Temperature-dependent downhill unfolding of ubiquitin. I. Nanosecond-to-millisecond resolved nonlinear infrared spectroscopy

• Published in: Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2008-07, Vol.72 (1), p.474-487
• Main Authors: Chung, Hoi Sung, Tokmakoff, Andrei
• Format: Article
• Language: English
• Subjects: Animals; Cattle; dispersed vibrational echo spectroscopy; Kinetics; Protein Folding; Protein Structure, Secondary; protein unfolding dynamics; Spectrophotometry, Infrared; Temperature; temperature jump; thermal unfolding; Thermodynamics; Time Factors; Ubiquitin - chemistry; Ubiquitin - metabolism; Vibration
• ISSN: 0887-3585; 1097-0134
• DOI: 10.1002/prot.22043

Transient thermal unfolding of ubiquitin is investigated using nonlinear infrared spectroscopy after a nanosecond laser temperature jump (T‐jump). The abrupt change in the unfolding free energy surface and the ns time resolution allow us to observe a fast response on ns to μs time‐scales, which we attribute to downhill unfolding, before a cross‐over to ms kinetics. The downhill unfolding by a sub‐population of folded proteins is induced through a shift of the barrier toward the native state. By adjusting the T‐jump width, the effect of the initial (Ti) and final (Tf) temperature on the unfolding dynamics can be separated. From the amplitude of the fast downhill unfolding, the fractional population prepared at the unfolding transition state is obtained. This population increases with both Ti and with Tf. A two‐state kinetic analysis of the ms refolding provides thermodynamic information about the barrier height. By a combination of the fast and slow unfolding and folding parameters, a quasi‐two‐state kinetic analysis is performed to calculate the time‐dependent population changes of the folded state. This calculation coincides with the experimentally obtained population changes at low temperature but deviations are found in the T‐jump from 67 to 78°C. Using temperature‐dependent barrier height changes, a temperature Φ value analysis is performed. The result shows a decreasing trend of ΦT with temperature, which indicates an increase of the heterogeneity of the transition state. We conclude that ubiquitin unfolds along a well‐defined pathway at low temperature which expands with increasing temperature to include multiple routes. Proteins 2008. © 2008 Wiley‐Liss, Inc.