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

## 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.

## Key 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 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.