discussed: i) the equilibrium thermal denaturation of ribonuclease A, a model for the use of DSC by following the temperature-unfolding of the proteins, a monomolecular transition; ii) the equilibrium thermal dissociation of a DNA double helix in two strands, an example of how DSC is used to follow a bimolecular process; iii) an example of the use of DSC for studying the melting of unimolecular and tetramolecular DNA quadruple-helices.

Thermal stability and energetics of 15‐mer DNA duplex interstrand crosslinked by trans‐diamminedichloroplatinum(II)

The effect of the location of the interstrand cross-link formed by trans-diamminedichloroplatinum(II) (transplatin) on the thermal stability and energetics of 15-mer DNA duplex has been investigated. The duplex containing single, site-specific cross-link, thermodynamically equivalent model structures (hairpins) and nonmodified duplexes were characterized by differential scanning calorimetry, temperature-dependent uv absorption, and circular dichroism. The results demonstrate that the formation of the interstrand cross-link of transplatin does not affect pronouncedly thermodynamic stability of DNA: the cross-link induces no marked changes not only in enthalpy, but also in "reduced" (concentration independent) monomolecular transition entropy. These results are consistent with the previous observations that interstrand cross-links of transplatin structurally perturb DNA only to a relatively small extent. On the other hand, constraining the duplex with the interstrand cross-link of transplatin results in a significant increase in thermal stability that is primarily due to entropic effects: the cross-link reduces the molecularity of the oligomer system from bimolecular to monomolecular. Importantly, the position of the interstrand cross-link within the duplex modulates cooperativity of the melting transition of the duplex and consequently its thermal stability.

The secondary structure of guide RNA molecules from Trypanosoma brucei.

RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is characterized by the insertion and deletion of uridine nucleotides into incomplete mRNAs. Key molecules in the process are guide RNAs which direct the editing reaction by virtue of their primary sequences in an RNA-RNA interaction with the pre-edited mRNAs. To understand the molecular details of this reaction, especially potential RNA folding and unfolding processes as well as assembly phenomena with mitochondrial proteins, we analyzed the secondary structure of four different guide RNAs from Trypanosoma brucei at physiological conditions. By using structure-sensitive chemical and enzymatic probes in combination with spectroscopic techniques we found that the four molecules despite their different primary sequences, fold into similar structures consisting of two imperfect hairpin loops of low thermodynamic stability. The molecules melt in two-state monomolecular transitions with Tms between 33 and 39 degrees C and transition enthalpies of -32 to -38 kcal/mol. Both terminal ends of the RNAs are single-stranded with the 3' ends possibly adopting a single-stranded, helical conformation. Thus, it appears that the gRNA structures are fine tuned to minimize stability for an optimal annealing reaction to the pre-mRNAs while at the same time maximizing higher order structural features to permit the assembly with other mitochondrial components into the editing machinery.

Thermodynamics of DNA hairpins: contribution of loop size to hairpin stability and ethidium binding.

A combination of calorimetric and spectroscopic techniques was used to evaluate the thermodynamic behavior of a set of DNA hairpins with the sequence d(GCGCTnGCGC), where n = 3, 5 and 7, and the interaction of each hairpin with ethidium. All three hairpins melt in two-state monomolecular transitions, with tm's ranging from 79.1 degrees C (T3) to 57.5 degrees C (T7), and transition enthalpies of approximately 38.5 kcal mol-1. Standard thermodynamic profiles at 20 degrees C reveal that the lower stability of the T5 and T7 hairpins corresponds to a delta G degree term of +0.5 kcal mol-1 per thymine residue, due to the entropic ordering of the thymine loops and uptake of counterions. Deconvolution of the ethidium-hairpin calorimetric titration curves indicate two sets of binding sites that correspond to one ligand in the stem with binding affinity, Kb, of approximately 1.8 x 10(6) M-1, and two ligands in the loops with Kb of approximately 4.3 x 10(4) M-1. However, the binding enthalpy, delta Hb, ranges from -8.6 (T3) to -11.6 kcal mol-1 (T7) for the stem site, and -6.6 (T3) to -12.7 kcal mol-1 (T7) for the loop site. Relative to the T3 hairpin, we obtained an overall thermodynamic contribution (per dT residue) of delta delta Hb = delta(T delta Sb) = -0.7(5) kcal mol-1 for the stem sites and delta delta Hb = delta(T delta Sb) = -1.5 kcal mol-1 for the loop sites. Therefore, the induced structural perturbations of ethidium binding results in a differential compensation of favorable stacking interactions with the unfavorable ordering of the ligands.

I.R. spectroscopic characterization of zeolite catalysts for the shape selective conversion of polynuclear aromatics

1- and 2-methylnaphthalene were reacted over acid zeolites with different pore widths, viz. HZSM-5, HZSM-12 and HY. The catalytic conversion was carried out in a flow-type i.r. cell. So, changes in the acid OH groups of the zeolites and the formation of carbonaceous deposits could be monitored while the catalysts were working. On HY at 300°C, isomerization and transalkylation occur while high molecular coke with a polynuclear aromatic structure is built up. On HZSM-12, all reactions which require bulky transition states are suppressed on account of the space constraints inside the pores. Hence, isomerization via monomolecular transition states is the predominant reaction in HZSM-12. The carbonaceous deposits in HZSM-12 seem to retain the methylnaphthalene building block up to ca. 300°C. In HZSM-5 with its relatively narrow pores, all reactions are absent which lead to higher molecular weight products (e.g., dimethylnaphthalenes, coke). Very low conversions of the methylnaphthalenes indicate that, even at high temperatures, diffusion of the polynuclear feed hydrocarbons in HZSM-5 is severely impeded.

Singlet oxygen in biological systems

Electronically excited molecular oxygen ( 2Δ g O 2) is of increasing interest in biology and medicine. Several methods have been employed for detection and identification, including chemical trapping and the continuous and non-invasive monitoring of low-level chemiluminescence in the visible and infrared spectral region due to the bi- and monomolecular transition of the excited molecules to the ground state. Performing spectral analysis and measuring the effect of D 2O and/or 1O 2 quenchers, evidence for the involvement of this reactive oxygen species in a variety of non-enzymatic and enzyme-catalyzed reaction pathways of biological and medical importance has been provided. The use of the intact perfused rat liver allowed direct monitoring of the metabolism of hydroperoxides and quinones by measuring the photoemission from the organ surface. Occurrence of 1O 2 in cells, generated under physiological conditions or under conditions of oxidative stress, has implications on biological functions at molecular and cellular level. Besides reaction with lipids, inactivation of several enzymes by means of amino acid modification has been demonstrated; also damage of constituents of DNA has been reported. Recently the effect of 1O 2 on the biological activity of intact DNA was investigated, showing the loss of transforming activity of pBR 322 in E. coli upon 1O 2 exposure. Quantification of 1O 2 has been performed during these measurements; quantification of steady states of 1O 2 in more complex systems remains the topic of present work.

Metal binding to yeast aminopeptidase I.

Binding of Zn(II) and Co(II) to homogeneous dodecameric aminopeptidase I from yeast was studied. Apparent binding constants were estimated from the dependence of enzyme activity on metal levels in solution as established by metal buffer systems. The binding curves were sigmoidal with Hill coefficients of 1.2-1.6, depending on the metal and on pH. Metal concentrations at half-saturation were 28 nM with Zn(II), and 390 nM with Co(II) at pH 7.0, they increased with decreasing pH. Equilibrium binding studies yielded binding stoichiometries of 0.5-0.6 mol metal/mol subunit for both Zn(II) and Co(II). However, after oxidation of Co(II)-substituted enzyme with H2O2 almost stoichiometric amounts of cobalt (greater than 0.9 mol/mol subunit) were found. The oxidized enzyme was inactive and did not exchange cobalt with the solvent. Native aminopeptidase I was also affected by H2O2, however only after prolonged incubation and at a much lower rate. Apoenzyme modified by ethoxyformic anhydride could not be reconstituted by Zn(II) or Co(II). On the other hand, either metal afforded full protection against ethoxyformic anhydride. This finding, and the pH dependence of stability constants suggest that histidine residues are involved in metal binding. Both the reconstitution of active enzyme from apoprotein and metal and its inactivation due to loss of metal are slow processes with half-times in the minute range. The rate of reconstitution did not depend on Zn(II) concentration. The rates of metal loss were not enhanced by chelating agents containing carboxylate functions. In contrast, chelators coordinating via nitrogen atoms and 2,3-dimercaptopropanol accelerated dissociation. A model is proposed that accounts for the substoichiometric metal contents of aminopeptidase I and for the slow time course of reconstitution. It is assumed that reconstitution involves a slow, monomolecular transition, leading to an active enzyme conformation with lower affinity for metal ions.

Formation of hairpin helices in uridylic acid oligomers

The behavior of uridylic acid oligomers under heating has been studied by equilibrium and kinetic experiments, either in 0.5 M CsCl or in presence of spermine. The absorption melting curves can be accounted for by a cooperative monomolecular transition. Temperature-jump results are consistent with the melting of double stranded structure. it is concluded that uridylic acid oligomers can form hairpin helices.