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One of the planet's more understudied ecosystems is the deep biosphere, where organisms can experience high hydrostatic pressures (30–110 MPa); yet, by current estimates, these subsurface and deep ocean zones host the majority of the Earth's microbial and animal life. The extent to which terrestrially relevant pressures up to 100 MPa deform most globular proteins—and which kinds—has not been established. Here, we report the invention of an experimental apparatus that enables structural proteomic methods to be carried out at high pressures for the first time. The method, called high-pressure limited proteolysis (Hi-P LiP), involves performing pulse proteolysis on whole cell extracts brought to high pressure. The resulting sites of proteolytic susceptibility induced by pressure are subsequently read out by sequencing the peptide fragments with tandem liquid chromatography–mass spectrometry. The method sensitively detects pressure-induced structural changes with residue resolution and on whole proteomes, providing a deep and broad view of the effect of pressure on protein structure. When applied to a piezosensitive thermophilic bacterium, , we find that approximately 40% of its soluble proteome is structurally perturbed at 100 MPa. Proteins with lower charge density are more resistant to pressure-induced deformation, as expected; however, contrary to expectations, proteins with lower packing density (i.e., more voids) are also more resistant to deformation. Furthermore, high pressure has previously been shown to preferentially alter conformations around active sites. Here, we show this is also observed in Hi-P LiP, suggesting that the method could provide a generic and unbiased modality to detect binding sites on a proteome scale. Hence, data sets of this kind could prove useful for training emerging artificial intelligence models to predict cryptic binding sites with greater accuracy. Published by the American Physical Society2024 

Hydrostatic pressure increases with depth in the ocean, but little is known about the molecular bases of biological pressure tolerance. We describe a mode of pressure adaptation in comb jellies (ctenophores) that also constrains these animals’ depth range. Structural analysis of deep-sea ctenophore lipids shows that they form a nonbilayer phase at pressures under which the phase is not typically stable. Lipidomics and all-atom simulations identified phospholipids with strong negative spontaneous curvature, including plasmalogens, as a hallmark of deep-adapted membranes that causes this phase behavior. Synthesis of plasmalogens enhanced pressure tolerance inEscherichia coli, whereas low-curvature lipids had the opposite effect. Imaging of ctenophore tissues indicated that the disintegration of deep-sea animals when decompressed could be driven by a phase transition in their phospholipid membranes. 

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNA Lys3 , and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U–A and G–C base pairs of tRNA Lys3 . HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states. 

Sampling and genomic efforts over the past decade have revealed an enormous quantity and diversity of life in Earth's extreme environments. This new knowledge of life on Earth poses the challenge of understandingits molecular basis in such inhospitable conditions, given that such conditions lead to loss of structure and of function in biomolecules from mesophiles. In this review, we discuss the physicochemical properties of extreme environments. We present the state of recent progress in extreme environmental genomics. We then present an overview of our current understanding of the biomolecular adaptation to extreme conditions. As our current and future understanding of biomolecular structure–function relationships in extremophiles requires methodologies adapted to extremes of pressure, temperature, and chemical composition, advances in instrumentation for probing biophysical properties under extreme conditions are presented. Finally, we briefly discuss possible future directions in extreme biophysics. 

Biological small-angle X-ray solution scattering (BioSAXS) is now widely used to gain information on biomolecules in the solution state. Often, however, it is not obvious in advance whether a particular sample will scatter strongly enough to give useful data to draw conclusions under practically achievable solution conditions. Conformational changes that appear to be large may not always produce scattering curves that are distinguishable from each other at realistic concentrations and exposure times. Emerging technologies such as time-resolved SAXS (TR-SAXS) pose additional challenges owing to small beams and short sample path lengths. Beamline optics vary in brilliance and degree of background scatter, and major upgrades and improvements to sources promise to expand the reach of these methods. Computations are developed to estimate BioSAXS sample intensity at a more detailed level than previous approaches, taking into account flux, energy, sample thickness, window material, instrumental background, detector efficiency, solution conditions and other parameters. The results are validated with calibrated experiments using standard proteins on four different beamlines with various fluxes, energies and configurations. The ability of BioSAXS to statistically distinguish a variety of conformational movements under continuous-flow time-resolved conditions is then computed on a set of matched structure pairs drawn from the Database of Macromolecular Motions (http://molmovdb.org). The feasibility of experiments is ranked according to sample consumption, a quantity that varies by over two orders of magnitude for the set of structures. In addition to photon flux, the calculations suggest that window scattering and choice of wavelength are also important factors given the short sample path lengths common in such setups.