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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Protein Diffusion in the Membrane01:24

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Proteins show rotational as well as lateral diffusion across the membrane. The lateral diffusion of proteins was confirmed through the cell fusion experiment where mouse and human cells were fused, resulting in hybrid cells. When the human and mouse cells fused, the specific membrane proteins on human and mouse cells were marked with the red and green-fluorescent markers, respectively. Initially, the red and green fluorescence was located on the respective hemisphere of the cell. As time...
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Membrane Fluidity01:26

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Membrane fluidity is explained by the fluid mosaic model of the cell membrane, which describes the plasma membrane structure as a mosaic of components—including phospholipids, cholesterol, proteins, and carbohydrates—that gives the membrane a fluid character.
Mosaic nature of the membrane
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Mechanisms of Membrane-bending01:15

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The living membranes are flexible due to their fluid mosaic nature; however, their bending into different shapes is an active process regulated by specific lipids and proteins. The membrane bending can be transient as seen in vesicles or stable for a long time as in microvilli. Cells regulate the size, location, and duration of the membrane curvature.
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Correlating membrane-protein dynamics with function: Integrating bioinformatics, molecular dynamics, and

Hugh R Higinbotham1, Christine A Arbour2, Barbara Imperiali2

  • 1Department of Biology and Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Biorxiv : the Preprint Server for Biology
|June 12, 2025
PubMed
Summary

We developed a new method combining structural bioinformatics, molecular simulation, and single-molecule FRET microscopy to observe how integral membrane proteins change shape when binding to ligands, aiding drug discovery.

Keywords:
glycoconjugate biosynthesismembrane proteinmolecular dynamicsnon-canonical amino acid mutagenesissingle-molecule FRETsubstrate specificity

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Area of Science:

  • Structural biology and biophysics of membrane proteins.
  • Prokaryotic glycoconjugate biosynthesis pathways.
  • Protein-ligand interactions and conformational dynamics.

Background:

  • Integral membrane proteins play crucial roles in biological processes, and understanding their structure-function relationships is vital for drug development.
  • Bacterial glycoconjugate biosynthesis pathways are attractive targets for novel antibiotics due to their essential roles and unique biochemical properties.
  • Characterizing these systems presents challenges due to the complex interplay of proteins, lipids, and carbohydrates.

Purpose of the Study:

  • To investigate the ligand-dependent conformational dynamics of small monotopic phosphoglycosyl transferase (SmPGT) superfamily members.
  • To correlate structural features with observed dynamics and validate their role in ligand binding.
  • To establish a versatile platform for studying protein dynamics in a native-like membrane environment.

Main Methods:

  • Integration of structural bioinformatics, all-atom molecular simulations, and single-molecule Förster Resonance Energy Transfer (smFRET) microscopy.
  • Development of a platform using selective cysteine labeling, non-canonical amino acid mutagenesis, and click chemistry for dual-labeling PglC variants.
  • Solubilization of modified proteins into styrene maleic acid liponanoparticles (SMALPs) to mimic native membrane environments.

Main Results:

  • Identification of substrate-specific structural features within the SmPGT superfamily.
  • Correlation of these features with ligand-dependent conformational dynamics observed in molecular simulations.
  • Experimental validation of protein motion's role in ligand binding using smFRET-SMALP technology.
  • Demonstration that PglC conformational changes upon inhibitor binding correlate with inhibitor potency.

Conclusions:

  • The developed smFRET-SMALP strategy effectively monitors in situ conformational dynamics of integral membrane proteins.
  • This approach provides insights into the mechanism of ligand binding and inhibitor potency for the SmPGT superfamily.
  • The methodology is adaptable for studying other SmPGT members with varying substrate specificities, supporting structure-based drug design.