tracking-single-proteins-lipid-bilayers-using-fluorescence

Tracking Single Proteins in Lipid Bilayers Using Fluorescence Microscopy

Direct observation is a fundamental method for scientific discovery and understanding. Time-lapse single-molecule fluorescence imaging allows the ...

Washington State University

Cytochrome P450 reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. It is also believed that CPR is an integral membrane protein exclusively. Herein, we report that, contrary to this belief, CPR can exist as a peripheral membrane protein in the absence of NADPH and will transition to an integral membrane protein in the presence of stoichiometric amounts of NADPH or greater. All experiments were performed in a solid-supported cushioned lipid bilayer that closely matched the chemical composition of the human endoplasmic reticulum and served as an ER biomimetic. The phase characteristics and fluidity of the ER biomimetic was characterized with fluorescence micrographs and temperature-dependent fluorescence recovery after photobleaching. The interactions of CPR with the ER biomimetic were directly observed by tracking single CPR molecules using time-lapse single-molecule fluorescence imaging and subsequent analysis of tracks. These studies revealed dramatic changes in diffusion coefficient and the degree of partitioning of CPR as a function of NADPH concentration.

Single-Protein Tracking Reveals That NADPH Mediates the Insertion of Cytochrome P450 Reductase into a Biomimetic of the Endoplasmic Reticulum.

Measuring the kinetics that govern ligand-receptor interactions is fundamental to our understanding of pharmacology. For ligand-gated ion channels, binding of an agonist triggers allosteric motions that open an integral ion-permeable pore. By mathematically modeling stochastic electrophysiological responses with high temporal resolution (ms), previous single channel studies have been able to infer the rate constants of ligands binding to these receptors. However, there are no reports of the direct measurement of the single-molecule binding events that are vital to how agonists exert their functional effects. For the first time, we report these direct measurements, the rate constants, and corresponding free energy changes, which describe the transitions between the different binding states. To achieve this, we use the super resolution technique: points accumulation for imaging in nanoscale topography (PAINT) to observe binding of ATP to orthosteric binding sites on the P2X1 receptor. Furthermore, an analysis of time-resolved single-molecule interactions is used to measure elementary rate constants and thermodynamic forces that drive the allosteric motions. These single-molecule measurements unequivocally establish the location of each binding states of the P2X1 receptor and the stochastic nature of the interaction with its native ligand. The analysis leads to the measurement of the forward and reverse rates from a weak ligand-binding state to a strong ligand binding state that is linked to allosteric motion and ion pore formation. These rates ( = 1.41 sec and  = 0.32 sec) were then used to determine the free energy associated with this critical mechanistic step (3.7 kJ/mol). Importantly, the described methods can be readily applied to all ligand-gated ion channels, and more broadly to the molecular interactions of other classes of membrane proteins.

Direct Measurement of Single-Molecule Ligand-Receptor Interactions.

Cytochrome P450 reductase (CPR) is a NADPH-dependent membrane-bound oxidoreductase found in the endoplasmic reticulum (ER) and is the main redox partner for most cytochrome P450 enzymes. Presented are the measured thermodynamic driving forces responsible for how strongly CPR partitions into a biomimetic ER with the same lipid composition of a natural ER. Using temperature-dependent fluorescence correlation spectroscopy and fluorescence single-protein tracking, the standard state free energies, enthalpies, and entropies of the CPR insertion process were all measured. The results of this study demonstrate that the thermodynamic driving forces are dependent on the redox states of CPR. In particular, the partitioning of CPR into a biomimetic ER is an exothermic process with a small positive change in entropy, while CPR partitioning is endothermic with a large positive change in entropy. Both resulted in negative free energies and strong association to the biomimetic ER, but the  of CPR insertion is measurably smaller than that of CPR. Using this new information and known results from literature sources, we also present a phenomenological model that accounts for membrane-protein interactions, protein orientation relative to the membrane, and protein conformation as a function of the redox state.

Article	Year
Single-Protein Tracking Reveals That NADPH Mediates the Insertion of Cytochrome P450 Reductase into a Biomimetic of the Endoplasmic Reticulum. Journal of the American Chemical Society\| PubMed ID: 28347139	2017
Direct Measurement of Single-Molecule Ligand-Receptor Interactions. The journal of physical chemistry. B\| PubMed ID: 32790373	2020
Thermodynamic Driving Forces of Redox-Dependent CPR Insertion into Biomimetic Endoplasmic Reticulum Membranes. The journal of physical chemistry. B\| PubMed ID: 35171619	2022

Michael J. Martinez

Biography

JoVE Journal Publications

Other Publications