Biomolecular dynamics study, from ensemble to single-molecule, from in vitro to in vivo

  • Posted on: 28 July 2020
  • By: liuk

Study Biomolecular dynamics using SAXS

The small-angle X-ray scattering (SAXS) can obtain the information on the structure and dynamics of biomolecules in solution. Through the SAXS approach, we found the K63-diUb exist two  conformation states in solution, which can be cross-validated with NMR results.
Reference: eLife, 2015, 4:e05767.

We integrated SAXS, smFRET and CXMS to get the ensemble structure of  K63-diUb from different dimensions.
Reference: Biochemistry, 2018, 57:305-313.

Integrate the experiment in tube and in silico

All-atom molecular dynamics simulation on the ultra-weak protein complex verify the effect of mutation on protein interactions
Reference: Angew Chem Int Ed, 2014, 53, 11501-11505.

Steered MD simulation indicate the relationship between subtle dynamics and ligand dissociation
Reference: Biochmeistry, 2014, 53, 1403-1409

MD simulation reveal from the atomic scale that the two structural states (relaxed state and retracted state) of ubiquitin after phosphorylation modification show different levels of protonation. Different levels of protonation of different stats show various structural stability.
Reference: Proc Natl Acad Sci, USA, 2017, 114:6770-6775.

We performed all-atom molecular dynamics simulation of membrane protein GPR17 dimer in lipid membrane environment. The key amino acids of the dimerization interaction interface were determined by simulation and verified with in vivo experiments.
Reference: J Mol Biol, 2020, doi: 10.1016/j.jmb.2020.06.009.

CXMS capture protein dynamics

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues. Protein dynamics can be manifested from cross-links, as illustrated here with the open-closed domain movement for a two-domain protein
Reference: J Biol Chem, 2017, 292, 1187-1196

We analyze the experimental cross-links for lysine or acidic residues and show the conformational preference of chemical cross-linkers determines the cross-linking probability of reactive protein residues.
In addition, we present a new format of CXMS distance restraints, which works by modifying the cross-linked residue with a rigid extension derived from the cross-linker. The use of tighter restraints not only afford better-resolved structures but also uncover protein dynamics.
Reference: J Phy Chem B, 2020, 123:4446-4453
                   Structure, 2020, in press

Study on biomolecule dynamics at single molecule level

Single-molecule FRET(smFRET) can provide the dynamic structure information of biological macromolecules from the single-molecule level. Owing to the FRET efficiency is inversely proportional to the sixth power of the distance between two fluorescent probe pairs (donor and acceptor), we can accurately obtain the dynamic information on the time scale of microseconds to seconds.
We have established the single molecule fluorescence experiment platform. Based on the PicoQuant picosecond pulsed laser, Nikon A1 confocal microscope and the TCSPC detection unit, as well as the self-made algorithm, we can calculate the FRET efficiency and count its distribution information. Besides, we introduced Bayesian information criterion (BIC), Akaike information criterion (AIC), and correlation coefficient R2 to evaluate the statistical FRET efficiency. These methods can accurately assess the dynamic structure of the protein while avoiding the occurrence of overfitting.

Study protein dynamics in situ and in vivo

By staining with chemical fluorescent probes or fusing fluorescent proteins, the proteins in cells can be imaged by CLSM with high resolution. In this way, the location of the protein in cell can be imaged. The related properties of the protein in cell can be captured and analyzed.

Based on the CLSM technology, we have carried out research on the phase separation of biological macromolecules. The phase separation process by real-time can be captured. The fluidity of the phase separation system can be analyzed using the FRAP analysis. Finally, the microscopic mechanism of phase separation can be explained by combined with other vitro experiments.

We present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (split-GFP). Based on split-GFP labeling scheme and TCSPC FLIM (Fluorescence Lifetime Imaging Microscopy) system,FLIM-FRET methods are used to detect the Class-A GPCR Dimer in the Cell Membrane.