CD spectrum from 400 to 800 nm of PS-I solubilized with 5mM lipopeptide C14DK.

<p>The concentration of PS-I is 0.233µmol/L (equal to 20µg Chl/ml).</p

PS-I solubility at the ambient temperature.

<p>The values were determined by optical absorption spectroscopy, in the presence of different lipopeptides and conventional surfactants DDM and FC14. All surfactant concentrations were fixed at 5.0 mM. The absorption maximum at 680 nm was utilized to calculate PS-I solubility in surfactant solution, with a molar extinction coefficient of 16.4µM^-1·cm^-1. Each bar represents the average of 2 experiments.</p

Molecular models of the designed lipopeptides.

<p>The hydrophilic heads of these surfactants vary through the pair-wise combination of hydrophilic amino acids Gly (G), Asp (D) and Lys (K), and their hydrophobic tails consist of the alkyl chains of lauric acid, myristic acid or palmitic acid, as indicated by C12, C14 and C16, respectively. Each row lists the lipopeptides with the same hydrophobic tails but different hydrophilic heads, whereas each column lists those with the same hydrophilic heads but different hydrophobic tails. Color code: gray, carbon; red, oxygen; blue, nitrogen; and white, hydrogen.</p

77K fluorescence emission spectra of PS-I solubilized with different surfactants.

<p>The surfactants tested include lipopeptides C14DK and C16DK, DDM and FC14 as indicated and the concentrations of surfactants were kept the same as under RT. The concentration of PS-I added was 0.117 µmol/L (equal to 10 µg Chl/ml).</p

Solubilization and Stabilization of Isolated Photosystem I Complex with Lipopeptide Detergents

<div><p>It is difficult to maintain a target membrane protein in a soluble and functional form in aqueous solution without biological membranes. Use of surfactants can improve solubility, but it remains challenging to identify adequate surfactants that can improve solubility without damaging their native structures and biological functions. Here we report the use of a new class of lipopeptides to solubilize photosystem I (PS-I), a well known membrane protein complex. Changes in the molecular structure of these surfactants affected their amphiphilicity and the goal of this work was to exploit a delicate balance between detergency and biomimetic performance in PS-I solubilization via their binding capacity. Meanwhile, the effects of these surfactants on the thermal and structural stability and functionality of PS-I in aqueous solution were investigated by circular dichroism, fluorescence spectroscopy, SDS-PAGE analysis and O₂ uptake measurements, respectively. Our studies showed that the solubility of PS-I depended on both the polarity and charge in the hydrophilic head of the lipopeptides and the length of its hydrophobic tail. The best performing lipopeptides in favour of PS-I solubility turned out to be C14DK and C16DK, which were comparable to the optimal amphiphilicity of the conventional chemical surfactants tested. Lipopeptides showed obvious advantages in enhancing PS-I thermostability over sugar surfactant DDM and some full peptide amphiphiles reported previously. Fluorescence spectroscopy along with SDS-PAGE analysis demonstrated that lipopeptides did not undermine the polypeptide composition and conformation of PS-I after solubilization; instead they showed better performance in improving the structural stability and integrity of this multi-subunit membrane protein than conventional detergents. Furthermore, O₂ uptake measurements indicated that PS-I solubilized with lipopeptides maintained its functionality. The underlying mechanism for the favorable actions of lipopeptide in PS-I solubilization and stabilization is discussed.</p> </div

Oxygen uptake activity of PS-I solubilized with different surfactants.

<p>The surfactants tested include lipopeptides C14DD, C14DK and C16DK, DDM and FC14 as indicated. The concentration of PS-I added was 0.117 µmol/L (equal to 10 µg Chl/ml). As a control, oxygen uptake of working solution without PS-I was also tested.</p

Thermostability of PS-I measured by following ellipticity at 687nm versus temperature.

<p>The measurements were conducted in the presence of C12DK, C14DK, C16DK, FC14 and DDM as indicated. The concentrations of PS-I and surfactants were 0.233µmol/L (equal to 20µg Chl/ml) and 5 mM, respectively. The measurement was performed after samples were incubated for 30min at RT. Temperature was increased at a rate of 1K/min. Data points were normalized and fitted with equation 1. An obvious two-stage response was observed in the presence of C14DK and C16DK. Each stage was fitted with equation 1, separately. The best fit curves are as shown in red and blue.</p

Deep-Learning-Enhanced Diffusion Imaging Assay for Resolving Local-Density Effects on Membrane Receptors

G-protein-coupled receptor (GPCR) density at the cell surface is thought to regulate receptor function. Spatially resolved measurements of local-density effects on GPCRs are needed but technically limited by density heterogeneity and mobility of membrane receptors. We now develop a deep-learning (DL)-enhanced diffusion imaging assay that can measure local-density effects on ligand–receptor interactions in the plasma membrane of live cells. In this method, the DL algorithm allows the transformation of 100 ms exposure images to density maps that report receptor numbers over any specified region with ∼95% accuracy by 1 s exposure images as ground truth. With the density maps, a diffusion assay is further established for spatially resolved measurements of receptor diffusion coefficient as well as to express relationships between receptor diffusivity and local density. By this assay, we scrutinize local-density effects on chemokine receptor CXCR4 interactions with various ligands, which reveals that an agonist prefers to act with CXCR4 at low density while an inverse agonist dominates at high density. This work suggests a new insight into density-dependent receptor regulation as well as provides an unprecedented assay that can be applicable to a wide variety of receptors in live cells

Deep-Learning-Enhanced Diffusion Imaging Assay for Resolving Local-Density Effects on Membrane Receptors

G-protein-coupled receptor (GPCR) density at the cell surface is thought to regulate receptor function. Spatially resolved measurements of local-density effects on GPCRs are needed but technically limited by density heterogeneity and mobility of membrane receptors. We now develop a deep-learning (DL)-enhanced diffusion imaging assay that can measure local-density effects on ligand–receptor interactions in the plasma membrane of live cells. In this method, the DL algorithm allows the transformation of 100 ms exposure images to density maps that report receptor numbers over any specified region with ∼95% accuracy by 1 s exposure images as ground truth. With the density maps, a diffusion assay is further established for spatially resolved measurements of receptor diffusion coefficient as well as to express relationships between receptor diffusivity and local density. By this assay, we scrutinize local-density effects on chemokine receptor CXCR4 interactions with various ligands, which reveals that an agonist prefers to act with CXCR4 at low density while an inverse agonist dominates at high density. This work suggests a new insight into density-dependent receptor regulation as well as provides an unprecedented assay that can be applicable to a wide variety of receptors in live cells