A Fluorescence-Based Temperature-Jump Apparatus for Illustrating Protein Dynamics on the Millisecond Time Scale
Liang-Che Kung, Li-Kang Chu
TL;DR
A new fluorescence-based tool was developed to study how proteins move on millisecond timescales when heated, using small sample amounts and precise temperature control.
Contribution
A novel T-jump apparatus using fluorescence and a diode laser to study protein dynamics on millisecond timescales with minimal sample.
Findings
BSA showed an apparent activation energy of 276 ± 23 kJ mol–1 for its dynamic response.
HSA did not show a dynamic component under the same conditions.
The method requires only ~1 μL of sample and provides millisecond-resolution data.
Abstract
A fluorescence-based temperature jump (T-jump) module was constructed to illustrate the large-domain motion of a given protein upon thermal stimulus on the millisecond time scale. The aqueous sample was readily heated by 5.0 °C in ca. 2 ms with a lasting high temperature plateau (>1 s) upon irradiation with the “optical Riemann sum” of the discrete infrared pulses of different energy sequences from a 1467 nm diode laser operated at 1k Hz. The temperature evolution was revealed by the time-evolved fluorescence intensity change of the dissolved tryptophan. Bovine serum albumin (BSA) and human serum albumin (HSA) were chosen as model proteins, and their fluorescence intensity evolutions were recorded at 36.6–39.9 °C upon T-jump from 35.0 °C, within the range of physiological temperatures. The observed protein dynamics of BSA was characterized with an apparent activation energy of 276 ± 23…
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Taxonomy
TopicsProtein Interaction Studies and Fluorescence Analysis · Protein Structure and Dynamics · Photosynthetic Processes and Mechanisms