Fluorescent Analogues of FRH Peptide: Cu(II) Binding and Interactions with ds-DNA/RNA

Four novel peptidoids, derived from the Phe-Arg-His (FRH) peptide motif, were prepared by replacing the histidine heterocycle with triazole and consequent triazole-fluorophore (coumarin) extension and also replacing arginine with less voluminous lysine. So the constructed Phe-Lys-Ala(triazole) (FKA(triazole)) peptidoids bind Cu2+ cations in water with a strong, nanomolar affinity comparable to the parent FRH and its known analogs, demonstrating that triazole can coordinate copper similarly as histidine. Moreover, even short KA(triazole)coumarin showed submicromolar affinity to Cu2+. Only FKA(triazole)coumarin with free amino groups and its shorter analog KA(triazole)coumarin showed strong induced CD spectra upon Cu2+ cation binding. Thus, KA(triazole)coumarin can be considered as the shortest peptidoid sequence with highly sensitive fluorescent and chiral CD response for Cu2+ cation, encouraging further studies with other metal cations. The FKA(triazole) coumarin peptidoids show biorelevant, 10 µM affinity to ds-DNA and ds-RNA, binding within DNA/RNA grooves. Intriguingly, only peptidoid complexes with Cu2+ strongly stabilize ds-DNA and ds-RNA against thermal denaturation, suggesting significant interactions of Cu2+ cation within the DNA/RNA binding site.

Equivalence of the Transition Heat Capacities of Proteins and DNA

It has been reported for many globular proteins that the native heat capacity at 25°C, per gram, is the same. This has been interpreted to indicate that heat capacity is a fundamental property of native proteins that provides important information on molecular structure and stability. Heat capacities for both proteins and DNA has been suggested to be related to universal effects of hydration/solvation on native structures. Here we report on results from thermal denaturation analysis of two well-known proteins, human serum albumin and lysozyme, and a short DNA hairpin. The transition heat capacities at the Tm for the three molecules were quantitatively evaluated by differential scanning calorimetry. When normalized per gram rather than per mol the transition heat capacities were found to be precisely equivalent. This observation for the transition heat capacities of the proteins is consistent with previous reports. However, an identical transition heat capacity for DNA has not been reported and was unexpected. Further analysis of the collected data suggested a mass dependence of hydration effects on thermal denaturation that is preserved at the individual protein amino acid and DNA base levels. Equivalence of transition heat capacities suggests the possibility of a universal role of hydration effects on the thermal stability of both proteins and DNA.

Conformational Consequences for Compatible Osmolytes on Thermal Denaturation

Compatible osmolytes are a broad class of small organic molecules employed by living systems to combat environmental stress by enhancing the native protein structure. The molecular features that make for a superior biopreservation remain elusive. Through the use of time-resolved and steady-state spectroscopic techniques, in combination with molecular simulation, insight into what makes one molecule a more effective compatible osmolyte can be gained. Disaccharides differing only in their glycosidic bonds can exhibit different degrees of stabilization against thermal denaturation. The degree to which each sugar is preferentially excluded may explain these differences. The present work examines the biopreservation and hydration of trehalose, maltose, and gentiobiose.

Denaturation of the SARS-CoV-2 spike protein under non-thermal microwave radiation

SARS-CoV-2, the virus that causes COVID-19, is still a widespread threat to society. The spike protein of this virus facilitates viral entry into the host cell. Here, the denaturation of the S1 subunit of this spike protein by 2.45 GHz electromagnetic radiation was studied quantitatively. The study only pertains to the pure electromagnetic effects by eliminating the bulk heating effect of the microwave radiation in an innovative setup that is capable of controlling the temperature of the sample at any desired intensity of the electromagnetic field. This study was performed at the internal human body temperature, 37 °C, for a relatively short amount of time under a high-power electromagnetic field. The results showed that irradiating the protein with a 700 W, 2.45 GHz electromagnetic field for 2 min can denature the protein to around 95%. In comparison, this is comparable to thermal denaturation at 75 °C for 40 min. Electromagnetic denaturation of the proteins of the virus may open doors to potential therapeutic or sanitation applications.

Site-Specific Synthesis of N4-Acetylcytidine in RNA Reveals Physiological Duplex Stabilization

N4-acetylcytidine (ac4C) is a post-transcriptional modification of RNA that is conserved across all domains of life. All characterized sites of ac4C in eukaryotic RNA occur in the central nucleotide of a CCG consensus sequence. However, the thermodynamic consequences of cytidine acetylation in this context have never been assessed due to its challenging synthesis. Here we report the synthesis and biophysical characterization of ac4C in its endogenous eukaryotic sequence context. First, we develop a synthetic route to homogenous RNAs containing electrophilic acetyl groups. Next, we use thermal denaturation to interrogate the effects of ac4C on duplex stability and mismatch discrimination in a native sequence found in human ribosomal RNA. Finally, we demonstrate the ability of this chemistry to incorporate ac4C into the complex modification landscape of human tRNA, and use duplex melting combined with sequence analysis to highlight a potentially unique enforcing role for ac4C in this setting. By enabling the analysis of nucleic acid acetylation in its physiological sequence context, these studies establish a chemical foundation for understanding the function of a universally-conserved nucleobase in biology and disease.

pH dependence of C·A, G·A and A·A mismatches in the stem of precursor microRNA-31

MicroRNAs (miRNAs) are important regulators of post-transcriptional gene expression. Mature miRNAs are generated from longer transcripts (primary, pri- and precursor, pre-miRNAs) through a series of highly coordinated enzymatic processing steps. The sequence and structure of these pri- and pre-miRNAs play important roles in controlling their processing. Both pri- and pre-miRNAs adopt hairpin structures with imperfect base pairing in the helical stem. Here, we investigated the role of three base pair mismatches (A·A, G·A, and C·A) present in pre-miRNA-31. Using a combination of NMR spectroscopy and thermal denaturation, we found that the three base pair mismatches displayed unique structural properties, including varying dynamics and sensitivity to solution pH. These studies deepen our understanding of how the physical and chemical properties of base pair mismatches influence RNA structural stability.

Kinetics And Thermodynamic Properties of Trametes Polyzona WRF03 Laccase

The effect of thermal treatment on the activity of laccase from Trametes polyzona WRF03 was studied at pH and temperature ranges of 3.0 to 6.5 and of 40 to 70 oC respectively. Kinetic data revealed that the heat inactivation of Trametes polyzona WRF03 laccase (TpL) was pH dependent and followed first-order kinetics. There was a positive correlation between activation energy (Ea) for thermal inactivation of TpL and the reaction pH. Highest activation energy, Ea, value of 175.49 kJ/mol was obtained at pH 6.0. On the contrary, the z-value decreased with a lowest value of 12.37 oC at pH 6.0. The high Ea value and low z-value were indicative of the thermo-stable nature of TpL which suggests that pH 6.0 had a compensatory stabilizing effect on TpL against its thermal denaturation. There was a gradual decrease in the enthalpy of denaturation (∆Ho) and Gibb’s free-energy with every 10 % rise in temperature within the investigated pH range, suggesting that TpL was more stable at 40 oC. Positive values of entropy of inactivation (ΔSº) at each temperature indicated that there was no aggregation during the inactivation processes. Thus, these results provided useful information about the behaviour of TpL under certain pH and temperature combination with respect to biotechnological application. Thus, the kinetic and thermodynamic data could be used to design a model to predict the thermal inactivation of TpL during industrial application.