Proteins are composed of amino acids and are the building blocks of life. They have flexible structures that thermally fluctuate with motion with respect to different biological functions. For the past years, several methods such as nuclear magnetic resonance spectroscopy have been used to demonstrate the multiple conformations of proteins regarding their functions. Many biological processes involving proteins take place at relatively slow timescale from micro- to milliseconds at room temperature. This indicates the usefulness of the collective motion of amino acid groups in protein functions. Considering the critical role of thermal energy in motion activation, insufficient thermal energy at low temperatures will, therefore, stop individual motion and hinder protein functions. As such, information on the thermodynamics based on enthalpy and entropy will be of great benefit in understanding protein functions.
Presently, calorimetric experiments for proteins have been used to provide thermodynamic parameters. Calorimetry can either be achieved through direct heat measurements or measured temperature equilibrium constants. Despite the advantages, additional calorimetry improvements are still considered essential. On the other hand, a non-invasive imaging technique based on radiation thermography using millimeter-wave radiation, which detects electromagnetic radiation emitted thermally from the object and relates it to the object temperature, is another thermometric technique suitable for use at low temperatures. However, thermography using millimeter waves has limited special resolution due to diffraction effects. By solving this problem, it is highly desirable owing to the rise in protein applications to develop effective techniques for observation of protein thermodynamics at low temperatures by radiation thermography.
Recently Japanese researchers: Manabu Ishino, Dr. Akio Kishigami, Dr. Hiroyuki Kudo, Dr. Jongsuck Bae, and Dr. Tatsuo Nozokido developed a passive millimeter-wave microscope for near-field imaging of thermal radiation with subwavelength spatial resolution below the diffraction limit. The feasibility of the microscope system was tested at temperatures below room temperature, where passive infrared imaging systems are normally ineffective. Their main objective was to observe protein thermodynamics in ice using the microscopy system. The work is currently published in Journal of Infrared, Millimeter, and Terahertz Waves.
The system allowed thermal radiation imaging at low temperatures below room temperature. This property in combination with low thermal emission property of water ice in the millimeter-wave region enabled the authors to characterize thermal radiation from the proteins. The low temperature equally led to the trapping of conformation intermediates in the proteins. For the experiment conducted at a millimeter-wave frequency of 50 GHz in a temperature ranging from 130-270K, the displacement between two conformal states was observed.
Generally, the presented thermometric technique enabled observation of proteins in frozen-aqueous solution at low temperatures thus terminating the slow timescale motions in proteins. Besides, this method exhibited several advantages: noninvasive measurements, free information from ice and the ability to provide two-dimensional thermal images. These advantages will offer solutions to the most common problems encountered in protein calorimetry. Altogether, the developed microscope system based on the freeze-trapping method will provide noninvasive thermal imaging even at relatively low temperatures. This will further enable the development of new high-throughput calorimetry for effective study of different protein functions.
Ishino, M., Kishigami, A., Kudo, H., Bae, J., & Nozokido, T. (2019). Observation of Protein Thermodynamics in Ice by Passive Millimeter-Wave Microscopy. Journal of Infrared, Millimeter, and Terahertz Waves, 40(5), 585-594.Go To Journal of Infrared, Millimeter, and Terahertz Waves