11.02.2013

Life under Pressure

DORIS explores protein interactions under deep sea conditions

Researchers from Technische Universität Dortmund (Germany) have gained new insights into how organisms may cope with the immense water pressures governing the depths of the oceans. With DESY’s x-ray lightsource DORIS III, the scientists found striking similarities between the stability of isolated proteins and protein-protein interactions under high pressure. Such interactions may play a role in stabilizing proteins in deep sea organisms as well. The study, originally published in the journal Angewandte Chemie (International Edition), has been featured in the latest DESY Photon Science Highlights and Annual Report.

Transparent deep-sea squid, about four-inches across. The highest amounts of TMAO are found in muscles of crustaceans, squids and cartilaginous fishes. Image: NOAA/Edie Widder

Life in the deep sea is exposed to extreme and hostile environmental conditions. At depths impenetrable for sunlight, oxygen levels are low and water temperatures are frigid. However, in the vicinity of hydrothermal vents, temperatures can surge to several hundred degrees Celsius. Deep sea water pressure can be over a thousand times greater than the atmospheric pressure at sea level.

Organisms are able to balance external stresses such as pressure by varying the cellular concentration of soluble compounds known as osmolytes, thereby adjusting the osmotic pressureinside the cell. Cartilaginous fishes such as sharks and rays, for example, contain large amounts of the solute urea. On the downside, urea is known to destabilize the three-dimensional structure of proteins and perturb their vital functions in the organism. Cartilaginous fishes therefore use the highly efficient osmolyte TMAO (trimethylamine-N-oxide), which counteracts the destructive effects of urea and pressure. In deep sea organisms, TMAO is most efficient in its dual role when a urea-to-TMAO ratio of 2:1 is met. In studies of isolated proteins, the same ratio has been found to protect protein structures from the denaturing agent urea. 

Little is known about the underlying mechanisms protecting cells from pressure. Cells, the fundamental units of all life, contain a dense mixture of intracellular components including a large variety of proteins. Hence, protein-protein interactions likely play a significant role in a cell’s response to pressure. “In our study, we simulated the crowded conditions inside cells by using highly concentrated protein solutions,” says DESY scientist Martin A. Schroer, who performed the experiments as a postgraduate at Technische Universität Dortmund. “We then added osmolytes at concentrations occurring in deep sea animals and investigated the pressure-dependence of protein-protein interactions in the solution.”

At the DORIS III beamline BW4, the scientists varied the pressure in protein solutions between one bar, the atmospheric pressure at sea level, and four thousand bars, which is four times greater than the water pressure at the bottom of the Mariana trench, the deepest part of the world’s oceans. At any pressure point, the researchers examined the sample’s interior with DORIS’ intense x-ray beam using the small-angle x-ray scattering (SAXS) technique. “With SAXS, we are able to determine a protein’s overall structure as well as the interaction strength between proteins in the solution,” Schroer explains.

In a previous SAXS study, Schroer and his colleagues had already demonstrated that changes in protein-protein interactions under pressure are related to pressure-dependent alterations of the water structure around the proteins. “When we studied proteins in a buffer without osmolytes, we saw that the interaction strength continually decreased when we increased the pressure to approximately two thousand bars,” Schroer says. “This trend is intuitive because the increasing pressure compresses the protein solution. As proteins are charged, compression increases their mutual repulsion. Above two thousand bars, however, the interaction strength increased again, which we attributed to a collapse of the second hydration sphere of water.” Water molecules arrange in several spherical layers around proteins. Under high pressures, the second layer is pushed towards the first one surrounding the protein, leading to an effective screening of the protein charges and a reduction of protein-protein repulsion.

How do osmolytes affect these protein-protein interactions and how do they impact the water structure? The researchers addressed these questions in the present study. “When we added urea, we observed the same pressure-dependent trend for the protein interaction strength as for the osmolyte-free solution. Urea does not markedly affect the water structure,” Schroer says. “TMAO, in contrast, alters the pressure-dependence, and the water structure changes.” By adding TMAO, the collapse of the second water layer is largely prevented. “TMAO hardens the water structure,” Schroer explains. “It moves the second hydration shell around the proteins a little bit outward, whereas the pressure pushes in the opposite direction. Therefore, TMAO works against the collapse." A similar stabilizing mechanism may take place inside the cells of deep sea organisms.

To further relate their research to the conditions found in deep sea creatures, the scientists analyzed the pressure-dependence of protein-protein interactions in solutions containing urea and TMAO with a 2:1 ratio. The researchers found that protein-destabilizing urea alone reduced the mutual attraction of proteins, whereas protein-stabilizing TMAO enhanced it. For the mixture of osmolytes, the pressure-dependence was comparable to the osmolyte-free protein solution. Apparently, TMAO compensates urea’s effect on protein-protein interactions.  “A mixture of urea and TMAO, as it is found in nature, has an impact on the interaction between proteins under pressure similar to the mixture’s known influence on protein stability,” says Schroer.

These results may help improve our understanding of how organisms protect their vital protein functions against the large pressures of the deep sea. The researcher’s findings suggest that particular osmolytes are able to control protein-protein interactions and therefore also protein stabilization inside the organisms’ cells.

Reference: Exploring the Piezophilic Behavior of Natural Cosolvent Mixtures; Martin A. Schroer et al.; "Angewandte Chemie International Edition"; DOI: 10.1002/anie.201104380