A Fluorescence-Based Temperature-Jump Apparatus for Illustrating Protein Dynamics on the Millisecond Time Scale

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 kJ mol^–1, whereas HSA did not manifest the dynamic component. In this measurement, only a tiny amount of sample, ca. 1 μL, was required due to the conjugation of the microspot objective, and the initial temperature was readily controlled by a homemade thermostatic pad. This millisecond-resolution technique is advantageous for illustrating the large-domain dynamics of the targeted protein, bridging the characterizations of the localized protein dynamics on nanosecond to microsecond time scales using the fast techniques and the steady-state protein conformational features by conventional methods, such as Fourier-transform infrared and circular dichroism spectroscopies.