Measuring the Adapted Low Temperature Resilience of Cold Shock Proteins - using Single Molecule Force Spectroscopy and Nuclear Magnetic Resonance

Cold Shock Proteins (CSPs) are produced when organisms experience cold-shock, a sudden
drop to roughly 7-10 °C below the optimum temperature, and are thought to aid
the melting of non-functional RNA secondary structures. For this function CSPs are
required to stay exible at temperatures determined by the optimum environment of
the organism. While the thermodynamic properties of these proteins vary depending on
the optimum temperature of the source organism, their structures are highly conserved,
making this a good model system to explore non-structural adaptations to various environments.
Single molecule force spectroscopy was used to measure the mechanical properties of the
CSP from Bacillus Subtilis while in the functional state, i.e. bound to nucleic acids. This
binding was shown to stabilise the protein, without increasing the rigidity, even when
the temperature of the complex was reduced.
The affinity of the CSP binding to ssDNA was measured and this was compared to
the affinity to ssDNA of a CSP from an Arctic soil bacterium, Psychrobacter sp6. The
CSP from the organism adapted to colder environments was found to have a lower binding
affinity to ssDNA and the binding strength of ssDNA to both CSPs increased with
decreasing temperature. When the binding of ssDNA was measured at the respective
cold-shock temperatures of each organism the affinities were of the same order of magnitude,
suggesting that the strength of this interaction is tuned to the native environment.
To explore how functionality is maintained in organisms adapted to extremely cold environments,
the dynamics of the CSP from Psychrobacter sp6 was measured at low,
physiologically relevant temperatures using a range of NMR techniques. Results showed
evidence of slow exchange with a more disordered state at low temperatures together with fast dynamics at the active site. These were not seen in measurements of the
CSP from Bacillus Subtilis at low temperatures, indicating they are part of the protein
adaptation to cold environments.