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 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.

Thermodynamic Driving Forces of Redox-Dependent CPR Insertion into Biomimetic Endoplasmic Reticulum Membranes.

The family of group XIV rhodamine zwitterions are fluorescence probes with carbon, silicon, germanium, or tin substituted in the 10-position of the xanthene ring. Because of their inherent near-infrared fluorescence, photostability and high quantum yields in aqueous solutions, the Si and Ge containing fluorophores in this class have become increasingly important for fluorescent labeling of proteins and biological molecules. This study fully characterizes photophysical rates derived from a model consisting of a singlet ground state, the lowest singlet excited state, and the lowest triplet excited state for two exemplar group XIV rhodamine zwitterions, one containing Si and the other Ge. Within a simple Jablonski diagram, all radiative and non-radiative rates, including intersystem crossing and triplet depopulation rates, were measured as a function of oxygen concentration. It was shown that the triplet depopulation rates are intrinsically fast in comparison with traditional xanthene containing fluorophores, probably due to the increased spin-obit coupling from the Si and Ge substitution in the xanthene ring. Dissolved oxygen increases both the intersystem crossing and triplet depopulation rates. Stern-Volmer analysis was conducted to estimate rates of quenching by oxygen. The experimental data was used to estimate the initial rates for reactive oxygen production by Si and Ge containing fluorophores in aqueous solutions containing different concentrations of dissolved O. These estimates showed a significantly slower initial rate of reactive oxygen production in comparison with rhodamine 6G. This goes a long way to explaining their inherent photostability. Spectroscopic experiments were also conducted in 77 K viscous aqueous glasses where it was observed that the fluorescence spectra remained unchanged, and the quantum yields increased from 0.53 to 0.84 and from 0.52 to 0.89 for the Si and Ge containing fluorophores respectively; no phosphorescence was observed. All intersystem crossing and triplet depopulation rates were measured using fluorescence correlation spectroscopy (FCS) and analyzed using a new method that extrapolated the power dependence of the FCS curves to optical saturation. This method was verified using published data.

Photophysical Rate Constants and Oxygen Dependence for Si and Ge Rhodamine Zwitterions.

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, , along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of -coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of -coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased  with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein.

Human aquaporin-4 (AQP4) is a water-channel protein crucial for water-ion homeostasis in the central nervous system. Dysregulation of AQP4 function is linked to neurological conditions like brain edema, neuromyelitis optica, and epilepsy. AQP4 has two isoforms, M1 and M23. The M23 isoform forms large structures known as Orthogonal Arrays of Particles (OAPs), while M1 coats the outer surface of OAPs. The dynamics of OAP aggregation may modulate water permeability and ion homeostasis in the brain. This study shows that the palmitoylation state of M1 influences OAP self-assembly, with depalmitoylated M1 producing OAPs 20 % larger than those formed by palmitoylated M1 at the same concentration. This, along with prior research on M1 aggregation, suggests palmitoylated M1 forms a single outer layer arounf OPAs, whereas depalmitoylated M1 creates a double layer. Single-particle tracking was used to study M23 OAP formation in lipid bilayers under equilibrium conditions. Our data support the idea that M1 regulates M23 OAP size, showing that average OAP size decreases as M1 concentration increases. This study explores the inhibitory effects of M1 on AQP4 M23 assembly and how M1's palmitoylation state affects OAP size regulation. These insights could lead to new therapeutic approaches for managing AQP4 assembly and function in related conditions.

Article	Year
Thermodynamic Driving Forces of Redox-Dependent CPR Insertion into Biomimetic Endoplasmic Reticulum Membranes. The journal of physical chemistry. B\| PubMed ID: 35171619	2022
Photophysical Rate Constants and Oxygen Dependence for Si and Ge Rhodamine Zwitterions. The journal of physical chemistry. A\| PubMed ID: 36689273	2023
Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein. International journal of molecular sciences\| PubMed ID: 38891840	2024
Size of aquaporin-4 orthogonal arrays of particles are affected by the palmitoylation state of the M1 isoform. Biochimica et biophysica acta. General subjects\| PubMed ID: 41077234	2025

Eric P. Jacobo

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