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This work introduces a technology that combines fluorescence anisotropy decay with microscale-volume viscometry to investigate the compaction and dynamics of ribosome-bound nascent proteins. Protein folding in the cell, especially when nascent chains emerge from the ribosomal tunnel, is poorly understood. Previous investigations based on fluorescence anisotropy decay determined that a portion of the ribosome-bound nascent protein apomyoglobin (apoMb) forms a compact structure. This work, however, could not assess the size of the compact region. The combination of fluorescence anisotropy with microscale-volume viscometry, presented here, enables identifying the size of compact nascent-chain subdomains using a single fluorophore label. Our results demonstrate that the compact region of nascent apoMb contains 57–83 amino acids and lacks residues corresponding to the two native C-terminal helices. These amino acids are necessary for fully burying the nonpolar residues in the native structure, yet they are not available for folding before ribosome release. Therefore, apoMb requires a significant degree of post-translational folding for the generation of its native structure. In summary, the combination of fluorescence anisotropy decay and microscale-volume viscometry is a powerful approach to determine the size of independently tumbling compact regions of biomolecules. This technology is of general applicability to compact macromolecules linked to larger frameworks.

In recent years, it has become clear that assembly processes based on liquidliquid phase separation (LLPS) of protein and protein-nucleic acid mixtures, acting as membrane-less organelles, play an important role in cellular selfassembly processes. We discuss the combined effects of temperature, pressure, crowding and natural cosolvents on LLPS phenomena of proteins, with a particular focus on the effect of pressure. We studied various systems undergoing LLPS, including lysozyme, crystallin, elastin, LAF1, FUS, and proteins mimicking postsynaptic densities. X-ray and light scattering, microscopy and various spectroscopies were employed to reveal structural changes and mesoscopic phase states of the systems. Corresponding pressure-jump relaxation studies informed about the kinetics of protein droplet formation and their mechanism. Strikingly, for most cases studied thus far, LLPSs are more sensitive to pressure than the folding of proteins, suggesting that organisms inhabiting the deep sea and sub-seafloor sediments, under pressures up to 1 kbar and beyond, have to mitigate this pressure-sensitivity to avoid unwanted destabilization of their functional biomolecular condensates. We found that crowding and particular osmolytes, such as trimethylamine-N-oxide, an osmolyte upregulated in deep-sea fish, can significantly stabilize protein droplets under pressure. We then briefly discuss the effects of aqueous two-phase systems on the pressure dependence of ligand binding and enzymatic reactions as well as the conformational dynamics of nucleic acid structures. These findings are relevant for understanding cellular processes of piezophiles and might have significant bearings on biotechnological applications.

No abstract

Downstream regulatory element antagonist modulator (DREAM) is a member of the neuronal calcium sensors family and is expressed in the hippocampus. DREAM is involved in memory, learning, pain sensitivity, kinetics of potassium channels, calcium homeostasis, gene expression and enzymatic activity of presenilin. It has also been linked to pathologies such as Parkinson and Alzheimer's disease. The multifunctionality of this protein is due to its ability to interact with several intracellular partners such as presenilin, calmodulin, DNA and Kv channels. These interactions depend on the oligomerization state changes in DREAM which have been shown to be allosterically modulated upon Ca 2þ binding. The molecular mechanism of Ca 2þ regulation of DREAM interactions is yet to be determined, previous molecular dynamics data proposed that a hydrophobic residues network connecting the C-and N-terminal domains is involved interdomain communication since several hydrophobic residues sidechains reposition upon Ca 2þ binding, acting as molecular switch between the apo and the Ca 2þ bound state. The Trp169 residue may be a key residue in this interdomain communication as its position in the hydrophobic cavity between the N-and C-terminal domain and it is highly conserved among NCS proteins. To investigate the role of Trp169 in allosteric communication, we have mutated it to Ala and carry out molecular dynamics studies in the presence and absence of Ca 2þ using classical molecular dynamics and accelerated Gaussian molecular dynamic. These simulations show a different positioning of residues in the hydrophobic networks such as Tyr174 and Phe218, as well as an impact on the formation of the Glu 165-Lys 87 salt bridge, suggesting the crucial role of W169 residue in the reorientation of the amino acids in the hydrophobic network connecting the N-and Cterminal.