Microsecond protein folding events revealed by time-resolved fluorescence resonance energy transfer in a microfluidic mixer.
We demonstrate the combination of the time-resolved fluorescence resonance energy transfer (tr-FRET) measurement and the ultrarapid hydrodynamic focusing microfluidic mixer. The combined technique is capable of probing the intermolecular distance change with temporal resolution at microsecond level and structural resolution at Angstrom level, and the use of two-photon excitation enables a broader exploration of FRET with spectrum from near-ultraviolet to visible wavelength. As a proof of principle, we used the coupled microfluidic laminar flow and time-resolved two-photon excitation microscopy to investigate the early folding states of Cytochrome c (cyt c) by monitoring the distance between the tryptophan (Trp-59)-heme donor-acceptor (D-A) pair. The transformation of folding states of cyt c in the early 500 μs of refolding was revealed on the microsecond time scale. For the first time, we clearly resolved the early transient state of cyt c, which is populated within the dead time of the mixer (<10 μs) and has a characteristic Trp-59-heme distance of ∼31 Å. We believe this tool can find more applications in studying the early stages of biological processes with FRET as the probe.
†HKUST Jockey Club Institute for Advanced Study, ‡Department of Electronic and Computer Engineering, §Department of Chemistry, ⊥Department of Mechanical and Aerospace Engineering, and ∇Division of Biomedical Engineering, Hong Kong University of Science and Technology, Hong Kong.
Liguo
L
Jiang
†HKUST Jockey Club Institute for Advanced Study, ‡Department of Electronic and Computer Engineering, §Department of Chemistry, ⊥Department of Mechanical and Aerospace Engineering, and ∇Division of Biomedical Engineering, Hong Kong University of Science and Technology, Hong Kong.
Yan
Y
Zeng
†HKUST Jockey Club Institute for Advanced Study, ‡Department of Electronic and Computer Engineering, §Department of Chemistry, ⊥Department of Mechanical and Aerospace Engineering, and ∇Division of Biomedical Engineering, Hong Kong University of Science and Technology, Hong Kong.
Qiqi
Q
Sun
†HKUST Jockey Club Institute for Advanced Study, ‡Department of Electronic and Computer Engineering, §Department of Chemistry, ⊥Department of Mechanical and Aerospace Engineering, and ∇Division of Biomedical Engineering, Hong Kong University of Science and Technology, Hong Kong.
Yueru
Y
Sun
†HKUST Jockey Club Institute for Advanced Study, ‡Department of Electronic and Computer Engineering, §Department of Chemistry, ⊥Department of Mechanical and Aerospace Engineering, and ∇Division of Biomedical Engineering, Hong Kong University of Science and Technology, Hong Kong.
Zhihong
Z
Guo
†HKUST Jockey Club Institute for Advanced Study, ‡Department of Electronic and Computer Engineering, §Department of Chemistry, ⊥Department of Mechanical and Aerospace Engineering, and ∇Division of Biomedical Engineering, Hong Kong University of Science and Technology, Hong Kong.
Jianan Y
JY
Qu
†HKUST Jockey Club Institute for Advanced Study, ‡Department of Electronic and Computer Engineering, §Department of Chemistry, ⊥Department of Mechanical and Aerospace Engineering, and ∇Division of Biomedical Engineering, Hong Kong University of Science and Technology, Hong Kong.
Shuhuai
S
Yao
D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation* / Q000379:methods*; D045304:Cytochromes c / Q000737:chemistry / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D006736:Horses; D044085:Microfluidics / Q000295:instrumentation*; D017510:Protein Folding
D045304:Cytochromes c