Determining small-molecule permeation through lipid membranes.

The passive permeability of cell membranes is of key importance in biology, biomedical research and biotechnology as it determines the extent to which various molecules such as drugs, products of metabolism, and toxins can enter or leave the cell unaided by dedicated transport proteins. The quantification of passive solute permeation is possible with radio-isotope distribution experiments, spectroscopic measurements and molecular dynamics simulations. This protocol describes stopped-flow fluorimetry measurements performed on lipid vesicles and living yeast cells to estimate the osmotic permeability of water and solutes across (bio)membranes. Encapsulation of the fluorescent dye calcein into lipid vesicles allows monitoring of volume changes upon osmotic shifts of the medium via (de)quenching of the fluorophore, which we interpret using a well-defined physical model that takes the dynamics of the vesicles into account to calculate the permeability coefficients of solutes. We also present analogous procedures to probe weak acid and base permeability in vesicles and cells by using the read-out of encapsulated or expressed pH-sensitive probes. We describe the preparation of synthetic vesicles of varying lipid composition and determination of vesicle size distribution by dynamic light scattering. Data on membrane permeation are obtained using either conventional or stopped-flow kinetic fluorescence measurements on instruments available in most research institutes and are analyzed with a suite of user-friendly MATLAB scripts ( https://doi.org/10.5281/zenodo.6511116 ). Collectively, these procedures provide a comprehensive toolbox for determining membrane permeability coefficients in a variety of experimental systems, and typically take 2-3 d.

Physiochemical Modeling of Vesicle Dynamics upon Osmotic Upshift.

We modeled the relaxation dynamics of (lipid) vesicles upon osmotic upshift, taking into account volume variation, chemical reaction kinetics, and passive transport across the membrane. We focused on the relaxation kinetics upon addition of impermeable osmolytes such as KCl and membrane-permeable solutes such as weak acids. We studied the effect of the most relevant physical parameters on the dynamic behavior of the system, as well as on the relaxation rates. We observe that 1) the dynamic complexity of the relaxation kinetics depends on the number of permeable species; 2) the permeability coefficients (P) and the weak acid strength (pKa-values) determine the dynamic behavior of the system; 3) the vesicle size does not affect the dynamics, but only the relaxation rates of the system; and 4) heterogeneities in the vesicle size provoke stretching of the relaxation curves. The model was successfully benchmarked for determining permeability coefficients by fitting of our experimental relaxation curves and by comparison of the data with literature values (in this issue of Biophysical Journal). To describe the dynamics of yeast cells upon osmotic upshift, we extended the model to account for turgor pressure and nonosmotic volume.

Mapping Multiple Distances in a Multidomain Protein for the Identification of Folding Intermediates.

The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation.

Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane.

We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). We observe a relation between the molecule permeability and the saturation of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, we conclude that the yeast plasma membrane is impermeable to lactic acid on timescales up to ∼2.5 h.

Single-Molecule FRET Measurements in Additive-Enriched Aqueous Solutions.

The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant, and the ionic strength can be affected. Here, we systematically evaluate the importance of solvent characteristics with respect to single-molecule FRET (smFRET) data. First, we present a confocal based method for the determination of fluorescence quantum yields to facilitate a fast characterization of smFRET-samples at sub-nM-concentrations. As a case study, we analyze smFRET data of structurally rigid, double-stranded DNA-oligonucleotides in aqueous buffer and in buffers with specific amounts of glycerol, guanidine hydrochloride (GdnHCl), and sodium chloride (NaCl) added. We show that the calculation of interdye distances, without taking into account solvent-induced spectral and photophysical changes of the labels, leads to deviations of up to 4 Å from the real interdye distances. Additionally, we demonstrate that electrostatic dye-dye repulsions are negligible for the interdye distance regime considered here (>50 Å). Finally, we use our approach to validate the further compaction of the already unfolded state of phosphoglycerate kinase (PGK) with decreasing denaturant concentrations, a mechanism known as coil-globule transition.

Accurate fluorescence quantum yield determination by fluorescence correlation spectroscopy.

Here, we present a comparative method for the accurate determination of fluorescence quantum yields (QYs) by fluorescence correlation spectroscopy. By exploiting the high sensitivity of single-molecule spectroscopy, we obtain the QYs of samples in the microliter range and at (sub)nanomolar concentrations. Additionally, in combination with fluorescence lifetime measurements, our method allows the quantification of both static and collisional quenching constants. Thus, besides being simple and fast, our method opens up the possibility to photophysically characterize labeled biomolecules under application-relevant conditions and with low sample consumption, which is often important in single-molecule studies.

Conformational state distributions and catalytically relevant dynamics of a hinge-bending enzyme studied by single-molecule FRET and a coarse-grained simulation.

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.

Inter-dye distance distributions studied by a combination of single-molecule FRET-filtered lifetime measurements and a weighted accessible volume (wAV) algorithm.

Förster resonance energy transfer (FRET) is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds is an inherent problem in FRET studies. By performing single-molecule FRET-filtered lifetime measurements, we are able to characterize the amplitude of the motions of fluorescent probes attached to double-stranded DNA standards by means of flexible linkers. With respect to previously proposed experimental approaches, we improved the precision and the accuracy of the inter-dye distance distribution parameters by filtering out the donor-only population with pulsed interleaved excitation. A coarse-grained model is employed to reproduce the experimentally determined inter-dye distance distributions. This approach can easily be extended to intrinsically flexible proteins allowing, under certain conditions, to decouple the macromolecule amplitude of motions from the contribution of the dye linkers.

CO rebinding kinetics and molecular dynamics simulations highlight dynamic regulation of internal cavities in human cytoglobin.

Cytoglobin (Cygb) was recently discovered in the human genome and localized in different tissues. It was suggested to play tissue-specific protective roles, spanning from scavenging of reactive oxygen species in neurons to supplying oxygen to enzymes in fibroblasts. To shed light on the functioning of such versatile machinery, we have studied the processes supporting transport of gaseous heme ligands in Cygb. Carbon monoxide rebinding shows a complex kinetic pattern with several distinct reaction intermediates, reflecting rebinding from temporary docking sites, second order recombination, and formation (and dissociation) of a bis-histidyl heme hexacoordinated reaction intermediate. Ligand exit to the solvent occurs through distinct pathways, some of which exploit temporary docking sites. The remarkable change in energetic barriers, linked to heme bis-histidyl hexacoordination by HisE7, may be responsible for active regulation of the flux of reactants and products to and from the reaction site on the distal side of the heme. A substantial change in both protein dynamics and inner cavities is observed upon transition from the CO-liganded to the pentacoordinated and bis-histidyl hexacoordinated species, which could be exploited as a signalling state. These findings are consistent with the expected versatility of the molecular activity of this protein.

Native and unfolded states of phosphoglycerate kinase studied by single-molecule FRET.

Single-molecule Förster resonance energy transfer (FRET) measurements with phosphoglycerate kinase from yeast were performed at different concentrations of guanidine hydrochloride. From these steady-state measurements we obtained FRET efficiency histograms characterizing structural properties of individual proteins at different stages between the native and the fully unfolded state. Native proteins exhibit a slightly more expanded structure under buffer conditions without denaturant as compared to conditions with denaturant. At 0.5 M GndHCl an unfolded state population that exhibits a significantly expanded structure as compared to the native state, emerges. The unfolded state is characterized by a pronounced broadening of the efficiency distribution, which indicates a large structural and/or dynamical heterogeneity within the population. At high denaturant concentrations, well above the unfolding transition at C(1/2)~0.7 M, we observe a progressive expansion of the protein structure, namely globule-coil transition.