Kinetic microscale thermophoresis

We established an extension of Microscale Thermophoresis (MST) to measure binding kinetics together with binding affinity in a single experimental run, by increasing the thermal dissipation of the sample. After the switch-off of an IR laser, that locally heated the sample, the temperature re-equilibrated within 250 ms. The kinetic relaxation fingerprints were extracted from the fluorescence changes back to thermodynamic equilibrium. We measured DNA hybridization on-rates and off-rates in the range between 104-106 M-1s-1 and 10-4-10-1 s‑1, respectively. We observed the expected exponential dependence of the DNA hybridization off-rates on salt concentration, strand length and inverse temperature. The measured on-rates showed a linear dependence on salt and weak if no dependence at all on length and temperature. For biological binding reactions with sufficient enthalpic contributions, Kinetic MST offers a robust and immobilization-free determination of kinetic rates and binding affinity and also in crowded solutions

Water cycles in a Hadean CO2 atmosphere drive the evolution of long DNA

HASH(0x7f8d29875448

Atomic Detail of Protein Folding Revealed by an Ab Initio Reappraisal of Circular Dichroism

Circular dichroism (CD) is known to be an excellent tool for the determination of protein secondary structure due to fingerprint signatures of α and β domains. However, CD spectra are also sensitive to the 3D arrangement of the chain as a result of the excitonic nature of additional signals due to the aromatic residues. This double sensitivity, when extended to time-resolved experiments, should allow protein folding to be monitored with high spatial resolution. To date, the exploitation of this very appealing idea has been limited, due to the difficulty in relating the observed spectral evolution to specific configurations of the chain. Here, we demonstrate that the combination of atomistic molecular dynamics simulations of the folding pathways with a quantum chemical evaluation of the excitonic spectra provides the missing key. This is exemplified for the folding of canine milk lysozyme protein

Atomic Detail of Protein Folding Revealed by an Ab Initio Reappraisal of Circular Dichroism

Circular dichroism (CD) is known to be an excellent tool for the determination of protein secondary structure due to fingerprint signatures of α and β domains. However, CD spectra are also sensitive to the 3D arrangement of the chain as a result of the excitonic nature of additional signals due to the aromatic residues. This double sensitivity, when extended to time-resolved experiments, should allow protein folding to be monitored with high spatial resolution. To date, the exploitation of this very appealing idea has been limited, due to the difficulty in relating the observed spectral evolution to specific configurations of the chain. Here, we demonstrate that the combination of atomistic molecular dynamics simulations of the folding pathways with a quantum chemical evaluation of the excitonic spectra provides the missing key. This is exemplified for the folding of canine milk lysozyme protein

Heated gas bubbles enrich, crystallize, dry, phosphorylate and encapsulate prebiotic molecules

Non-equilibrium conditions must have been crucial for the assembly of the first informational polymers of early life, by supporting their formation and continuous enrichment in a long-lasting environment. Here, we explore how gas bubbles in water subjected to a thermal gradient, a likely scenario within crustal mafic rocks on the early Earth, drive a complex, continuous enrichment of prebiotic molecules. RNA precursors, monomers, active ribozymes, oligonucleotides and lipids are shown to (1) cycle between dry and wet states, enabling the central step of RNA phosphorylation, (2) accumulate at the gas-water interface to drastically increase ribozymatic activity, (3) condense into hydrogels, (4) form pure crystals and (5) encapsulate into protecting vesicle aggregates that subsequently undergo fission. These effects occur within less than 30 min. The findings unite, in one location, the physical conditions that were crucial for the chemical emergence of biopolymers. They suggest that heated microbubbles could have hosted the first cycles of molecular evolution