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Probing the folding and unfolding processes of proteins as a function of temperature is a major challenge in biophysics. Here we examine the effects of temperature spikes that heat and cool proteins within tens of nanoseconds. Our results show these spikes are capable of causing irreversible changes sufficient to eliminate protein activity.
The folding and unfolding of proteins over milliseconds is commonly described using conventional chemical kinetics and transition-state theory (1). On submicrosecond timescales, it has been reported that proteins are simply too large to undergo the significant structural changes required for folding, and that the assumptions of conventional rate kinetics break down (1). “Speed limits” for protein folding of the order of 1 μsμs\mu{\rm s} have been reported (2,3). In general, smaller and less complex proteins fold and unfold more quickly than larger proteins, and most cases of folding near the “speed limit” involve polypeptides <100<100{<}100 residues in size.
In the case of unfolding, the opportunity exists to substantially increase the rates by increasing the temperature. “T-jump” experiments have observed unfolding of proteins on microsecond timescales (4,5), and significant structural change of small proteins in nanoseconds (6). Molecular dynamics simulations performed at high temperatures (100225100−−225∘100--225^{\circ}C) have predicted an unfolding “speed limit” of \sim0.1 ns (7), but so far no experiments have been able to show unfolding near this limit.
Temperature-jump experiments are restricted to temperatures below the boiling point of the solution, thus limiting therate at which unfolding may occur. Attempts to heat proteins to higher temperatures have been made (8), but these simultaneously induce nonthermal effects (extreme levels of electromagnetic fields and microbubbles) likely to affect proteins. Here, we use a technique that can heat proteins tocalibrated temperatures well above 100100∘100^{\circ}C without these potentially destructive effects. Our measurements achieve heat/ cool times of 40 ns, with significant cooling within 10 ns.
By measuring protein activity we monitor the active site of proteins without making assumptions about how much secondary structure remains intact away from the active site. In this article, we define unfolding to refer to a structural change significant enough to cause loss of protein activity.
All reports of thermally induced unfolding on a nanosecond timescale have used proteins that unfold reversibly. In this article, nanosecond temperature spikes are applied to two enzymes, horseradish peroxidase isozyme C (HRP-C) and catalase, which have previously been shown toirreversibly unfold when heated (9,10). Temperature spikesbelow 100100∘100^{\circ}C do not affect these enzymes, but at higher temperatures, irreversible thermal unfolding is induced using temperature spikes lasting only tens of nanoseconds.
We exploit three opportunities for the production of very brief temperature spikes in solution:
  • Heat rapidly diffuses away from very thin layers in a heat conductive medium. By solving the heat conduction equation, it is readily shown that confining a temperature rise to a small structure results in extremely fast cooling times.
  • Metals combine a high thermal conductivity with a large extinction coefficient for visible light. For example, a 150 nm gold film on glass transmits <0.1%<0.1%<0.1\% of incident laser light (Fig. 1 a). The film is also thin enough to deliver significant heat to the gold surface and the liquid medium in contact with it. In this way, “pure” conducted heat is delivered to macromolecules attached to the gold-liquid interface without the complicating effect of large electromagnetic fields, or photons with sufficient energy to break covalent bonds.
  • The boiling of a liquid on a surface requires the formation of active vapor nuclei. For a smooth water-metal interface at the temperatures and timescales we employ, the rate of vapor generation is negligible (11).
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Laser heating system used to deliver temperature pulses to proteins (purple ellipses). Temperature is monitored using the top reflecting beam (red). (b) Two typical temperature pulses comparing the calculated transient (dashed blue lines) and the measured temperature (red lines). Full width at half-maximum of both modeled spikes is 40 ns.