Erik Frotscher

A Fluorinated Detergent for Membrane-Protein Applications†

Surfactants carrying fluorocarbon chains hold great promise as gentle alternatives to conventional hydrocarbon-based detergents for the solubilization and handling of integral membrane proteins. However, their inertness towards lipid bilayer membranes has limited the usefulness of fluorinated surfactants in situations where detergent-like activity is required. We demonstrate that fluorination does not necessarily preclude detergency, as exemplified by a fluorinated octyl maltoside derivative termed F6 OM. This nonionic compound readily interacts with and completely solubilizes phospholipid vesicles in a manner reminiscent of conventional detergents without, however, compromising membrane order at subsolubilizing concentrations. Owing to this mild and unusual mode of detergency, F6 OM outperforms a lipophobic fluorinated surfactant in chaperoning the functional refolding of an integral membrane enzyme by promoting bilayer insertion in the absence of micelles.

Inhibitory glycinergic neurotransmission in the mammalian auditory brainstem upon prolonged stimulation: short-term plasticity and synaptic reliability

Short-term plasticity plays a key role in synaptic transmission and has been extensively investigated for excitatory synapses. Much less is known about inhibitory synapses. Here we analyze the performance of glycinergic connections between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) in the auditory brainstem, where high spike rates as well as fast and precise neurotransmission are hallmarks. Analysis was performed in acute mouse slices shortly after hearing onset (postnatal day (P)11) and 8 days later (P19). Stimulation was done at 37°C with 1–400 Hz for 40 s. Moreover, in a novel approach named marathon experiments, a very prolonged stimulation protocol was employed, comprising 10 trials of 1-min challenge and 1-min recovery periods at 50 and 1 Hz, respectively, thus lasting up to 20 min and amounting to >30,000 stimulus pulses. IPSC peak amplitudes displayed short-term depression (STD) and synaptic attenuation in a frequency-dependent manner. No facilitation was observed. STD in the MNTB-LSO connections was less pronounced than reported in the upstream calyx of Held-MNTB connections. At P11, the STD level and the failure rate were slightly lower within the ms-to-s range than at P19. During prolonged stimulation periods lasting 40 s, P19 connections sustained virtually failure-free transmission up to frequencies of 100 Hz, whereas P11 connections did so only up to 50 Hz. In marathon experiments, P11 synapses recuperated reproducibly from synaptic attenuation during all recovery periods, demonstrating a robust synaptic machinery at hearing onset. At 26°C, transmission was severely impaired and comprised abnormally high amplitudes after minutes of silence, indicative of imprecisely regulated vesicle pools. Our study takes a fresh look at synaptic plasticity and stability by extending conventional stimulus periods in the ms-to-s range to minutes. It also provides a framework for future analyses of synaptic plasticity.

Sparingly fluorinated maltoside-based surfactants for membrane-protein stabilization

Membrane proteins pose formidable challenges during in vitro investigations, as they require amphiphilic molecules for their solubilization, stabilization, and crystallization for structural characterization. Therefore, numerous, chemically diverse new amphiphiles have been developed for membrane-protein applications. Among these, both perfluorinated and hemifluorinated surfactants have long been known to stabilize membrane proteins, but the contribution of the fluorine content in the aliphatic chain has not yet been examined in detail. We have synthesized two new maltose-based fluorosurfactants bearing either a perfluoroethyl (F2H9) or a perfluorobutyl (F4H5) tip at the end of the chain and compared them with the common detergent dodecyl maltoside and a commercial highly fluorinated octyl maltoside derivative. We describe the physicochemical properties, aggregate morphologies, and micellization thermodynamics of these sparingly fluorinated surfactants as a function of the length of the fluorinated segment and evaluate their biochemical use for membrane-protein stabilization. Intriguingly, the surfactant carrying a perfluorobutyl (F4H5) tip trumps both nonfluorinated dodecyl maltoside and a more extensively fluorinated octyl maltoside derivative in conferring extraordinary long-term functional and colloidal stability to the model membrane protein bacteriorhodopsin.

Model-Free Analysis of Critical Micellar Concentrations for Detecting Demixing in Surfactant Mixtures

Aqueous mixtures of two or more surfactants are often employed for research or industrial purposes because such mixtures offer advantages over single-surfactant systems. This is particularly true for mixtures of fluorocarbon (FC) and hydrocarbon (HC) surfactants, which display a broad range of mutual miscibilities in mixed micelles. Unfortunately, the prediction and even the experimental elucidation of the micellar mixing behavior of surfactant mixtures remain challenging, as evidenced by conflicting results and conclusions derived from diverse, and often complex, mixing models. One of the most intriguing questions is whether certain combinations of FC and HC surfactants form only one type of mixed micelle or rather demix into two micelle populations, namely, FC-rich and HC-rich ones. Here, we demonstrate a novel approach to the model-free analysis of critical micellar concentrations (CMCs) of surfactant mixtures that is based on a fit of the experimental data with cubic splines using a stringent thermodynamic criterion for mixing. As a proof of principle, we analyze CMC values determined by isothermal titration calorimetry and confirm the conclusions with the aid of combined 1H- and 19F-NMR spectroscopy. Specifically, we show that aqueous mixtures of an FC maltoside and an HC maltoside conform with the assumption of only one type of micelle regardless of the mixing ratio, whereas combining the same FC surfactant with an HC surfactant carrying a zwitterionic phosphocholine headgroup gives rise to two coexisting micelle populations at high mole fractions of the FC maltoside.

Dissecting Nanosecond Dynamics in Membrane Proteins with Dipolar Relaxation upon Tryptophan Photoexcitation

The structural dynamics of proteins on the nanosecond time scale can be probed with dipolar relaxation in response to photoexcitation of intrinsic tryptophan (Trp) residues. For membrane proteins, however, the complexity due to overlapping contributions from the protein itself, the membrane mimic, and the aqueous solvent impairs detailed analysis and interpretation. To disentangle these contributions, we measured time-resolved emission spectra of Trp in the protein Mistic in detergent micelles of various polarities. By comparison with Trp analogues in water and micelles, we could dissect the contributions from hydration, micelle, and protein matrix to dipolar relaxation on the nanosecond time scale. Our results demonstrate that ultrafast, subnanosecond relaxation reports on the extent of Trp shielding from water, with micelle and protein moieties making additive contributions. By contrast, relaxation in the low nanosecond regime is due to dipolar rearrangement of micelle and protein moieties upon photoexcitation, thereby probing conformational dynamics around the intrinsic fluorophore.

Conformational Dynamics Govern the Free-Energy Landscape of a Membrane-Interacting Protein

The equilibrium stabilities and the folding rates of membrane-bound proteins are determined by hydrophobic and polar intermolecular contacts with their environment as well as by intramolecular packing and conformational dynamics. The contributions of these factors, however, remain elusive and might vary considerably among proteins. Mistic from Bacillus subtilis is a particularly intriguing example of an α-helical protein that associates with membranes in spite of its unusual hydrophilicity. In micelles, Mistic is stabilized by hydrophobic and polar interactions with detergents, but it is unclear whether and how these intermolecular contacts are coupled to structural and dynamic adaptations of the protein itself. Here, we investigated the packing and the conformational dynamics of Mistic as functions of detergent headgroup chemistry and chain length, employing single-molecule Förster resonance energy transfer spectroscopy and time-resolved intrinsic tryptophan fluorescence spectroscopy. Surprisingly, in nonionic detergents, more effective hydrophobic burial and, thus, greater protein stability with increasing hydrophobic micellar thickness were accompanied by a gradual loosening of the helical bundle. By contrast, Mistic was found to assume a stable, compact fold in zwitterionic detergents that allowed faster dynamics on the nanosecond timescale. Thus, intramolecular packing per se is insufficient for conferring high protein stability; instead, enhanced nanosecond dynamics and, consequently, greater conformational entropy in the compact folded state account for Mistic’s high equilibrium stability and fast folding rates in zwitterionic micelles even at the expense of less effective hydrophobic burial.